Blastocyst Stage Embryos Research Articles

P–582 High level of concordance between invasive and non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) at day5 and day6–7

Abstract Study question To explore ploidy concordance between invasive and non-invasive PGTA (niPGT-A) at different embryo culture time. Summary answer High level (>84%) of concordance rate for ploidy and sex, sensitivity (>88%), and specificity (76%) were obtained for both day6/7 samples and day5 samples. What is known already The analysis of embryo cell free DNA (cfDNA) that are released into culture media during in vitro embryo development has the potential to evaluate embryo ploidy status. However, obtaining sufficient quality and quantity of cfDNA is essential to achieve interpretable results for niPGT-A. More culture time is expected to be directly proportional to the release of more cfDNA. But embryo culture time is limited due to in-vitro embryo survival potential. Therefore, it is important to estimate the duration of the culture that will provide the maximum cfDNA that can be obtained without adversely affecting the development of the embryo. Study design, size, duration A total of 105 spent culture media (SCM) from day5-day7 blastocyst stage embryos have been included in this cohort study. The cfDNA of SCM samples were amplified and analyzed for niPGT-A by NGS analysis. The SCM samples were divided into 2 subgroups according the embryo culture hours (Day5 and Day6/7 group). The DNA concentration, informativity and euploidy results have then been compared with their corresponding embryos after trophectoderm biopsy (TE) and PGT-A analysis by NGS Participants/materials, setting, methods Embryos cultured until Day3 washed and cultured again in 20µl fresh culture media until embryo biopsy on Day5, 6, or 7. After biopsy SCM samples were immediately collected in PCR tubes and conserved at –20 °C until whole genome amplification by MALBAC® (Yicon Genomics). The TE and SCM samples were analyzed by next-generation sequencing (NGS) using Illumina MiSeq® System. NGS data analysis has been done by Bluefuse Multi Software 4.5 (Illumina) for SCM and TE samples Main results and the role of chance Only the SCM samples which have an embryo with a conclusive result were included in this cohort (n = 105). Overall 97.1% (102/105) of SCM samples gave a successful DNA amplification with a concentration ranging 32.4–128.5ng/µl. Non-informative (NI) results including a chaotic profile (>5 chromosome aneuploidies) were observed in 17 samples, so 83.3%(85/102) of SCM samples were informative for NGS data analysis. Ploidy concordance rate with the corresponding TE biopsies (euploid vs euploid, aneuploid vs aneuploid) was 84.7% (72/85). Sensitivity and specificity were 92,8% and 76,7%, respectively with no significant difference for all parameters for day 6/7 samples compared with day 5 samples. The false-negative rate was 3.5% (3/85), and false-positive rate was 11.7% (10/85). Limitations, reasons for caution The sample size is relatively small. Larger prospective studies are needed. As this is a single-center study, the impact of the variations in embryo culture conditions can be underestimated. Maternal DNA contamination risk cannot be revealed in SCM, therefore the use of molecular markers would increase the reliability. Wider implications of the findings: Non-invasive analysis of embryo cfDNA analyzed in spent culture media demonstrates high concordance with TE biopsy results in both early and late culture time. A non-invasive approach for aneuploidy screening offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population. Trial registration number Not applicable

O-099 TEAD4 regulates trophectoderm differentiation upstream of CDX2 in human preimplantation embryos

