IVF Embryos Research Articles

P–282 A time lapse analysis of 36,671 embryos to compare the incidence of early stage abnormal cleavage events in ICSI and IVF derived embryos

Abstract Study question Is this incidence of early stage abnormal cleavage events different between embryos created following ICSI compared with IVF? Summary answer Embryos derived from ICSI are more likely to exhibit abnormal cleavage compared with those from IVF. This difference is most marked in women ≥35 years. What is known already Time lapse imaging (TLI) has been instrumental in allowing detailed annotation of early embryo development to provide an objective aid for embryo selection in ART cycles. Amongst several abnormal cleavage events reported, rapid cleavage and multichotomous mitosis/direct cleavage, during the first days after fertilisation have been demonstrated to be associated with lower blastulation rates, reduced implantation potential, increased aneuploidy and poor pregnancy outcomes. With ICSI being utilised commonly, and being the insemination method of choice in some clinics, the incidence of abnormal cleavage was investigated in association with insemination method, ICSI or IVF. Study design, size, duration The incidence of abnormal cleavage events was evaluated in a large multicentre retrospective analysis of 36,671 embryos from 6689 patients treated in 8 IVF clinics enabled with time lapse imaging, between 2011 - 2019. This constituted 10931 IVF embryos and 25740 ICSI embryos. Participants/materials, setting, methods Following ICSI or after IVF fertilisation check, embryos were time-lapse imaged every 10 minutes and annotated using the EmbryoScope. Second cell cycle durations were calculated as follows: time to reach 3-cell (t3) from 2-cell (t2) (t3-t2 = cc2). These were analysed using a welch t-test as three groups of abnormal cleavage: direct cleavage/trichotomous mitosis (DC) - where cc2=0 hours(h), rapid cleavage within 2h (R2) - where 0<cc2<2h and rapid cleavage between 2–5h (R5) where 2h<cc2<5h. Main results and the role of chance The incidence of DC, R2 and R5 in the whole cohort of embryos was 5%, 8% and 9% respectively. In the subpopulation of IVF embryos the incidence of DC, R2 and R5 was 4%, 8% and 9% respectively. In the subpopulation of ICSI embryos the incidence of DC, R2 and R5 was 6%, 8% and 9% respectively. The incidence of DC was significantly higher in ICSI embryos compared with IVF (p < 0.001) whilst R2 and R5 were the same. ICSI derived embryos had a mean (± SE) cc2 value of 9.39 ± 0.03h, compared with 9.56 ± 0.05h for IVF embryos (p < 0.0038). Examination of data split by maternal age demonstrated that ICSI oocytes from women of advanced maternal age (≥35) also had significantly more embryos exhibiting rapid cleavages R2 and R5 than IVF oocytes (p < 0.007). There were no significant differences however, in rates of abnormal cleavages between ICSI and IVF in embryos from women aged <30 (p = 0.06). Male-factor diagnoses showed no significant differences in abnormal cleavage values between ICSI or IVF in all three abnormal cleavage categories. Limitations, reasons for caution This analysis could not control for all potential confounders therefore it is possible that the increased abnormal cleavages observed in this investigation are a result of another, or combination of factors. Despite quality assurance programs being in place across all clinics, there is a risk of annotation bias. Wider implications of the findings: There is a higher incidence of early abnormal cleavage in embryos derived from ICSI, particularly in those from women of increased age and this research may help elucidate the reasons for this and add to the debate regarding the appropriateness of the increasing use of ICSI. Trial registration number Not applicable

P–190 Can pregnancy be predicted according to mean infra-red attenuated total reflectance (MIR-ATR) spectrometry of incubation medium of embryos in IVF?: A proof of concept

Abstract Study question Can MIR-ATR spectrometry of embryo incubation medium be used to predict embryo quality and IVF treatment results. Summary answer MIR-ATR spectrometry is able to distinguish between good and poor embryo quality and may improve the prediction of pregnancy based on better embryo selection. What is known already Infra Red (IR) spectra enable to determine if certain chemical functional components are present in a molecule. Attenuated total reflection (ATR) spectroscopy is a well established, simple and rapid technique for objective classification of biological samples. ATR spectrometry was investigated and confirmed to serve as an additional diagnostic tool in oral and gynecologic cancer. Currently, embryo quality is assessed based on morphokinetics parameters from Time-laps incubator and/or final embryo’s grading Study design, size, duration Culture media of 227 embryos on cleavage stage and blastocyst stage were collected and analysed between January 1st, 2018 and December 31st 2020. Participants/materials, setting, methods The incubation medium liquid of embryos cultured in Time-lapse incubator were collected after transferring the embryos to their final destination. Infra-Red (IR) absorbance spectra was measured. Nominal resolution was 4 cm–1, wavenumber range was from 600 cm–1 to 5000 cm–1 and each absorbance spectrum was normalized to the height of amide I band at ∼1650cm–1. Machine learning techniques utilized to build discrimination models for the absorbance data. Results were correlated with clinical and pregnancy results. Main results and the role of chance This preliminary study demonstrates that ATR spectrometry differs in media of : Day 3 embryos compared to day 5 embryos, top quality compared to poor quality embryos and implanting embryos comparing to non-implanting embryos. We found that MIR-ATR spectrometry might predict pregnancy in accuracy rates of 85%. MIR-ATR spectrometry of incubation medium can be used as an additional tool for selection of embryos for transfer. Limitations, reasons for caution Additional study and collection of larger number of culture Media is requested to validate MIR-ATR spectrometry as an additional tool in clinical set up in IVF units and laboratories. Wider implications of the findings: MIR-ATR spectrometry can be used in the future as an additional tool for selection of embryos and prediction of cycles outcomes. Trial registration number NCT03317418

O-056 Evaluating the utility of screening human IVF embryos with polygenic risk scores for complex diseases

