IVF Embryos Research Articles

Use of artificial intelligence embryo selection based on static images to predict first-trimester pregnancy loss

Research questionCan an artificial intelligence (AI) embryo selection assistant predict the incidence of first trimester spontaneous abortion (SA) using static images of IVF embryos? DesignIn a blind, retrospective study, a cohort of 172 blastocysts from IVF cases with single embryo transfer and a positive biochemical pregnancy test was retrospectively ranked by the AI morphometric algorithm ERICA™. Making use of static embryo images from a light microscope, each blastocyst was assigned one of four possible groups (Optimal, Good, Fair, Poor), and linear regression was used to correlate the results with the presence or absence of a normal fetal heart rate (FHR) as an indicator of ongoing pregnancy or SA, respectively. Additional analyses included modelling for recipient age and chromosomal status established by preimplantation genetic testing (PGT-A). ResultsEmbryos grouped as Optimal/Good had a lower incidence of SA (16.1%) compared to embryos classified as Fair/Poor (25%, P=0.005, Odds ratio (OR)=0.46). The incidence of SA in chromosomally normal embryos (determined by PGTA) was 13.3% for Optimal/Good embryos and 20.0% for Fair/Poor embryos, although this difference was not statistically significant (P=0.531). There was a significant association between embryo rank and recipient age (P=0.018) in that the incidence of SA was unexpectedly lower in older recipients (21.3% in ≤35, 17.9% in 36-38, 16.4% in ≥39, P=0.0181, OR=0.354). Overall, these results support a correlation between risk of SA and embryo rank as determined by AI; the classification accuracy was calculated to be 67.4%. ConclusionsThis preliminary study suggests that AI (ERICA™), which was designed as a ranking system to assist with embryo transfer decisions and ploidy prediction, might also be useful in providing information for couples on the risk of SA. Future work will include larger sample size and karyotyping of miscarried pregnancy tissue.

BlastAssist: a deep learning pipeline to measure interpretable features of human embryos.

BlastAssist: a deep learning pipeline to measure interpretable features of human embryos.

IVF embryos in the bin, embryo-like structures in the spotlight

Embryo-like structures (ELS) are intended for the study of embryonic development without the use of human supernumerary embryos. Scientists working in countries that do not allow research on embryos hope that these structures will replace natural embryos. The interest in ELS is largely based on two misconceptions: the belief that there is a shortage of research embryos and the belief that research on ELS will make research on natural embryos redundant. This paper argues that research efforts should be refocused on natural embryos.

Association of early cleavage, morula compaction and blastocysts ploidy of IVF embryos cultured in a time-lapse system and biopsied for genetic test for aneuploidy

IVF embryos have historically been evaluated by morphological characteristics. The time-lapse system (TLS) has become a promising tool, providing an uninterrupted evaluation of morphological and dynamic parameters of embryo development. Furthermore, TLS sheds light on unknown phenomena such as direct cleavage and incomplete morula compaction. We retrospectively analyzed the morphology (Gardner Score) and morphokinetics (KIDScore) of 835 blastocysts grown in a TLS incubator (Embryoscope+), which were biopsied for preimplantation genetic testing for aneuploidy (PGT-A). Only the embryos that reached the blastocyst stage were included in this study and time-lapse videos were retrospectively reanalysed. According to the pattern of initial cleavages and morula compaction, the embryos were classified as: normal (NC) or abnormal (AC) cleavage, and fully (FCM) or partially compacted (PCM) morulae. No difference was found in early cleavage types or morula compaction patterns between female age groups (< 38, 38–40 and > 40 yo). Most of NC embryos resulted in FCM (≅ 60%), while no embryos with AC resulted in FCM. Aneuploidy rate of AC-PCM group did not differ from that of NC-FCM group in women < 38 yo, but aneuploidy was significantly higher in AC-PCM compared to NC-FCM of women > 40 yo. However, the quality of embryos was lower in AC-PCM blastocysts in women of all age ranges. Morphological and morphokinetic scores declined with increasing age, in the NC-PCM and AC-PCM groups, compared to the NC-FCM. Similar aneuploidy rates among NC-FCM and AC-PCM groups support the hypothesis that PCM in anomalous-cleaved embryos can represent a potential correction mechanism, even though lower morphological/morphokinetic scores are seen on AC-PCM. Therefore, both morphological and morphokinetic assessment should consider these embryonic development phenomena.

Avoidance Versus Elimination. How Can We Harm Less? How Can We Do Rather Good?

The present article focuses on hereditary genetic disorders which is specified and evaluated through comparative analysis. This comparison extends on highlighting a fewfactors in connection with avoidance through IVF embryo selection procedure and elimination, removal through germline gene editing. Focusing on the later listed factors the author introduces a new perspective of moral evaluation of the mentioned procedures. With the explanation of the dilemma, according to showing the factors and with the analysis of their effects the paper concludes that it is conceivable based on the factors discussed here that avoiding the appearance of genetic disease through IVF embryo selection is morally moreacceptable, given the current state of science and technology, than the direct modification, intervention into the germline.

Islamic Perspectives on Polygenic Testing and Selection of IVF Embryos (PGT-P) for Optimal Intelligence and Other Non-Disease-Related Socially Desirable Traits.

In recent years, the genetic testing and selection of IVF embryos, known as preimplantation genetic testing (PGT), has gained much traction in clinical assisted reproduction for preventing transmission of genetic defects. However, a more recent ethically and morally controversial development in PGT is its possible use in selecting IVF embryos for optimal intelligence quotient (IQ) and other non-disease-related socially desirable traits, such as tallness, fair complexion, athletic ability, and eye and hair colour, based on polygenic risk scores (PRS), in what is referred to as PGT-P. Artificial intelligence (AI) and machine learning-based analysis of big data sets collated from genome sequencing of specific human ethnic populations can be used to estimate an individual embryo's likelihood of developing such multifactorial traits by analysing the combination of specific genetic variants within its genome. Superficially, this technique appears compliant with Islamic principles and ethics. Because there is no modification of the human genome, there is no tampering with Allah's creation (taghyīr khalq Allah). Nevertheless, a more critical analysis based on the five maxims of Islamic jurisprudence (qawa'id fiqhiyyah) that are often utilized in discourses on Islamic bioethics, namely qaṣd (intention), yaqın̄ (certainty), ḍarar (injury), ḍarūra (necessity), and `urf (custom), would instead reveal some major ethical and moral flaws of this new medical technology in the selection of non-disease-related socially desirable traits, and its non-compliance with the spirit and essence of Islamic law (shariah). Muslim scholars, jurists, doctors, and biomedical scientists should debate this further and issue a fatwa on this new medical technology platform.