Abstract Study question What is the main pathway regulating trophectoderm (TE) differentiation during pre-implantation development in mouse versus human embryos? Summary answer TEAD4 is acting upstream of CDX2 and is involved in TE differentiation, as TEAD4-null human embryos exhibit compromised TE lineage differentiation. What is known already TEAD4 is the earliest transcription factor during early embryo development, required for the expression of TE-associated genes leading to successful TE differentiation and subsequent blastocoel formation in mouse. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination have shown that Tead4-null embryos do not express TE specific genes, including Caudal-Type Homeobox Protein 2 (Cdx2) and GATA Binding Protein 3 (Gata3), but expression of inner cell mass (ICM)-specific genes, remains unaffected. Furthermore, ablation of Tead4 compromises embryonic development and subsequent blastocoel formation in mouse. The role of TEAD4, during human pre-implantation development has not been functionally characterized yet. Study design, size, duration CRISPR-Cas9 was introduced in mouse zygotes and editing efficiency was evaluated by next-generation sequencing (NGS) on 4.5dpc embryos (n = 55). Developmental kinetics were monitored in CRISPR-Cas9 targeted (n = 83), sham-injected (n = 26) and non-injected media-control (n = 51) mouse embryos. Immunofluorescence analysis was performed in Tead4 targeted (n = 57) and non-injected media-control embryos (n = 94). The same methodology was applied in human donated in vitro matured (IVM) metaphase-II (MII) oocytes, which were CRISPR-Cas9 targeted (n = 74) during ICSI or used as media-Control (n = 33). Participants/materials, setting, methods A gRNA-Cas9 mixture targeting exon 2 of Tead4/TEAD4 was microinjected in respectively mouse 2PN (pronuclear) stage zygotes, or human IVM MII oocytes along with the sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. Embryonic development and morphology were assessed daily, followed by a detailed scoring at the late blastocyst stage. Successful targeting following CRISPR-Cas9 introduction was assessed by immunostaining and NGS analysis of the targeted locus. Main results and the role of chance In mouse, we confirmed previous findings, as the developmental capacity of Tead4 targeted embryos was significantly reduced starting from the morula stage and blastocyst formation rates were 8.97% in the targeted group, compared to 87.23% in the control and 87.50% in the sham group, respectively. Immunofluorescence analysis of late morula and blastocyst stage embryos confirmed the absence of Tead4, Cdx2 and Gata3, resulting from the successful interruption of the Tead4 locus (n = 57). Exon 2 of TEAD4 was successfully targeted in human. In total, 21 embryos from various developmental stages were successfully NGS analyzed and 90,48% (19 out of 21) of the embryos carried genetic modifications as a result of CRISPR-Cas9 genome editing and seven blastocysts were identified carrying exclusively frameshift mutations. In contrast to mouse, the developmental capacity of human targeted embryos (25%) did not differ significantly from the control group (23%). However, the blastocyst morphology and quality were compromised in the targeted group showing mostly grade C TE scores, containing very few cells. Immunofluorescence analysis of targeted blastocysts (n = 6) confirmed successful editing by complete absence of TEAD4 and its downstream TE marker CDX2. Limitations, reasons for caution CRISPR-Cas9 germline genome editing results in multiple editing outcomes with variable phenotypic penetrance, the mosaic nature of which complicates the phenotypic analysis and developmental behaviour of the injected embryos. Wider implications of the findings Elucidation of the evolutionary conserved molecular mechanisms that regulate self-renewal of the trophoblast lineage can give us fundamental insights on early implantation failure. Trial registration number Not Applicable

P-382 Pre-selected for an award: Association of extended culture to blastocyst and gestational trophoblastic disease risk following IVF/ICSI assisted reproduction cycles: An analysis of large UK National database

Abstract Study question Is there any association between stage of embryo at transfer based on extended in vitro culture and gestational trophoblastic disease risk during assisted reproduction? Summary answer No significant association between stages of embryo transfer from zygote stage to blastocyst stage was found after analysing 540376 cycles of IVF and ICSI. What is known already Gestational trophoblastic disease (GTD), commonly referred to as molar pregnancy, results from abnormal proliferation of the trophoblast with a reported incidence of ∼1 in 700 in the UK. Despite technological advances such as ICSI, PGT and selection of normally fertilised (2PN) embryos, there are reported cases of GTD following assisted reproduction. Blastocyst transfer is associated with higher pregnancy and live birth rates but evidence is lacking whether extended embryo culture to blastocyst stage influences implantation of an abnormal embryo or abnormal trophoblastic proliferation leading to GTD. Study design, size, duration A retrospective study was carried out using Human Fertilisation and Embryology Authority (HFEA) anonymised register data from 1999 to 2016. HFEA holds the longest running register for fertility treatment data in the world and is the national database for fertility treatment data in UK. A total of 540376 fresh IVF or ICSI assisted reproduction cycles were analysed. Participants/materials, setting, methods There were 1033588 treatment cycles during the study period but only 540376 cycles met the inclusion criteria of fresh IVF or ICSI. Cycles with incomplete data, frozen embryo transfers, donor treatment or surrogacy were excluded. A subgroup analysis of those with primary subfertility was performed after excluding subjects with secondary infertility in order to exclude an effect of a previous molar pregnancy. Multivariate regression analysis was used to adjust for possible confounders. Main results and the role of chance 78 molar pregnancies were reported in the original sample giving a prevalence of 4/10000 live births (78/228461), much lower than the prevalence given with natural pregnancies. Prevalence of molar pregnancy amongst the study population after meeting exclusion criteria was 4 /10000 livebirths (53/156683). Incidence of molar pregnancy was not statistically different between treatment type (0.0001 vs 0.00009). Significantly higher incidence of GTD was seen in the 40 to 42 age category compared to 18-34 category(OR 1.86(95% CI 8.7-3.75)),in par with known higher GTD risk in women above 40 in the general population. Of interesting note, although the incidence of molar pregnancy was significantly lower in women undergoing assisted reproduction increased risk with advancing age is not totally eliminated with treatment. There was no significant association between the occurrence of molar pregnancy with the type and cause for infertility and number of embryos transferred. Crude (1.06 (95% CI 0.852-1.31)) and adjusted odds ratios (1.07 (95% CI (0.857-1.32)) did not show any association between day of embryo transfer and molar pregnancy even after adjusting for age and excluding secondary infertility. Selection of blastocyst stage embryo after extended culture did not alter the likelihood of having a GTD compared to cleavage stage embryo. Limitations, reasons for caution The retrospective analysis of anonymised HFEA data limited adjustments for confounders such as smoking, previous history of GTD, ethnicity etc that predispose to GTD. Caution needs to be exercised for under-reporting of GTD to HFEA and lack of information on type of GTD identified. Wider implications of the findings Though GTD cannot be prevented by IVF/ICSI, the incidence is significantly low and extended culture is not associated with higher risk of abnormal trophoblastic proliferation or GTD occurrence with IVF/ ICSI treatment. These findings would aid informed implications counselling and reassurance of patients during assisted reproduction treatments. Trial registration number not applicable