Abstract Study question It is now feasible to screen human IVF embryos with “polygenic risk scores” for predicting complex disease risk. What is the expected risk reduction? Summary answer Under some conditions, prioritizing embryos based on polygenic risk scores can lead to substantial disease risk reductions. However, only excluding high-risk embryos is less effective. What is known already Recent genetic studies have identified numerous mutations associated with complex diseases, leading to the development of accurate polygenic risk scores (PRSs) for disease risk prediction. Given that genomes of human IVF embryos can now be sequenced with relative ease, it has become technically feasible to use PRSs for prioritization of embryos for transfer. Clearly, such use is associated with ethical and social concerns, from inequality to eugenics. Nevertheless, polygenic embryo screening is already offered to consumers, with little research so far on expected outcomes. Our previous evaluation of screening IVF embryos for polygenic traits showed little current utility. Study design, size, duration This is a theoretical/computational study based on statistical genetics theory and simulations. Participants/materials, setting, methods We used the liability threshold model to estimate the disease risk given the PRS. We considered screening for a single disease (with known prevalence and PRS accuracy), and assumed that n viable embryos are available. We calculated the risk of the child given these parameters and the prioritization strategy, either when parents are random or when their disease status is known. We also used simulations based on genomic data from a schizophrenia case-control study. Main results and the role of chance We modeled the disease risk of a hypothetical future child when the PRSs of embryos are used for prioritization, relative to random selection. When selecting an embryo at random among those who do not have an extremely high risk (typically, top 2% of the PRS distribution), the relative risk reduction (RRR) is limited, and is under 10% for currently realistic scenarios. In contrast, selecting the lowest risk embryo for implantation results in substantial RRRs of ∼20-50% already with n = 5 embryos and realistic disease parameters. For example, the RRR for schizophrenia is > 40% with current PRSs, a result we validated with simulated genomes of parents and children based on genotypes from a schizophrenia study. When one of the parents is known to be affected, selecting the lowest risk embryo out of n = 5 may restore the risk of the future child to nearly normal levels. Limitations, reasons for caution Our analytical modeling is based on several simplifying assumptions regarding the dependence of the risk on the PRS and the accuracy of the PRS. Further, the estimated risk reductions depend on the availability of n = 5 embryos that could lead to a live birth. Wider implications of the findings Under some conditions, prioritizing embryos for transfer based on polygenic risk scores could lead to substantial disease risk reductions. However, predicted outcomes vary considerably with prioritization strategies and disease and PRS parameters. The emerging ethical and social concerns and the challenges in communicating these results need to be urgently discussed. Trial registration number Not applicable

P–240 Human extracellular vesicles (EVs) secreted by aneuploid embryos potentiate development of non-invasive PGT-A RNA biomarkers and stimulate MUC1 up-regulation in primary endometrial stromal cells (ESCs)

Abstract Study question Could EVs secreted by aneuploid embryos a) serve for development of RNA biomarkers for PGT-A and b) elicit a relevant transcriptomic response in decidualised ESCs? Summary answer Aneuploid embryo EVs a) contain PPM1J, LINC00561, ANKRD34C and TMED10 in differential abundance from euploid EVs and b) induce up-regulation of MUC1 in decidualised ESCs. What is known already Embryo aneuploidy accounts for approximately 50% of all recurrent implantation failures in women >35 years old. PGT-A identifies euploid embryos to increase implantation probability but the technology is controversial as it requires an invasive embryo biopsy with an elusive long-term biosafety. The development of non-invasive methods to screen out aneuploid embryos is paramount. It is also critical to decode the embryo-endometrial dialog underlying implantation failure. We have previously reported that IVF embryos secrete EVs that can be internalised by ESCs, conceptualising that successful implantation to the endometrium is facilitated by EVs, which may additionally serve as biomarkers of ploidy status. Study design, size, duration Embryos destined for biopsy on days 5–7 for PGT-A were grown under standard conditions. Spent media (30μl) were collected from euploid (n = 175) and aneuploid embryos (n = 145) at both cleavage (days 1–3) and blastocyst (days 3–5) stage. Media samples from n = 35 cleavage embryos were pooled in order to obtain five euploid and four aneuploidy pools. Blastocyst media were pooled to create one euploid and one aneuploid pool. ESCs were obtained from five women undergoing diagnostic laparoscopy. Participants/materials, setting, methods The study was realised at a research hospital. EVs were isolated from euploid and aneuploid Day3 pools with differential ultracentrifugation and EV-RNA sequencing was performed following the SMARTer Stranded Total RNA-Seq approach. ESCs were decidualised (E2:10nM, P4:1uM, cAMP:0.5 mM twice every 48 hours) and treated for 24 hours with 50 ng/ml euploid or aneuploid EVs extracted from blastocyst media. RNA sequencing was performed on ESCs following the Truseq RNAseq protocol. Main results and the role of chance Aneuploid cleavage stage embryos (n = 4) secreted EVs that were less abundant in RNA fragments originating from the genes PPM1J (log2fc=–5.13, p = 0.011), LINC00561 (log2fc=–7.87, p = 0.010) and ANKRD34C (log2fc=–7.30, p = 0.017) and more abundant in TMED10 (log2fc=1.63 p = 0.025) compared to EVs (n = 5) from euploid embryos. Decidualisation per se induced downregulation of MUC1 (log2FC=–0.54, p = 0.0028) in ESCs as prerequisite for the establishment of receptive endometrium. The expression of MUC1 transcript in decidualised ESCs was significantly increased following treatment with aneuploid compared to euploid embryo-secreted EVs (log2FC=0.85, p = 0.0201). Limitations, reasons for caution The findings of the study may require validation utilising a second cohort of EVs samples. Wider implications of the findings: This discovery that the RNA cargo of EVs secreted from aneuploid cleavage stage embryos is diverse from that of euploid embryos potentiates the development of non-invasive methodology for PGT-A. The upregulation of MUC1 in decidualised ESCs following aneuploid embryo EV treatment proposes a new mechanism underlying implantation failure. Trial registration number NA