Islamic Viewpoints on Opportunistic Sex Selection of IVF Embryos upon doing Preimplantation Genetic Testing for Preventing Genetic Diseases.

In recent years, preimplantation genetic testing (PGT) of IVF embryos have gained much traction in clinical assisted reproduction for preventing various genetic defects, including Down syndrome. However, such genetic tests inevitably reveal the sex of IVF embryos by identifying the sex (X and Y) chromosomes. In many countries with less stringent IVF regulations, information on the sex of embryos that are tested to be genetically normal is readily shared with patients. This would thus present Muslim patients with unintended opportunities for sex selection based on personal or social biases without any pressing need or valid medical reason. Additionally, there are other patients who claim using PGT for preventing genetic defects as a pretext or "convenient excuse," with a secret intention to do sex selection when it is banned in their home country. Currently, non-medicalsex selection is a highly-controversial and hotly debated issue in Islam, because there is generally a strong preference for having sons over daughters due to widespread cultural norms of elderly parents depending on their sons for financial support, as well as the need for male heirs to continue the family lineage within the backdrop of local patriarchal cultures. There is a risk of gender imbalance and social disequilibrium occurring in Islamic societies due to prevalent sex selection. Hence, the question is whether opportunistic sex selection with PGT would contravene Islamic ethics and principles, which will thus be discussed here.

Pre-Vitrification and Post-Warming Variables of Vitrified-Warmed Blastocysts That Are Predictable for Implantation.

Human IVF embryos that are not used for fresh transfer are cryopreserved by vitrification for later embryo transfers. This study evaluates pre-vitrification and post-warming embryo characteristics that are suitable to predict the chance of clinical pregnancy in single vitrified blastocyst transfer (SVBT) cycles. In a multicenter observational trial (IMBOS trial), embryos were cultured in a time-lapse system before and after vitrification. Associations between clinical pregnancy, morphokinetic parameters, blastocyst collapse, KIDScore D5, pre-vitrification and post-warming Gardner scores, post-warming blastocyst size and re-expansion rates before SVBT were analyzed in 182 SVBTs which resulted in 89 clinical pregnancies. No association was found between clinical pregnancy after SVBT and the number of collapses or the maximal collapse size before vitrification. The multifactorial analysis of pre-vitrification Gardner scores showed a significant association with clinical pregnancy for trophectoderm grading but not for expansion/hatching status and inner cell mass grading. A significant association with clinical pregnancy was found for the time to reach a blastocyst after pronuclear fading (tB-tPNf), KIDScore D5 and post-warming size but not the rate of expansion or maximal expansion size. The selection of blastocysts for SVBT could benefit from using pre-vitrification parameters like tB-tPNf, trophectoderm grading and post-warming blastocyst size.

H19 Promoter DNA Methylation is Lower Among Early Abortion Patients Undergoing IVF Embryo Transfer.

H19 is the first long noncoding RNA (lncRNA) found to be associated with gene imprinting. It is highly expressed in the embryonic stage and may have important regulatory effects on human embryonic development. We investigated the differences between the levels of H19 promoter DNA methylation in the chorionic villi of patients who experienced spontaneous abortion (SA) following in vitro fertilization embryo transfer (IVF-ET) and those of patients with a normal early pregnancy (NEP). We also analyzed the associated DNA methyltransferase (DNMT) activity. Chorionic villus tissue from patients with SA and NEP were collected. The DNA methylation levels of two CpG islands in the promoter region of the H19 gene in the two groups were detected by bisulfite sequencing, and the mRNA expression of DNMTs was analyzed by real-time polymerase chain reaction. The sample size of each group was 32, and there were no significant differences in baseline data, including age, parity, and body mass index, between the two groups. Among the 7 CpG islands measured, the methylation rates of 3 CpG islands (CpG 1, 6, and 7) were significantly lower in the SA group than in the NEP group (P < 0.01). The methylation levels of the other 4 CpG islands were not significantly different between the two groups. There were no differences in the expression of DNMT1 between the two groups (P > 0.05), but DNMT3a and DNMT3b RNA levels were significantly lower in SA group than in the NEP group (P < 0.01). The lower H19 promoter DNA methylation levels found in the chorionic villi of patients with SA patients following IVF-ET may be explained by decreased expression of DNMT3a and DNMT3b.

Cumulative live birth rate and neonatal outcomes after early rescue ICSI: a propensity score matching analysis