P–752 Embryo morphological grading across several IVF centers is not consistent but an interactive training is useful to improve its consistency

Abstract Study question Are the embryologists across several IVF clinics concordant when evaluating embryo morphology? Summary answer Embryo morphological grading is sufficiently consistent among embryologists from the same center, while an interactive training was essential to improve its accuracy across several clinics. What is known already Embryo morphology, mostly at the blastocyst stage, is the strongest non-invasive embryological feature that associates with implantation potential. This association is confirmed also when euploid blastocysts are transferred. At present, several embryo grading schemes exist but is still unclear which is the most effective among them. Moreover, many IVF clinics adopt internal embryo grading scores, further limiting the transferability of this crucial prognostic information across different laboratories. With the aim of assessing the level of concordance in embryo grading within and between IVF clinics, the Italian Society of Embryology, Reproduction and Research (SIERR) conceived this study. Study design, size, duration We photographed 40 cleavage-stage and 40 blastocyst-stage embryos (3 focal-planes=240 photos). Two embryologists (senior and junior) from 65 Italian IVF clinics were invited to grade them. Their evaluations were blindly collected as Phase-I (January2020-March2020). Phase-II consisted of an interactive-training on Google-Classroom during which 6 selected experts found a Consensus on the morphological evaluation of the 80 embryos (April2020). As Phase III (May2020-July2020), a second set of 240 pictures was sent to senior participants and experts. Participants/materials, setting, methods Eighteen centers agreed to participate, and 36 embryologists were included. The embryo grading scheme adopted was the Alpha-ESHRE Istanbul Consensus (parameters: cleavage-stage blastomeres’ symmetry and fragmentation, blastocyst’s expansion, inner-cell-mass and trophectoderm quality), conventionally used in 50% of the centers (N = 9/18). The concordance within (junior versus senior) and between (senior versus experts) centers was calculated through the Cohen’s-k. The concordance between centers was compared before and after the interactive training on the two sets of pictures. Main results and the role of chance The centers and embryologists included were representative of the Italian IVF scenario: oocyte-retrievals per year:711±636,range100–2200; cycles with cleavage-stage embryo-transfer:322±339,0–1300; cycles with blastocyst-stage embryo-transfer:390±403,0–1100; operators per center:5.6±4.0,2–13; senior embryologists’ experience:14.8±7.4yr,7–30; junior embryologists’ experience:2.7±0.6yr,1–3. The intra-center concordance was (i)for blastomeres’ symmetry 82±15% (38–100%), k 0.59±0.27 (0.02–1), (ii)for blastomeres’ fragmentation 88±9% (65–100%), k 0.71±0.2 (0.29–1), (iii)for blastocysts’ expansion 80±16% (48–100%), k 0.66±0.27 (0.19–1), (iv)for inner-cell-mass quality 73±16% (35–95%), k 0.58±0.24 (0.07–0.92), (v)for trophectoderm quality 71±19% (38–95%), k 0.54±0.32 (0.01–0.97). Linear regressions showed no association of centers’ and embryologists’ characteristics with all k-values. Among clinics with the highest mean number of cycles per year and intra-center concordance, we selected 6 experts for the interactive-training. We then calculated the inter-center concordance as the agreement rate between senior embryologists and the experts for phase-I and phase-III: (i)for blastomeres’ symmetry 67±15% (30–85%) and 73±17% (15–90%;Wilcoxon-signed-ranks-test=0.06), k 0.33±0.22 (–0.29–0.58) and 0.42±0.33 (–0.56–0.77); (ii)for blastomeres’ fragmentation 81±17% (23–95%) and 83±14% (50–95%;Wilcoxon-signed-ranks-test=0.8), k 0.54±0.22 (–0.05–0.84) and 0.55±0.22 (0.17–0.81); (iii)for blastocysts’ expansion 59±16% (35–85%) and 67±17% (23–90%;Wilcoxon-signed-ranks-test=0.04), k 0.35±0.20 (0.06–0.73) and 0.44±0.22 (–0.10–0.7); (iv)for inner-cell-mass quality 60±14% (33–80%) and 69±11% (48–85%;Wilcoxon-signed-ranks-test=0.02), k 0.40±0.20 (0.01–0.69) and 0.51±0.18 (0.18–0.77); (v)for trophectoderm quality 55±12% (23–70%) and 63±10% 48–78%;Wilcoxon-signed-ranks-test<0.01), k 0.29±0.15 (–0.08–0.52) and 0.42±0.15 (0.21–0.66). Limitations, reasons for caution Only 28% (N = 18/65) of the Italian IVF centers invited to participate responded to the survey. The conventional adoption of grading schemes other than Istanbul-Consensus by 50% of the embryologists might have biased their evaluation. The experts were not fully-concordant in grading 13.8% of the embryos (N = 22/160), which required active discussions. Wider implications of the findings: Blastocyst-grading concordance was significantly improved after the training phase. Therefore, interactive consensus meetings and training platforms are keenly needed to standardize this practice across the centers. The “avant-garde” of artificial intelligence applied to embryo image analysis might help overcoming this issue in the future. Trial registration number N.A.