P-240 Human extracellular vesicles (EVs) secreted by aneuploid embryos potentiate development of non-invasive PGT-A RNA biomarkers and stimulate MUC1 up-regulation in primary endometrial stromal cells (ESCs)

Abstract Study question Could EVs secreted by aneuploid embryos a) serve for development of RNA biomarkers for PGT-A and b) elicit a relevant transcriptomic response in decidualised ESCs? Summary answer Aneuploid embryo EVs a) contain PPM1J, LINC00561, ANKRD34C and TMED10 in differential abundance from euploid EVs and b) induce up-regulation of MUC1 in decidualised ESCs. What is known already Embryo aneuploidy accounts for approximately 50% of all recurrent implantation failures in women >35 years old. PGT-A identifies euploid embryos to increase implantation probability but the technology is controversial as it requires an invasive embryo biopsy with an elusive long-term biosafety. The development of non-invasive methods to screen out aneuploid embryos is paramount. It is also critical to decode the embryo-endometrial dialog underlying implantation failure. We have previously reported that IVF embryos secrete EVs that can be internalised by ESCs, conceptualising that successful implantation to the endometrium is facilitated by EVs, which may additionally serve as biomarkers of ploidy status. Study design, size, duration Embryos destined for biopsy on days 5-7 for PGT-A were grown under standard conditions. Spent media (30μl) were collected from euploid (n = 175) and aneuploid embryos (n = 145) at both cleavage (days 1-3) and blastocyst (days 3-5) stage. Media samples from n = 35 cleavage embryos were pooled in order to obtain five euploid and four aneuploidy pools. Blastocyst media were pooled to create one euploid and one aneuploid pool. ESCs were obtained from five women undergoing diagnostic laparoscopy. Participants/materials, setting, methods The study was realised at a research hospital. EVs were isolated from euploid and aneuploid Day3 pools with differential ultracentrifugation and EV-RNA sequencing was performed following the SMARTer Stranded Total RNA-Seq approach. ESCs were decidualised (E2:10nM, P4:1uM, cAMP:0.5 mM twice every 48 hours) and treated for 24 hours with 50 ng/ml euploid or aneuploid EVs extracted from blastocyst media. RNA sequencing was performed on ESCs following the Truseq RNAseq protocol. Main results and the role of chance Aneuploid cleavage stage embryos (n = 4) secreted EVs that were less abundant in RNA fragments originating from the genes PPM1J (log2fc=-5.13, p = 0.011), LINC00561 (log2fc=-7.87, p = 0.010) and ANKRD34C (log2fc=-7.30, p = 0.017) and more abundant in TMED10 (log2fc=1.63 p = 0.025) compared to EVs (n = 5) from euploid embryos. Decidualisation per se induced downregulation of MUC1 (log2FC=-0.54, p = 0.0028) in ESCs as prerequisite for the establishment of receptive endometrium. The expression of MUC1 transcript in decidualised ESCs was significantly increased following treatment with aneuploid compared to euploid embryo-secreted EVs (log2FC=0.85, p = 0.0201). Limitations, reasons for caution The findings of the study may require validation utilising a second cohort of EVs samples. Wider implications of the findings This discovery that the RNA cargo of EVs secreted from aneuploid cleavage stage embryos is diverse from that of euploid embryos potentiates the development of non-invasive methodology for PGT-A. The upregulation of MUC1 in decidualised ESCs following aneuploid embryo EV treatment proposes a new mechanism underlying implantation failure. Trial registration number NA

P–222 Can we optimise the time that we perform the fertilisation check in the lab? Lessons learnt from time-lapse incubation

Abstract Study question Can time-lapse data be used to identify the optimum time to perform the fertilisation check for oocytes cultured in standard incubation? Summary answer The optimum time to perform fertilisation checks for oocytes cultured in standard incubation is 16.5hpi+/–0.5h. What is known already Time-lapse incubation allows the embryologist to retrospectively review collated images of oocytes and embryos to capture important embryological observations that may have otherwise been missed. This is a luxury not available to embryologists when oocytes or embryos are cultured in standard incubation. Traditionally, the optimum time to perform the fertilisation check is 17 hours post insemination (hpi) +/- 1 hour. It was hypothesised that this could be fine-tuned ensuring the maximum number of fertilised oocytes were observed, thereby increasing the number of usable embryos for the patient. Study design, size, duration This was a retrospective, multicentre analysis including data from 27,022 ICSI derived embryos cultured in time-lapse incubation between January 2011 to November 2019. Participants/materials, setting, methods The time of pronuclei appearance and disappearance was recorded using the time-lapse incubation software. The number of oocytes exhibiting normal fertilisation (defined as the presence of two pronuclei) during 30 minute intervals from 15hpi to 20hpi was determined. Main results and the role of chance Between 15–17.5hpi the average number of oocytes exhibiting normal fertilisation was 98.19% with most oocytes having visible pronuclei at 16–16.5hpi (98.32%). At 18–18.5hpi the number of visible pronuclei falls to 95.53% and continues to fall to 87.02% at 19.5–20hpi meaning that over 3000 (11%) normally fertilised oocytes, within this cohort, would not be identified. Limitations, reasons for caution The conclusions of this investigation cannot be effectively extrapolated to IVF embryos as only ICSI embryos were used for the determination of the results. Wider implications of the findings: The optimum time to perform fertilisation checks for oocytes cultured in standard incubation is 16.5hpi+/–0.5h. However, without the use of time-lapse incubation, the fertilisation of at least 2% of embryos that create a fetal heart will be missed, even if the fertilisation check is performed in the optimum window (16.5hpi+/–0.5h). Trial registration number Not applicable