Abstract STUDY QUESTION Is early rescue ICSI (E-RICSI) an effective and safe technique compared to conventional ICSI? SUMMARY ANSWER Despite the higher multi-pronucleus (PN) rate compared to conventional ICSI, E-RICSI did not add extra risks to clinical and neonatal outcomes. WHAT IS KNOWN ALREADY Based on the finding that the second polar body was released in 80% of fertilized oocytes by 4 h after exposure to spermatozoa and in ∼90% of fertilized oocytes by 6 h, E-RICSI brings forward the timing of rescue ICSI to 6 h after initial insemination, and effectively prevents oocyte aging and embryo-uterus asynchrony. However, some researchers still voice concerns about the efficacy and safety of E-RICSI, and comparative studies are limited. STUDY DESIGN, SIZE, DURATION A retrospective cohort study was conducted on patients who underwent conventional ICSI or E-RICSI treatment between January 2015 and December 2020 at a university-affiliated hospital. Using 1:1 propensity score matching, 1496 cases entered each group. PARTICIPANTS/MATERIALS, SETTING, METHODS In total, 1496 couples undergoing conventional ICSI oocyte retrieval cycles and 1496 undergoing E-RICSI oocyte retrieval cycles were enrolled in this study, and basic clinical characteristics, embryologic data, clinical outcomes and neonatal data were compared between groups. The embryos in the E-RICSI group were divided into two subgroups: those fertilized by iIVF (IVF subgroup) and those fertilized by E-RICSI (E-RICSI subgroup); the embryologic data, clinical outcomes, and neonatal data for these subgroups were also compared with the conventional ICSI group. Logistic regression was used for statistical analysis with potential confounder adjustment. MAIN RESULTS AND THE ROLE OF CHANCE The 2PN rate, blastocyst formation rate, and viable blastocyst formation rate of the E-RICSI group were significantly lower compared to the conventional ICSI group (2PN rate: P &amp;lt; 0.001; blastocyst formation rate: P &amp;lt; 0.001; viable blastocyst formation rate: P = 0.004), and the multi-PN rate in the E-RICSI group was significantly higher than the conventional ICSI group (P &amp;lt; 0.001). However, the number of 2PN embryos, normal cleavage embryo rate, Day 3 high-quality cleavage embryo rate, and high-quality blastocyst rate were similar between groups. When considering the IVF embryos and E-RCSI embryos in the E-RICSI group independently, the 2PN rate of the conventional ICSI group was significantly lower than E-RICSI subgroup but higher than the IVF subgroup, whereas the blastocyst formation rate and viable blastocyst formation rate were higher than E-RICSI embryos but comparable to IVF embryos. As for the clinical and neonatal outcomes, the implantation rate of the E-RICSI subgroup was significantly lower than the IVF subgroup but comparable to the conventional ICSI group, while the low birthweight (LBW) rate was significantly lower compared with the conventional ICSI group but similar with the IVF subgroup. No other differences were observed among the three groups for cumulative clinical pregnancy rate, cumulative live birth rate, and the pregnancy outcomes per transfer including clinical pregnancy, ectopic pregnancy, miscarriage, and live birth, either in fresh or frozen embryo transfer cycles. Furthermore, neonatal outcomes, including cesarean section, sex ratio, LBW, preterm birth, and macrosomia, were similar among groups. LIMITATIONS, REASONS FOR CAUTION This study is limited by the retrospective design, limited sample size, and short follow-up period. However, our study underlies the need for large-scale, multi-center randomized controlled trials with long-term follow-up. WIDER IMPLICATIONS OF THE FINDINGS Short-term insemination (3 h) combined with E-RICSI may be a safe and effective method to prevent the occurrence of total fertilization failure, and patients with normal or borderline sperm could be encouraged to try IVF first. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the National Key &amp; Development Program of China (No. 2021YFC2700603) and the National Natural Science Foundation of China (No. 81801443). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER N/A.

Pregnancy and neonatal outcomes in infertile patients with positive tuberculin skin test results

Research questionDo infertile women with positive tuberculin skin test (TST) results have a higher risk of adverse pregnancy outcomes after IVF or intracytoplasmic sperm injection and embryo transfer (ICSI–ET) and does preventive anti-tuberculosis treatment applied to infertile women with positive TST results before IVF/ICSI–ET affect pregnancy and neonatal outcomes? DesignThis was a retrospective cohort analysis of 6283 infertile women who underwent IVF/ICSI–ET treatment for the first time at the Reproductive Hospital affiliated to Shandong University from November 2016 to September 2022. None of the participants had prior tuberculosis or active tuberculosis. According to their TST results, 5947 patients who had never received preventive anti-tuberculosis treatment were divided into a TST-positive group (1704 cases) and a TST-negative group (4243 cases). A total of 504 patients with TST (+++) results (using the 20 mm sclerosis threshold) were divided into a treated TST (+++) group (336 cases) and an untreated TST (+++) group (168 cases) according to whether they received preventive anti-tuberculosis treatment before IVF/ICSI–ET. The outcome measures were pregnancy outcomes and neonatal outcomes. ResultsThere were no significant differences in pregnancy or neonatal outcomes between the TST-positive group and the TST-negative group (P > 0.05). In the TST (+++) group, there were no significant differences in pregnancy or neonatal outcomes between the treated TST (+++) group and the untreated TST (+++) group (P > 0.05). ConclusionsFor infertile women undergoing IVF/ICSI–ET without prior tuberculosis or active tuberculosis, positive TST results and preventive anti-tuberculosis treatments prior to IVF/ICSI–ET do not affect pregnancy or neonatal outcomes.

O-180 What is the true potential of 1PN embryos? A report on utilisation and live birth rates following PGT-A and biparental testing