P–501 Deep in the Maze: The psychosocial trajectory and decision making of Women with recurrent implantation failure of IVF

Abstract Study question To understand the psychosocial trajectory of Chinese women who have experienced recurrent implantation failure (RIF) of IVF and their decision making accordingly. Summary answer Chinese women experience despair, doubt, and disorientation along with the cumulative failure cycles of IVF, and stick to IVF as the ultimate option. What is known already Recurrent implantation failure, the absence of implantation after repeated embryo transfers is a stressful event for people undergoing treatment for infertility. Numerous researches have focused on the psychological wellness of women undertaking IVF, but pay less attention to the subgroup who have undergone repeated failures. Current studies have shown that women after repeated unsuccessful IVF might endure anxiety, depression, and other psychosocial distress; however, the feelings brought by the different times of failure are unlike, and these experiences will affect their treatment decisions accordingly, which is rarely studied. Study design, size, duration Semi-structured interview was adopted with sixteen Chinese women from March 2020 to July 2020. The interview lasted 90 minutes. A follow-up survey was conducted three months after the interview. Driven by grounded theory, data is analyzed by thematic analysis. Participants/materials, setting, methods Convenience sampling was used to recruit female participants who have failed to achieve clinical pregnancy after two consecutive cycles of fresh or frozen IVF embryo transfers with a cumulative number of transferred embryos of four or more cleavage-stage embryos or two or more blastocysts. Chinese-speaking women who were undertaking IVF treatment in the reproductive center of hospital in Shenzhen City were recruited by pamphlets and doctors’ referral. Main results and the role of chance Chinese women with recurrent implantation failures experience the following psychosocial trajectory during the cumulative failure cycles of IVF: despair, doubt, and disorientation. Despair usually comes after the first failure: the high expectation for success rates makes the initial failure exceptionally shocking and desperate. Huge distress brings two kinds of decisions: start a new IVF cycle quickly to welcome the positive results in the imagination, or wait for a period of time to avoid the pain of failure again. Doubt usually appears after the second failure. In addition to doubting the health function of their body, recurrent failure makes the patients particularly doubt the efficacy of IVF and doctors’ clinical judgment. Some patients would do ‘hospital shopping’ and consider change clinics. Disorientation can be seen in patients who have experienced more than three cycles of failure. Past experience and meaning cannot help them understand and solve the current dilemma. The inherent concept of fertility continues to strengthen their belief of having a baby as ultimate goal. The follow-up survey found that most women still choose to continue IVF treatment after repeated failure. They are like being in the maze of fertility, wandering for a long time but unable to get out. Limitations, reasons for caution Several limitations are identified: self-selection bias due to convenience sampling; narrow sampling approach may limits the generalizability; the exclusion of men may ignore the marital interdependence during the infertility treatment. Wider implications of the findings: Patients who have experienced recurrent implantation failure demand promising intervention during and after infertility treatment. The findings demonstrate the need for both supportive and implication counseling to facilitate them cope with the psychosocial distress, and make value-based decision making, so as to enhance their self-agency. Trial registration number Not applicable

P–174 Globulin-rich protein supplements improve blastulation efficiency in culture and promote implantation in vitro

Abstract Study question Can globulin-rich compared to albumin (HSA) supplements improve blastulation and support embryo development towards post implantation? Summary answer Yes, globulin supplements with clinical-grade quality increase blastulation efficiency by 20% (50% in older mothers) and support the transition of embryos towards post-implantation development. What is known already During embryonic development at the morula stage there is a metabolic transition towards glycolysis as demand from outsourced energy increases. Therefore as cleavage proceeds, the demand for nutrients in the embryo increases accordingly. With few exceptions, HSA from human plasma or recombinant origin has been the main an only protein supplement used in almost all IVF-procedures. Globulin rich supplements are available but their use is not widespread and little is known about their efficiency in post-implantation development. Study design, size, duration We have cultured more than 600 mouse embryos in continuous media containing a protein supplement#1 (PS#1), from 1-cell up to blastocyst stage. At blastocyst stage embryos were replaced into fresh media containing protein supplement#2 (PS#2). The embryos were allowed to hatch naturally and then transferred into a proprietary matrix for further development and implantation for an additional 48h. Participants/materials, setting, methods: The blastulation rate, measured for HSA-supplemented embryo cohort was compared with embryos cultured in PS#1. Hatching efficiency was reported for embryos cultured in transfer media including PS#2. Once embedded in the matrix, advanced label-free imaging techniques and custom algorithms to measure matrix implantation strength were used. Key molecular markers (i.e. OCT4, CDX2) for correct post-implantation lineage patterning were documented by conventional 3D confocal immunofluorescence imaging. Main results and the role of chance Embryos supplemented with PS#1 reached blastocyst with overall 21% higher efficiency than embryos supplemented by HSA. When separated by age cohorts, embryos obtained from older females (ex-colony breeders, >14 weeks old) reached blastocyst stage with 55% higher efficiency than the same type of embryos cultured in the presence of HSA. Embryos obtained from females at optimal reproductive age reached blastocyst stage 10% more efficiently under PS#1 supplementation than with HSA. Hatching efficiency was 45% higher for embryos cultured with PS#2 than embryos supplemented with HSA. For every variable tested (e.g.% of arrested or degenerated embryos) or condition implemented (e.g. mouse basal media, human basal media from different brands, etc.) PS#1 and PS#2 outperformed, without exception, the supplementation with HSA. When embedded in the implantation matrix, the embryos cultured with PS#1 (cleavage) and transferred to PS#2 at blastocyst stage showed a remarkable implantation ability as measured by trophoblast outgrowth and matrix deformations. The embryos in PS#2 medium exerted stronger force into the matrix and also survived longer times than the embryos in HSA. PS#2 supported the transition of blastocyst towards post-implantation stages of development showing the correct lineage patterning of embryonic and extraembryonic molecular markers, including Oct4, CDx2, EOMES or GATA4. Limitations, reasons for caution This is a study based on an animal model. These observations need to be confirmed by ongoing experiments with human embryos. Wider implications of the findings: This work constitutes a proof-of-concept for the use of globulin-rich supplements as higher performance substitute of albumin in the culture of IVF embryos, both as (i) a standard protein source for culture media and (ii) as a supplement for transfer media to capacitate the embryo for implantation. Trial registration number Not applicable

A novel test for annexin A5 M2 haplotyping in in vitro fertilization patients and preimplantation embryos

To develop a test for evaluating the annexin A5 M2 haplotype in invitro fertilization patients and preimplantation embryos. Test performance was measured by comparing Sanger sequencing of parental blood DNA and quantitative real-time polymerase chain reaction (qPCR) of saliva DNA, 3 fibroblast cell line 7-cell aliquots and their corresponding purified DNA, 123 trophectoderm biopsy samples, and DNA isolated from 1 embryonic stem cell line along with the Mendelian inheritance expectations, embryo Sanger sequencing, and single-nucleotide polymorphism (SNP) microarray-based linkage analysis. Preimplantation genetic testing laboratory research on IVF patient and embryo DNA. An assay was developed for the detection of the M2 haplotype on saliva samples of 6 invitro fertilization patients. In addition, 13 patients who underwent preimplantation genetic testing with data on parental and embryo biopsy DNA available for research use were evaluated. None. The concordance rates between Sanger sequencing, SNP array-based linkage analysis, and Mendelian inheritance expectations with qPCR. The concordance rate between Sanger sequencing and qPCR was 100% on parental blood DNA and saliva DNA. The sample concordance rate between all replicates of 7-cell aliquots was 100%. The sample concordance rate between 3 cell lines used to prepare 7-cell aliquots and purified genomic DNA was 100%. The concordance rate between qPCR and Sanger sequencing results from a single trophectoderm biopsy and isolated embryonic stem cell line was 100%. The concordance rate of trophectoderm biopsy qPCR results and expectations from Mendelian inheritance rules was 97%; however, when SNP array-based linkage analysis was included, the concordance rate reached 100%. This study resulted in the development of a convenient saliva collection method and qPCR-based genotyping method to screen for the M2 haplotype. In addition, a novel method for testing preimplantation embryos has been established, providing an alternative to the use of low molecular weight heparin, through selection of embryos without the M2 haplotype.

HEREDITARY THROMBOPHILIA IN RECURRENT IVF FAILURE

Background: The predominant part of IVF cycles results in embryo transfer, but only about one third of all cycles reach clinically achieved pregnancy. This is evidence that most embryos failed in an early stage of pregnancy achievement. Recurrent implantation failure after IVF procedures emphasize the clinical importance of this crucial step in assisted reproductive technology. Repeated unsuccessful IVF attempts force efforts to investigate the firm mechanism of the implantation and to find approach to increase pregnancy outcome success. Plenty of factors have been recognized to affect either success, or failure rate of IVF embryo transfer. Maternal side factors include age, parity, hormonal levels before stimulation, antral follicles count, endometrial thickness and quality of transformed endometrium. Other factors, having functions in coagulation and fibrinolysis cascades, were found to be connected with the transformation processes in the endometrium during the implantation.Aim of work: The aim from this study is to evaluate the relation between hereditary thrombophilia and recurrent IVF failure. This will be done by comparing the prevalence of thrombophilia factors mutation in patients with repeated IVF failure and in normal population.Methods: Thirty patients with recurrent IVF failure underwent genetic testing of thrombophilia markers by PCR: Factor II prothrombin mutation, Factor V leiden mutation (FVL), and Methylene tetrahydrofolate reductase (MTHFR) mutation.Results: 13.3% were tested positive for factor V mutation G1619A, 56.7% were positive for MTHFR c667t mutation. While in healthy normal Mediterranean population the percentage of mutation were 3.7% for FVL mutation and 11.1% for MTHFR mutation.Conclusion: hereditary thrombophilia may be considered as a factor in recurrent IVF failure, proper management is to be considered.

Corrigendum. First PGT-A using human in vivo blastocysts recovered by uterine lavage: comparison with matched IVF embryo controls.

Corrigendum. First PGT-A using human in vivo blastocysts recovered by uterine lavage: comparison with matched IVF embryo controls.

Effect of parental and ART treatment characteristics on perinatal outcomes.