Abstract Study question What is the developmental potential of monopronuclear (1PN) embryos, and does allowing their use only after confirming biparental inheritance offer a clinical solution for them? Summary answer 1PN embryos demonstrate clear utilisation, euploidy, and live birth rates, that warrant their routine culture, and use following biparental inheritance testing, in the clinical setting. What is known already Embryos only displaying a single pronuclei (1PN) are widely regarded to be abnormal and not suitable for clinical use. It is therefore common practice to discard these embryos following fertilisation check. However, 1PN embryos have shown the ability for normal blastocyst development, albeit at a decreased rate, and the ability to produce live births. To bridge the gap between discarding an “abnormal” 1PN embryo, and the knowledge that it can result in a live birth, our clinic utilises PGT-A with biparental inheritance testing to assess the fitness of a 1PN embryo for transfer. Study design, size, duration A retrospective analysis was performed of 5010 1PN embryos derived from IVF (2612) or ICSI (2398) insemination at Genea clinics across Australia between January 2017 and November 2022. Blastocyst development, euploidy, parental inheritance, pregnancy, and live birth rates were analysed. Participants/materials, setting, methods Embryos were defined as being 1PN by the appearance of one pronuclei at fertilisation check 16-18 hours post insemination. Time lapse footage was reviewed on day 3 of culture to eliminate the late appearance of a second pronucleus. Suitable embryos underwent trophectoderm biopsy for PGT-A and biparental inheritance testing. Only euploid embryos with confirmed biparental inheritance were available to patients for frozen embryo transfer. Main results and the role of chance 1PN embryos derived from ICSI insemination were found to have a significantly lower blastocyst development potential of 17.4% compared to 33.7% for IVF embryos (p &amp;lt; 0.0001). The utilisation rate was also significantly lower at 8.1% for ICSI vs 21.2% for IVF (p &amp;lt; 0.0001). The IVF embryos showed a higher rate of euploidy following PGA-T testing, 50.9% for IVF vs 37.9% for ICSI (p &amp;lt; 0.0001). Uniparental inheritance, an indication the 1PN appearance was caused by parthenogenesis, was only observed in 6.7% of the total embryos tested, with IVF embryos having a considerably lower rate of 1.8% vs 20.5% for ICSI (p &amp;lt; 0.0001). There was no significant difference between the 2 groups for pregnancy rate or live births, following single embryo transfer, with 58.6% for IVF vs 62.9% for ICSI (p = 0.6) and 39.6% for IVF vs 45.7% for ICSI (p = 0.5) respectively. These levels were comparable to normally fertilised (2PN) euploid embryos across Genea clinics in the same time period. During the study period there have been 175 1PN embryos transferred that has resulted in 69 live births from embryos that otherwise would have been discarded. Limitations, reasons for caution The use of timelapse imaging from the time of insemination for ICSI embryos, as opposed to 16-18 hours post insemination for IVF embryos may play a role in the type of 1PN embryos that are being identified in each group. Wider implications of the findings This study demonstrates that not all 1PN embryos are abnormal and their routine use in the clinical IVF lab in conjunction with appropriate testing should be re-evaluated. Trial registration number not-applicable

O-018 Impact of atypical bacterial semen infections on spermocytogram parameters and Spermatozoa DNA fragmentation and denaturation

Abstract Study question Does Spermocytogram parameters and Spermatozoa-DNA-Fragmentation (SDF) / Denaturation (SDD) levels are affected by atypical-bacterial semen infections in North-African men during the period between 2013-2022? Summary answer SDD presented positive-correlations with the presence of 3/5 atypical bacteria in the semen. Various Spermocytogram-parameters were also found to be negatively associated with atypical-bacterial-semen-infection. What is known already Atypical-Bacterial semen infections such as infections by Chlamydia-trachomatis (CT), Mycoplasma-hominis (MH) and genitalium (MG), Ureaplasma urealyticum (UU) and parvum (UP), were shown to be associated with poor sperm function (Prabha.et.al.,2009; Agarwal.et.al.,2012). In infections caused by CT, SDF was observed to be increased significantly and associated with male infertility (Karinen.et.al.,2004; Gallegos.et.al.,2008). In addition, MH and UU appeared to cause induction of nuclear decondensation and SDD, which damages sperm and has effects on sperm parameters. However, other studies failed to show any effect of MH, UU and CT on sperm parameters and SDD (Lee.et.al.,2013; Gdoura.et.al.,2008; Huang.et.al.,2015; Farsimadan and Motamedifar,2020). Study design, size, duration Retrospective study, including Sperm-culture-analyzes, Spermocytograms and SDF/SDD tests performed between January,2013 and December,2022 of 10.386 patients from North-Africa aged between 19 and 73 years-old (y.o) (average-age: 43.2 y.o ± 7.16). Patients were classified in 2 main groups according to the presence (Group-1: n = 613) or not (Group-2: n = 9773) of atypical-bacteria in the semen and 5 subgroups of monobacterial-infected-semen which were extracted from Group-1 as: CT (n = 14), MH (n = 61), MG (n = 10), UU (n = 147) and UP (n = 73) subgroups. Participants/materials, setting, methods 8201-Tunisian-patients(79%), 1142-Algerians(11%) and 1038-Libyans(10%) presented for semen-analyzes. Samples were analyzed according to World-Health-Organization (WHO) guidelines. Bacterial-semen-infection was detected by real-time-polymerase-chain-reaction(RT-PCR). Spermocytogram analyzes were performed according to WHO2010-recommendations using Sperm-class-analyzer-software (SCA5/6(CASA-system)) and SCA-scope (Microptic®). SDF analyzed with terminal-uridine-nucleotide-end-labeling(TUNEL)/sperm chromatin dispersion(SCD) techniques. SDD analyzed with the Aniline-Blue-staining-method. Statistical-analyzes were performed using SPSS22.0 for Windows-software. Kolmogorov–Smirnov-test for normality-analysis and comparisons by Student-t-test/Mann–Whitney U-test, as appropriate. Pearson/Spearman’ tests for correlations were used as appropriate, P-value&amp;lt;0.05 was considered as significant. Main results and the role of chance Sperm concentration was significantly lower in atypical-bacterial-semen-infected-group (Group-1) compared to controls (Group-2) with 11.39[0-311] vs 20.06[0-694]x106spz/mL, (p = 0.014); respectively, as well as progressive motility (14.6%[0%-64.47%] vs 21.89%[0%-81.35%], (p = 0.002); respectively) and typical morphology (5.34%[0%-18%] vs 9.29[0%-21%] (p &amp;lt; 0.001); respectively). However, Leucocytes concentration and SDD presented significant higher levels in Group-1 compared to Group-2 with 1.8[0.1-8.5] vs 0.5[0.1-2.45]x106 leucocytes/mL, (p &amp;lt; 0.001) and 21%[4%-60%] vs 15%[2%-75%], (p = 0.04); respectively. Similar significant differences were observed with 3/5 of the monobacterial-semen-infected-subgroups in comparison with Group-2 (MH, UU and UP-subgroups). No significant differences were observed in MG and CT-subgroups in except of SDD which presented significant higher levels in CT-subgroup in comparison with Group-2 (52%[4%-85%] vs 15%[2%-75%], (p = 0.002);respectively). Correlation-analyzes showed that SDD was positively correlated to MH (r = 0.069; p = 0.022) and UU (r = 0.084; p = 0.005) which is in accordance with the study of .Lee.et.al.(2013). Leucocytes concentration was also correlated positively to MH, CT, UU and UP (r = 0.047; p&amp;lt;0.001; r = 0.035; p=0.005; r = 0.074; p&amp;lt;0.001; r = 0.045; p=0.05; respectively). Sperm concentration, Progressive motility and Typical morphology presented Negative correlations with MH (r=-0.038; p=0.034; r=-0.048; p=0.05; r=-0.047; p=0.05; respectively), UU (r=-0.034;p=0.003; r=-0.04; p&amp;lt;0.001; r=-0.067; p=0.007; respectively) and UP (r=-0.072; p&amp;lt;0.001; r=-0.078; p=0.018; r=-0.057; p&amp;lt;0.05; respectively). Our results are in accordance with other studies such as of Prabha.et.al.(2009) and Agarwal. et al. (2012). However, SDF did not show any significant variation between our studied groups. Limitations, reasons for caution Our study is a retrospective-statistical survey that included patients presenting to the laboratory for fertility diagnosis or for a diagnosis due to a pathology and/or inflammation of the genital tract. Meta-analysis studies in addition to more prospective-randomized-controlled-trials in collaboration with IVF or other medical-analyzes-laboratories are necessary to confirm/refute our results. Wider implications of the findings These results should interest urologists, microbiologists, gynecologists, reproductive science fundamentalists, epidemiologists and embryologists who want to improve the investigations on the relationship between bacteria and semen parameters to the reasons of infertility, recurrent miscarriages and poor quality embryos in IVF. Trial registration number Not applicable