STUDY QUESTIONDo parental characteristics and treatment with ART affect perinatal outcomes in singleton pregnancies?SUMMARY ANSWERBoth parental and ART treatment characteristics affect perinatal outcomes in singleton pregnancies.WHAT IS KNOWN ALREADYPrevious studies have shown that singleton pregnancies resulting from ART are at risk of preterm birth. ART children are lighter at birth after correction for duration of gestation and at increased risk of congenital abnormalities compared to naturally conceived children. This association is confounded by parental characteristics that are also known to affect perinatal outcomes. It is unclear to which extent parental and ART treatment characteristics independently affect perinatal outcomes.STUDY DESIGN, SIZE, DURATIONAll IVF clinics in the Netherlands (n = 13) were requested to provide data on all ART treatment cycles (IVF, ICSI and frozen-thawed embryo transfers (FET)), performed between 1 January 2000, and 1 January 2011, which resulted in a pregnancy. Using probabilistic data-linkage, these data (n = 36 683) were linked to the Dutch Perinatal Registry (Perined), which includes all children born in the Netherlands in the same time period (n = 2 548 977).PARTICIPANTS/MATERIALS, SETTING, METHODSAnalyses were limited to singleton pregnancies that resulted from IVF, ICSI or FET cycles. Multivariable models for linear and logistic regression were fitted including parental characteristics as well as ART treatment characteristics. Analyses were performed separately for fresh cycles and for fresh and FET cycles combined. We assessed the impact on the following perinatal outcomes: birth weight, preterm birth below 37 or 32 weeks of gestation, congenital malformations and perinatal mortality.MAIN RESULTS AND THE ROLE OF CHANCEThe perinatal outcomes of 31 184 out of the 36 683 ART treatment cycles leading to a pregnancy were retrieved through linkage with the Perined (85% linkage). Of those, 23 671 concerned singleton pregnancies resulting from IVF, ICSI or FET. Birth weight was independently associated with both parental and ART treatment characteristics. Characteristics associated with lower birth weight included maternal hypertensive disease, non-Dutch maternal ethnicity, nulliparity, increasing duration of subfertility, hCG for luteal phase support (compared to progesterone), shorter embryo culture duration, increasing number of oocytes retrieved and fresh embryo transfer. The parental characteristic with the greatest effect size on birth weight was maternal diabetes (adjusted difference 283 g, 95% CI 228–338). FET was the ART treatment characteristic with the greatest effect size on birth weight (adjusted difference 100 g, 95% CI 84–117) compared to fresh embryo transfer. Preterm birth was more common among mothers of South-Asian ethnicity. Preterm birth was less common among multiparous women and women with ‘male factor’ as treatment indication (compared to ‘tubal factor’).LIMITATIONS, REASONS FOR CAUTIONDue to the retrospective nature of our study, we cannot prove causality. Further limitations of our study were the inability to adjust for mothers giving birth more than once in our dataset, missing values for several variables and limited information on parental lifestyle and general health.WIDER IMPLICATIONS OF THE FINDINGSMultiple parental and ART treatment characteristics affect perinatal outcomes, with birth weight being influenced by the widest range of factors. This highlights the importance of assessing both parental and ART treatment characteristics in studies that focus on the health of ART-offspring, with the purpose of modifying these factors where possible. Our results further support the hypothesis that the embryo is sensitive to its early environment.STUDY FUNDING/COMPETING INTEREST(S)This study was funded by Foreest Medical School, Alkmaar, the Netherlands (grants: FIO 1307 and FIO 1505). B.W.M. reports grants from NHMRC and consultancy for ObsEva, Merck KGaA, iGenomics and Guerbet. F.B. reports research support grants from Merck Serono and personal fees from Merck Serono. A.C. reports travel support from Ferring BV. and Theramex BV. and personal fees from UpToDate (Hyperthecosis), all outside the remit of the current work. The remaining authors report no conflict of interests.TRIAL REGISTRATION NUMBERN/A.

Embryo biosensing by uterine natural killer cells determines endometrial fate decisions at implantation.

Decidualizing endometrial stromal cells (EnSC) critically determine the maternal response to an implanting conceptus, triggering either menstruation‐like disposal of low‐fitness embryos or creating an environment that promotes further development. However, the mechanism that couples maternal recognition of low‐quality embryos to tissue breakdown remains poorly understood. Recently, we demonstrated that successful transition of the cycling endometrium to a pregnancy state requires selective elimination of pro‐inflammatory senescent decidual cells by activated uterine natural killer (uNK) cells. Here we report that uNK cells express CD44, the canonical hyaluronan (HA) receptor, and demonstrate that high molecular weight HA (HMWHA) inhibits uNK cell‐mediated killing of senescent decidual cells. In contrast, low molecular weight HA (LMWHA) did not attenuate uNK cell activity in co‐culture experiments. Killing of senescent decidual cells by uNK cells was also inhibited upon exposure to medium conditioned by IVF embryos that failed to implant, but not successful embryos. Embryo‐mediated inhibition of uNK cell activity was reversed by recombinant hyaluronidase 2 (HYAL2), which hydrolyses HMWHA. We further report a correlation between the levels of HYAL2 secretion by human blastocysts, morphological scores, and implantation potential. Taken together, the data suggest a pivotal role for uNK cells in embryo biosensing and endometrial fate decisions at implantation.

Kantian Ethics And The Hesc Research: A Philosophical Exploration

The scientific reports on the successful use of Human Embryonic Stem cells to cure many sicknesses as provoked a long-standing controversy about the ethics of research involving human embryos. This controversy arises from sharply differing moral views regarding the use of embryos for research purposes. Indeed, an earnest international scholarly debate continues till today over the ethical, legal, and medical issues that arise in this arena. Immanuel Kant (1724-1804) had given a moral guideline that ethical decisions should be made by considering the nature of the act itself, not its consequences. Furthermore, Kant has warned that persons (autonomous agents) have a special moral worth or dignity, which is the basis for the respect that is owed to them. Thus, respect for persons, means never using persons merely as means to our ends, but always treating them also as ends in themselves. Some philosophers like Richard Doerflinger, Michael Novak, Gilbert Meilaender, and Robert P. George have used the Kantian formula of humanity to criticize the argument that spare IVF embryos can be used for stem cell research given their inevitable death and thus lack of properties for future life. However, the purpose of this paper is to take a critical look at the Human Embryonic Stem cells subject matter to investigate the concept of “personhood’, with the maxim of ‘never treating a person as a means’. This paper argues that if we accepts the definition of a person to possess capacities such as ‘rational’ ‘will’ and ‘self-determination’, then IVF embryos is not a person and can therefore be researched upon, used to derive human embryonic stem cells. Hence, Human Embryonic Stem cells research can be carried out within the ambiance of Kant Categorical Imperative without moral conflict.

Aggregation of Leopardus geoffroyi hybrid embryos with domestic cat tetraploid blastomeres.