O-212 Screening IVF embryos with polygenic risk scores: risk reduction estimates, population impact, and attitudes of patients and clinicians

Abstract Study question IVF embryos can now be screened for complex diseases. What is the expected risk reduction? What are the attitudes of patients and clinicians? Summary answer Substantial risk reductions can be achieved, but only in the best-case scenario. The views of patients and clinicians regarding the technology are not fully aligned. What is known already It recently became possible to screen IVF embryos for a variety of complex diseases using polygenic risk scores. Prioritization of embryos for transfer based on these scores may lead to reduced disease risk in offspring. Preliminary risk reduction modeling suggested that substantial risk reductions are possible, particularly when screening for a single disease. However, multiple factors remain unexplored, such as the effect of implantation failure and assortative mating on risk estimates and the impact of embryo prioritization on population allele frequencies. The attitudes of IVF patients and clinicians towards polygenic embryo screening are also unknown. Study design, size, duration We estimated the expected risk reduction under various settings using statistical modeling, based on the liability threshold model and other quantitative and population genetic models. To learn about attitudes of patients and clinicians, we performed in-depth, semi-structured interviews with 26 USA patients and 27 USA clinicians over a period of about one year. Participants/materials, setting, methods We used R to numerically estimate risk reductions and implemented a web app in Shiny. To recruit patients and clinicians for the interviews, we used flyers (for patients), emails on publicly available lists (for clinicians), snowball sampling, and convenience sampling. Interviews were conducted over Zoom for one hour each by a single interviewer (D.B.). The study was approved by Baylor College of Medicine's IRB committee. We applied deductive and inductive thematic analysis using a team-based approach. Main results and the role of chance Statistical genetic modeling suggested that with five viable embryos, risk reductions of up to 50% can be achieved when screening for a single common disease. We developed a risk reduction estimator web app, allowing users to examine the effect of the number of embryos, the accuracy of the score, the selection strategy, and the score percentile or disease status of the parents. Assortative mating and modeling of implantation failures are expected to only slightly decrease risk reductions. Risk allele frequencies are expected to change very little across generations even if embryo selection becomes widespread. In interviews, patients accepted screening for health conditions, particularly severe diseases. Many were concerned about costs, and some were interested in screening for informative purposes. Most patients did not oppose screening for traits. In contrast, about half of clinicians were unwilling to offer screening due to concerns over utility, implementation, harm to patients, and lack of scientific validity, although all were willing to reconsider it in the future for specific conditions. Many clinicians considered screening for traits a non-medical procedure. About half agreed that screening should be regulated. Limitations, reasons for caution The risk reduction estimates may depend on the assumptions of the statistical models. As with any qualitative research, interview findings may not be generalizable. Furthermore, the design of the semi-structured interview guide may have influenced participants’ expressed attitudes. Wider implications of the findings In the best-case scenario, prioritizing IVF embryos based on polygenic scores may achieve substantial risk reductions. Several initial concerns seem unfounded. IVF patients are broadly interested in the technology but may be unaware of its many limitations. Clinicians have major concerns regarding clinical implementation and harm to patients. Trial registration number not applicable

P-541 Does the IVF embryo remain at the site of deposition in successful IVF cycles?