Heterospecific embryo transfer of an endangered species has been carried out using recipients from related domestic females. Aggregation of an embryo from an endangered species with a tetraploid embryo from the species to be transferred could improve the development of pregnancy to term. The main objective of the present study was to analyze embryo aggregation in domestic cat model using hybrid embryos. For this purpose, we compared in vitro development of synchronic (Sync) or asynchronic (Async) and asynchronic with a tetraploid (Async4n) aggregation of domestic cat IVF embryos. Furthermore, aggregated blastocyst quality was analyzed by evaluation of the total cell number, cell allocation by mitotrackers staining of embryonic cells, expression of Oct4, Nanog, Sox2, Cdx2 genes, number of OCT4+ nuclei, and presence of DNA fragmentation. Additionally, the developmental rates of Async4n aggregation of domestic cat with Leopardus geoffroyi hybrid (hLg) embryos were evaluated. Async aggregation increased blastocyst cell number and the number of OCT4+ nuclei as compared to non-aggregated diploid (2n) and tetraploid (4n) embryos. Moreover, blastocysts produced by Async4n aggregation showed reduced rates of fragmented DNA. No differences were found in the expression of the pluripotent genes, with exception of the Cdx2 expression, which was higher in 4n and aggregated embryos as compared to the control group. Interestingly, hybrids embryos derived by Async4n aggregation with domestic cat embryos had similar rates of blastocysts development as the control. Altogether, the findings support the use of two-cell-fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos.

442 Low ovarian response to stimulation and adverse perinatal outcome in fresh IVF embryo transfer cycles

Unlike maternal age, it is unclear whether the ovarian reserve alone has an impact on the obstetric outcome of pregnancies resulting from assisted reproductive technologies. We aimed to study the association between low ovarian response to gonadotropin stimulation and adverse perinatal outcome in singleton gestations. A retrospective cohort study of 382 women who underwent in-vitro fertilization and fresh embryo transfer and delivered a singleton neonate at a single center (pre-implantation genetic testing excluded). We examined the association between low ovarian response and adverse obstetric/perinatal outcomes. Patients were classified as: low responders - daily FSH≥300IU yielding ≤4 oocytes; normal responders 5-15 oocytes aspirated; high responders >15 oocytes aspirated; and sub-optimal treatment-daily FSH<300IU yielding ≤4 oocytes. Primary outcome was a composite of pre-eclampsia, gestational diabetes, small-for-gestational-age and delivery under 37 weeks. We compared maternal and neonatal outcomes between low responders and other groups. 27 women(7%) were categorized as low responders, 355(93%) as normal, high responders, or sub-optimal treatment. Maternal baseline characteristics were similar between groups except for increased maternal age and cycle number in low responders (36.15±3.74vs.30.7±4.9,p=<.0001, 4.52±2.76vs.3.04±2.66,p=0.0006, respectively). There were no differences in gestational age at delivery, birthweight, and the rates of preeclampsia, gestational diabetes, cesarean delivery and post-partum hemorrhage, or the composite outcome. Interestingly, low responders were more likely to give birth to males (74.07% vs. 49.86%,p=0.01522). Using multivariate logistic regression, the only two parameters found to be nearly-significantly associated with the composite outcome were low endometrial thickness on the day of ovulation triggering (p=0.0538) and nulliparity (p=0.0533). Low ovarian response was not associated with adverse maternal or neonatal outcome, despite a higher maternal age in this group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Exogenous progesterone for LH surge prevention is redundant in ovarian stimulation protocols

During ovarian stimulation for IVF–embryo transfer treatment, a premature LH surge may lead to progesterone elevation that disrupts endometrial maturation and affects the probability of pregnancy following fresh embryo transfer. Preventing this LH surge and progesterone elevation using gonadotrophin-releasing hormone (GnRH) analogues is considered a standard practice. The same policy applies to cycles in which the ‘freeze-all’ protocol has been selected from the outset (e.g. donors), but the need for this has not been discussed. Moreover, in ‘freeze-all’ cycles, exogenous progesterone administration tends to replace GnRH antagonists, without reducing efficacy after embryo transfer in frozen-thawed cycles. Nevertheless, as exogenous progesterone is expected to have the same impact on the endometrium as endogenous progesterone, it is clear that, unlike in fresh cycles, in ‘freeze-all’ cycles an endogenous LH surge prevention does not seem necessary. Therefore, both GnRH antagonists and exogenous progesterone appear to be redundant in ‘freeze-all’ cycles, and in this context the indications for the use of GnRH analogues in ovarian stimulation protocols need to be revisited.

Histone Demethylase KDM4D Could Improve the Developmental Competence of Buffalo (Bubalus Bubalis) Somatic Cell Nuclear Transfer (SCNT) Embryos.

Somatic cell nuclear transfer (SCNT) holds vast potential in agriculture. However, its applications are still limited by its low efficiency. Histone 3 lysine 9 trimethylation (H3K9me3) was identified as an epigenetic barrier for this. Histone demethylase KDM4D could regulate the level of H3K9me3. However, its effects on buffalo SCNT embryos are still unclear. Thus, we performed this study to explore the effects and underlying mechanism of KDM4D on buffalo SCNT embryos. The results revealed that compared with the IVF embryos, the expression level of KDM4D in SCNT embryos was significantly lower at 8- and 16-cell stage, while the level of H3K9me3 in SCNT embryos was significantly higher at 2-cell, 8-cell, and blastocyst stage. Microinjection of KDM4D mRNA could promote the developmental ability of buffalo SCNT embryos. Furthermore, the expression level of ZGA-related genes such as ZSCAN5B, SNAI1, eIF-3a, and TRC at the 8-cell stage was significantly increased. Meanwhile, the pluripotency-related genes like POU5F1, SOX2, and NANOG were also significantly promoted at the blastocyst stage. The results were reversed after KDM4D was inhibited. Altogether, these results revealed that KDM4D could correct the H3K9me3 level, increase the expression level of ZGA and pluripotency-related genes, and finally, promote the developmental competence of buffalo SCNT embryos.