Abstract Study question Does the placement of the IVF embryo differ from the implantation site? Summary answer At five weeks the implanted embryo has not moved in the lateral plane of the endometrial cavity but is slightly lower in the vertical plane. What is known already We previously showed that vertical placement of embryos approximately 9 mm from the fundus and equidistant from the tubal ostia has the highest implantation rate. Whether the embryo migrates from the point where the droplet is deposited before implantation is controversial. We used 3D ultrasound to compare the point of placement with the location of the gestational sac (GS) at 5 weeks gestational age (GA). Study design, size, duration Prospective analysis of 292 transfers of single egg donation blastocysts (2018-2020) and 139 resultant pregnancies in which the position of the GS in the coronal plane at 5 weeks was compared to that of the embryo flash immediately post transfer. A Voluson 6 with a 4D RAB4-8 transabdominal probe imaged the embryo transfer and a 4D RIC-5-9 transvaginal probe imaged the pregnancy scan (GE Healthcare, Austria). Participants/materials, setting, methods Consecutive patients with a normal uterine cavity undergoing egg donation in a single private IVF Center. Embryo transfers were performed under 2D US guidance using a soft Wallace Catheter. A 3D volume of the uterus was acquired immediately post-transfer. A second 3D scan was performed at 5 weeks GA. Distances of the embryo flash and the center of GS from the fundus, ostia and internal cervical os (ICO) were recorded and compared using SPSS. Main results and the role of chance Of 292 transfer procedures, 139 pregnancies who had the first ultrasound in our Center were included and analysed. The mean distance between the embryo flash and the uterine fundus was significantly less than the distance between the center of the GS and the uterine fundus [(9,49±3,03 vs 11,02±3,9 mm respectively (p &amp;lt; 0,05)]. The mean distances between the embryo flash and the right and left ostium were 16,66 ± 4,96 and 16,69 ± 4,61 mm respectively, with no significant differences from the same distances taken from the center of the GS. Limitations, reasons for caution As the image is of the embryo flash and not the embryo, this might have been misleading. Wider implications of the findings These findings extend and support the proposal of an optimal point of embryo placement for pregnancy. The apparent vertical difference in the implantation of the embryo could be due to uterine growth or it could be consequence of uterine peristalsis. This bears further study. Trial registration number not applicable

P-235 Influence of female karyotype polymorphic variants on oocyte quality and embryo development

Abstract Study question Do polymorphic variants in the karyotype of women undergoing an IVF cycle directly affect oocyte and embryo laboratory parameters? Summary answer The presence of certain polymorphic variants negatively affects the oocyte survival rate and the blastocyst quality in an IVF cycle. What is known already In the recent years, there has been a growing interest in the study of polymorphic variants in infertile patients because their incidence, compared to the fertile population, is increased. However, most research has focused on the male patient study. Several studies have reported information about clinical outcomes, but the influence they may have on IVF laboratory procedures and embryo development has rarely been studied up to the blastocyst stage. In addition, it is very rare to find publications that show the study of polymorphisms according to their type or combination. Study design, size, duration Retrospective evaluation of a cohort of women who underwent autologous IVF cycles and karyotyping. The sample included 424 IVF cycles performed between July 2017-December 2021: control group (CG) with normal karyotype (211) and study group (SG) with polymorphisms (213). We studied the correlation between karyotype polymorphisms and laboratory outcomes in terms of: Number of Oocytes Retrieved, Fresh Oocyte Maturity (MII), Oocyte Survival after Thawing (TS), Fertilization (FZ), Oocyte Degeneration (OD) and Non-viable embryo rates. Participants/materials, setting, methods Analysis of the women karyotype, prior to the IVF cycle, was performed using the guidelines of the International System for Human Cytogenetic Nomenclature (ISCN). Differences between the different study groups (presence or absence of different chromosomal polymorphisms) were assessed with the appropriate statistical test according to the normality or non-normality of the variable distribution. Statistical analysis was performed using R statistical software (v.4.2.0) and SPSS (v.23.0, Chicago, IL, USA). Main results and the role of chance Average female age was 37.72 ± 3.98 (CG) and 36.73 ± 3.59 (SG). Average number of mature oocytes (MII) was: 6.45 ± 4.96(CG) and 7.59 ± 5.10 (SG). Statistically significant differences were found regarding the number of oocytes retrieved between the CG (8.14 ± 5.90) and the SG (9.53 ± 6.63) (p = 0.036), increasing when the ps+ variant was present (9.75 ± 7.21) (p = 0.045). However, no statistically significant differences were found between the presence of polymorphism (SG) and the CG in terms of: number of MII: 79.85% vs. 80.48% (p = 0.447), FZ: 73.76% vs. 70.49% (p = 0.352) and OD: 8.76% vs. 8.36% (p = 0.563) rates. In addition, the TS rate was statistically significant when there was the ps+ variant (82.95%) (p = 0.010) and/or combinations of more than one polymorphic variant (87.80%) (p = 0.044), compared to the CG (93.51%). Finally, according to embryo quality there was an increase in the non-viable embryos rate, on day 5 and/or day 6 of development, between the CG (34.17%) and the SG (43.02%) (p &amp;lt; 0.001), increasing when the ps+ variable was present (45.57%) (p &amp;lt; 0.001) and/or combinations of more than one polymorphic variant (p = 0.045). 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 Other variables that have not been analyzed may influence the outcomes. Larger prospective studies including homogeneous cohorts are needed in order to corroborate our initial results. Wider implications of the findings The polymorphic variants in the female karyotype, especially the “ps+” variant and the combination of multiple variants, could influence certain parameters in the laboratory. Therefore, it is important to request a karyotype to all patients before starting IVF treatment. Trial registration number NOT APPLICABLE

O-132 Comparison of pregnancy rates in antagonist cycles after luteal support with GnRH-agonist versus progesterone – prospective randomized study

Abstract Study question Are pregnancy rates in GnRH-antagonist cycles triggered with hCG and luteal phase support (LPS) with intranasal GnRH-agonist or vaginal progesterone comparable? Summary answer Nasal GnRH-agonist for LPS is associated with higher pregnancy rates compared with standard progesterone support in antagonist-based cycles triggered with hCG What is known already Regardless of its mechanism of action, the use of GnRH-a during the luteal phase in addition to progesterone, either in a single or multiple doses, has become a common practice as a part of ART protocols, based on its association with higher pregnancy and live birth rates . Its use as a sole agent for LPS has not gained the same popularity although studies supporting non-inferiority and even higher live birth rates with GnRH-a as a sole LPS agent have been published. Study design, size, duration A single-center, prospective, randomized study. A total of 150 patients underwent 164 GnRH-antagonist-based IVF cycles triggered with hCG and fresh embryo transfer. The study was conducted between June 9th, 2020, and May 5th, 2022. Participants/materials, setting, methods The study was conducted in the IVF clinic at University-affiliated tertiary medical center. Patients meeting the inclusion criteria who underwent antagonist-based IVF cycles and hCG triggering were enrolled. Computer-based randomization (1:1 ratio) was performed on the day of oocyte pickup (OPU). In both groups LPS was initiated on the evening of the OPU day with either GnRH-agonist nasal spray (Nafareline, nasal spray 200 mcg twice daily)or vaginal micronized progesterone (Utrogestan 300 mg, 3 times daily). Main results and the role of chance A total of 150 patients underwent 164 cycles, 127 cycles of which were included in the study cohort. Of them, 64 (50.4%) and 63 (49.6%) cycles were treated with GnRH-agonist or progesterone respectively as sole luteal phase support. A significantly higher pregnancy rate was found in the GnRH-a group compared with the progesterone group (43.8% versus 25.4% respectively; P = 0.030). After adjustment of several potential confounders such as age, body mass index (BMI), past obstetric history, number of IVF cycles, oocytes retrieved and embryos transferred, GnRH-agonist was still associated with a higher pregnancy rate (odds ratio 3.4, 95% confidence interval 1.4-8.3). Ovarian hyperstimulation syndrome (OHSS) rates were similar between the groups. Significantly higher progesterone levels were measured at β-hCG testing day in the GnRH-a group, both for the cleavage stage embryos (129.1±23.7 nmol/L vs. 97.7±48.9 nmol/L, P = 0.024) and for the blastocysts (127.2±0.1 nmol/L vs. 75.3±49.1 nmol/L, P = 0.034). Limitations, reasons for caution This study is not powered for analysis of clinical pregnancy rates, live birth rates and pregnancy outcomes. Larger studies are needed for research of these outcomes. All patients participating were at low risk for OHSS, therefore the study is underpowered to find significant differences in early or late OHSS.Wider implications of the findings Our findings suggest that intranasal GnRH-agonist administration as a sole luteal support in antagonist-based IVF cycles triggered with hCG results in higher pregnancy rates in comparison with traditional irritating vaginal preparations while not mandating additional support in the first weeks of pregnancy. Trial registration number Clinicaltrials.gov - NCT05484193

O-063 Accurate mitochondrial DNA quantification clarifies the clinical value of measuring mtDNA in trophectoderm biopsy specimens

Abstract Study question Can accurate mitochondrial DNA (mtDNA) quantification of trophectoderm (TE) biopsy specimens provide insights into the biology and viability of blastocyst-stage human embryos? Summary answer mtDNA quantity in TE cells is correlated with embryo morphology and shows alterations associated with aneuploidy. However, measurement does not significantly improve embryo viability assessment. What is known already Mitochondria are essential organelles, responsible for producing ATP. Changes in the amount of mtDNA in blastocysts biopsy specimens have been reported in association with embryo implantation potential, leading to proposals that mtDNA quantification might serve as a useful biomarker of embryo viability. However, results from clinical studies to explore this possibility have yielded contradictory data, due in part to deficiencies of the molecular methods used for mtDNA measurement. We sought to clarify what quantification of mtDNA can tell us about embryo biology and viability by developing and applying a method that we believe to be the most accurate ever devised. Study design, size, duration This study involved the analysis of samples collected during the course of routine preimplantation genetic testing for aneuploidy (PGT-A). The IVF treatments and embryo biopsies were undertaken at two different clinics, while chromosomal analyses were carried out at a single reference laboratory. Mitochondrial data was subsequently analysed in a university setting. The embryos analysed were derived from a broad population of patients referred for PGT-A (average age 38.7 years; range 25-47). Participants/materials, setting, methods 651 blastocysts from 133 couples underwent trophectoderm biopsy on day-5 or day-6. The specimens were analysed using a highly validated real-time PCR method, which was used to quantify three distinct sites in the mtDNA and 198 loci in the nuclear genome. The measurement of multiple independent loci provided outstanding sensitivity and accuracy. The nuclear loci were used to normalise the mtDNA data, adjusting for differences in the number of cells in the biopsy specimens. Main results and the role of chance The method developed displayed extraordinary sensitivity and accuracy when quantifying mtDNA. Lower mtDNA quantities were associated with day-6 biopsy (p &amp;lt; 0.0001), extent of blastocyst expansion (p &amp;lt; 0.0001), and superior TE morphology, although the latter was not statistically significant (p = 0.09). mtDNA levels were higher in aneuploid embryos (p &amp;lt; 0.0001), independent on patient age. However, the difference was not sufficient to be considered diagnostic. There was no correlation between mtDNA level and the chances of blastocyst implantation in this dataset. Lower mtDNA levels, previously reported to be associated with higher probabilities of embryo implantation, were most often observed in embryos of excellent morphological grade and likely reflect the increased TE cell numbers of such embryos. Little if any mtDNA replication occurs during preimplantation development and consequently the mtDNA content is divided amongst an ever-growing number of cells, meaning less mtDNA per cell. In this context, mtDNA quantification of blastocyst biopsy specimens provides a highly sensitive measure of TE cellularity, but probably provides little additional benefit for embryo selection beyond conventional morphological grading. However, the fact that higher mtDNA quantities were observed in aneuploid embryos, may indicate that subtle differences in TE cellularity exist in abnormal embryos, which are not fully captured by traditional morphological assessment. Limitations, reasons for caution Previous studies suggested that some blastocysts have greatly elevated mtDNA levels and that such embryos are not viable. In this study, only 5% of embryos were considered outliers in terms of mtDNA quantity. Unfortunately, none of these embryos were transferred, so the potential of these embryos could not be assessed. Wider implications of the findings The quantification of mtDNA in trophectoderm biopsies has sometimes been used for the prioritisation of embryos for transfer. While our results confirm existence of biologically interesting associations between mtDNA and aneuploidy, and a relationship with certain aspects of embryo morphology, measurement of mtDNA seems unlikely to significantly improve embryo selection. Trial registration number not applicable

O-263 Effect of clindamycin and a live biotherapeutic containing lactobacilli on the reproductive outcomes of IVF patients with abnormal vaginal microbiota: a double-blind, placebo-controlled multicentre trial