16 Embryo aggregation and adipose-derived mesenchymal donor cells in bovine somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) is a powerful tool, but its efficiency remains low. The use of less differentiated donor cells or the embryo aggregation (EA) strategy improves the SCNT rates in several species. It remains unexplored whether the combined use of both strategies results in a synergistic effect that improves SCNT efficiency in bovine. To evaluate that, we assessed the optimal time of EA using IVF embryos (aim 1) and we evaluated whether the use of adipose-derived mesenchymal stem cells (ASC) as donor for SCNT together with EA improves the blastocyst rates and quality (aim 2). For aim 1, cumulus–oocyte complexes (COCs) were collected from slaughterhouse ovaries, invitro matured (TCM-199), fertilized (16×106 spermatozoa mL−1 for 5h) and cultured (synthetic oviductal fluid media in a humidified gas mixture at 39°C). After IVF, the zona pellucida was enzymatically removed and zona-free (ZF) embryos were cultured individually (1X) or 2 embryos placed together within a microwell (2X) (Day 0, n=70). This procedure was performed at Days 3, 4, 5, 6, or 7 (n=76, 78, 94, 96, 90, respectively) and blastocyst rate was assessed at Day 8. Contribution of both embryos to the 2X blastocyst was confirmed by staining Day 0 IVF embryos either with green or red Mitotracker (ThermoFisher Scientific) before EA. For aim 2, fibroblast (FB) and ASC cells were isolated from the skin and subcutaneous adipose tissue of the same adult animal, respectively. Cloned embryos were produced by ZF enucleation, fusion of one ASC or FB cell, and activation with 5μM ionomycin/6-(dimethylamino)purine (6DMAP). After activation, cloned embryos were aggregated (FB2X or ASC2X) or individually cultured (FB1X or ASC1X). Blastocyst rates were recorded at Day 7 of invitro culture. Three biological replicates were evaluated for each aim. Embryo developmental differences were determined using Fisher’s exact test. Relative expression of OCT4, SOX2, and KRT18 was measured by RTqPCR at the blastocyst stage and analysed by Kruskal–Wallis statistical test. Regarding aim 1, no differences for developmental rates were found for Day 0, 3, 4, and 5 groups (57%, 60%, 61.5%, 61%), but the blastocyst rate was only improved in Day 0 and Day 3 relative to their respective 1X controls (Day 0 2X 54.2% vs. Day 0 1X 25.5% and Day 3 2X 52.6% vs. Day 3 1X 25.3%). No aggregation occurred in Day 6 and Day 7 groups. All blastomeres were homogeneously distributed in the 2X blastocyst. Regarding aim 2, no effect of the donor cell was observed on the blastocyst rate (FB1X 26.8%, n=82; ASC1X 21.7%, n=198; FB2X 39.7%, n=126; ASC2X 33%, n=204), whereas EA improved the blastocyst rate of ASC-derived embryos (ASC1X 21.7% vs. ASC2X 33%). Overall, no synergistic effect of the use of both strategies was observed. Relative expression of KRT18 was significantly different between ASC1X and ASC2X embryos. Although OCT4 and SOX2 expression did not differ between groups, EA tended to bring the values closer to that of an IVF embryo. No effect of the donor cell was observed on the embryo relative expression. Our results suggest that EA at Day 0 improves the blastocyst rate in bovine SCNT and IVF embryos. EA of 2 ASC-derived embryos seemed to normalise the embryo quality and may improve post-implantation development.

75 Effect of mycobacterium cell wall fraction immunostimulant on conception rates in heifer recipients

In cattle, the average pregnancy for IVF embryos around Days 45 to 90 is 35% (Ealy et al. 2019 J. Anim. Sci. 97, 2555–2568). Among other factors, uterine contamination with pathogens from the vagina during embryo transfer may affect pregnancy success. Prophylactic antibiotic treatment for this purpose in embryo recipients is considered malpractice. An alternative to antibiotics is the use of mycobacterium cell wall fraction (MCWF) immunostimulant (Amplimune, NovaVive Inc.) against these pathogens. Therefore, the objective was to determine the effect of MCWF on conception rates in heifer recipients. For this purpose, Holstein heifers were synchronized with 2 injections of prostaglandin F2α 14 days apart and randomly allocated to 2 groups: (1) MCWF (n=292), single intramuscular injection of 5mL of Amplimune on the day of pretransfer oestrus; (2) Control (n=296): no Amplimune treatment. Embryo transfers using quality 1 frozen IVF embryos were performed in all recipients 7 to 8 days after oestrus and with a corpus luteum ≥22mm, using a double sanitary sheath technique. Data were analysed using the generalized linear mixed model (GLMM). Results are shown in Table 1. In conclusion, treatment with MCWF increased the number of recipients to be used for embryo transfer and improved the conception rate at Day 60. Importantly, MCWF treatment was more efficient for getting a recipient pregnant by Day 60 per total of synchronized recipients. Table 1. Effect of mycobacterium cell wall fraction (MCWF) on pregnancy in heifer recipients transferred frozen embryos (quality 1) Variable MCWF Control Utilisation rate 227/292a 174/296b (No. of transferred recipients/no. of synchronized recipients) (78%) (59%) Pregnancy Day 30 144/227 94/174 (No. of pregnant recipients at Day 30/no. of transferred recipients) (63%) (54%) Pregnancy Day 60 131/227a 83/174b (No. of pregnant recipients at Day 60/no. of transferred recipients) (58%) (48%) Embryo mortality 13/144 11/94 (No. of embryo loss at Day 60/no. of pregnant recipients at Day 30) (9%) (12%) Pregnancy Day 60 per total of synchronized 131/292a 83/296b (No. of pregnant recipients/no. of synchronized recipients) (45%) (28%) a,bValues with different superscripts within a row are different (P ≤ 0.05).