Abstract Study question Does treatment of abnormal vaginal microbiota improve the reproductive outcomes of IVF patients? Summary answer Data is being analysed and will be presented at ESHRE 2023. What is known already An increasing number of studies reported an association between abnormal genital tract microbiota and adverse reproductive outcomes in IVF patients. One hypothesis could be that vaginal microbiota ascend to the endometrium where the microbiota hampers implantation. To the best of our knowledge, this is the first study powered to investigate causality between abnormal genital tract microbiota - in this study defined by a bacterial vaginosis like vaginal microbiota - and clinical pregnancy rate in IVF patients. Study design, size, duration Double-blind, placebo-controlled multicentre trial in IVF patients diagnosed with abnormal vaginal microbiota and subsequently randomised into three parallel groups 1:1:1. The first group received clindamycin 300 mg ×2 daily for 7 days followed by vaginal Lactobacillus crispatus until the clinical pregnancy scan. The second group received clindamycin and placebo, whereas the third group received placebo/placebo. A total of 1518 patients were screened, and 338 patients were randomised. The study duration was from 2017 to 2023. Participants/materials, setting, methods IVF patients with any cause of infertility embarking on their first, second or third IVF stimulation cycle or embryo transfer were approached for inclusion. At a minimum 12 days prior to embryo transfer, patients were screened for abnormal vaginal microbiota defined by a qPCR assay, targeting high quantitative loads of Fannyhessea vaginae and Gardnerella spp. Patients were excluded if they had intrauterine malformations like polyps, septum, and fibroma. Main results and the role of chance Data and safety monitoring board has decided not to open the randomization code yet. The final dataset is being completed. Data is being analysed and will be presented at ESHRE 2023. Limitations, reasons for caution Data is being analysed and will be presented at ESHRE 2023. Wider implications of the findings The present drug intervention study used clindamycin and a live biotherapeutic containing Lactobacillus crispatus. The results of the study could have a worldwide important clinical impact on daily IVF practice as currently there is no evidence that screening and treating IVF patients with abnormal vaginal microbiota improves the reproductive outcome. Trial registration number EudraCT 2016-002385-31

P-741 Morphokinetic Goldilocks: assessing the morphokinetic range to identify embryos with the optimal chance of being euploid

Abstract Study question Can CHLOE-EQ, an AI embryo assessment support tool, automatically identify the optimal time-range of morphokinetic events where chance of euploidy is maximized? Summary answer Embryos that are within the normal morphokinetic range have increased chances of being euploid compared to embryos developing at a pace outside the optimal range. What is known already The introduction of time-lapse technologies in IVF has revealed quantitative and qualitative morphokinetic parameters that predict embryo viability (ESHRE Workshop group, 2020), but their assessment is time-consuming and subjective. Artificial Intelligence (AI) based tools, such as CHLOE-EQ (Fairtility), are ideally suited to automatically annotate morphokinetics as part of a range of tools to quantify embryo quality and detect abnormalities. There have been several attempts in the literature to predict ploidy with morphokinetics. We postulated that embryos that develop at a normal pace (not too fast and not too slow) would be more likely to be euploid. Study design, size, duration Retrospective case-controlled study of 1328 time-lapse videos collected in 2022 from IVF and ICSI embryos from a private single fertility clinic. 142 of those were biopsied and genetically tested by NGS. The embryos were automatically assessed by CHLOE-EQ (Fairtility), an AI embryologist support tool. Participants/materials, setting, methods Time-lapse videos were automatically annotated using CHLOE-EQ(Fairtility) for morphokinetics, number of pronucleates and anomalies. The frequency distribution for each morphokinetic parameter was compared between euploid and aneuploid embryos to establish ranges for optimal euploidy rate. The ranges between optimal (maximum euploidy rate) and sub-optimal (outside optimal range) were compared (t-test). Efficacy of blastocyst, utilisation and ploidy prediction by CHLOE blast score at 68hpi and CHLOE-EQ score were assessed using the area under the curve (AUC). Main results and the role of chance For each morphokinetic event, an optimal range for identification of euploids was identified (tPNf:21.37-25.78; t2:24.01-28.6; t3:34.07-39.20; t4:35.5-40.64; t5:46.12-53.92; t6:48.77-55.63; t7:50.22-57.45; t8:52-60.21; t9:67.35-75.55; tM:78.49-89.08; tsB:92.20-102.39; tB:99.54-109.83; tEB:106.42-120.38). Optimal range of euploid embryos was smaller than the total range for all embryos (p &amp;lt; 0.001): tPNf (0.27 vs 152.36), t2(5.52 vs 158.96), t3(22.7 vs 159.29), t4(30.38 vs 167.96), t5(32.02 vs 168.29), t6(35.58 vs 155.44), t7(41.04 vs 157.65), t8(41.37 vs 158.06), t9(48.85 vs 158.39), tM(56.4 vs 163.89), tSB(84.74 vs 173.26), tB(93.01 vs 168.62); tEB(95.96 vs 164). Embryos with optimal ranges across morphokinetic events had a higher euploidy rate than embryos with suboptimal ranges [50% (11/20), 35.35% (35/99), NS]. CHLOE-BLAST Score at 68hpi was predictive of blastulation (AUC=0.86), whilst CHLOE-EQ Score was predictive of utilisation (AUC 0.88) and euploidy (AUC=0.64) and CHLOE Ranking was predictive of utilisation (AUC=0.91) and selection for transfer (AUC=0.80). Limitations, reasons for caution This is a single-center, retrospective study, where only the blastocysts deemed suitable for biopsy were assessed for ploidy. Therefore, the ploidy rate of non-blastocysts or inferior quality embryos is unknown, creating a potential bias regarding the lower cutoff threshold for optimal ranges. Wider implications of the findings CHLOE-EQ can identify the optimal morphokinetic time range to maximise the chance of an embryo being euploid, a potentially valuable biomarker for embryo assessment, selection, managing patients expectations down to individual embryos, and helping reduce the chance of viable embryos being discarded. Trial registration number Not applicable