The Mare: A Pertinent Model for Human Assisted Reproductive Technologies?

Simple SummaryArtificial reproduction techniques (ART) are used widely in human medicine to overcome infertility, with about one in seven couples being concerned in the Western world. Due to ethical concerns, animal models are needed to develop new methodologies. Although laboratory animals are seminal in this context, they have a short lifespan and are usually fertile. Horses are long-lived domestic animals that are bred until old age, often after they have had a career being used for equestrian activities. Their reproductive functions become altered after 20 years, in a similar way to humans, although there is no menopause per se in horses. There is also a concern for rising overweight and obesity concerns in these species. In addition, embryo transfer and ART are developed to overcome infertility, as for humans. This review details similarities and differences in the reproductive cycle, ART, and fertility concerns in women and mares and discusses the opportunity of using the horse as an appropriate model for ART in humans.Although there are large differences between horses and humans for reproductive anatomy, follicular dynamics, mono-ovulation, and embryo development kinetics until the blastocyst stage are similar. In contrast to humans, however, horses are seasonal animals and do not have a menstrual cycle. Moreover, horse implantation takes place 30 days later than in humans. In terms of artificial reproduction techniques (ART), oocytes are generally matured in vitro in horses because ovarian stimulation remains inefficient. This allows the collection of oocytes without hormonal treatments. In humans, in vivo matured oocytes are collected after ovarian stimulation. Subsequently, only intra-cytoplasmic sperm injection (ICSI) is performed in horses to produce embryos, whereas both in vitro fertilization and ICSI are applied in humans. Embryos are transferred only as blastocysts in horses. In contrast, four cells to blastocyst stage embryos are transferred in humans. Embryo and oocyte cryopreservation has been mastered in humans, but not completely in horses. Finally, both species share infertility concerns due to ageing and obesity. Thus, reciprocal knowledge could be gained through the comparative study of ART and infertility treatments both in woman and mare, even though the horse could not be used as a single model for human ART.

Is a freeze-all policy the optimal solution to circumvent the effect of late follicular elevated progesterone? A multicentric matched-control retrospective study analysing cumulative live birth rate in 942 non-elective freeze-all cycles.

Is late follicular elevated progesterone (LFEP) in the fresh cycle hindering cumulative live birth rates (CLBRs) when a freeze only strategy is applied? LFEP in the fresh cycle does not affect the CLBR of the frozen transfers in a freeze only approach, nor the embryo freezing rate. Ovarian stimulation promotes the production of progesterone (P) which has been demonstrated to have a deleterious effect on IVF outcomes. While there is robust evidence that this elevation produces impaired endometrial receptivity, the impact on embryo quality remains a matter of debate. In particular, previous studies have shown that LFEP is associated with a hindered CLBR. However, most clinical insight on the effect of progesterone on embryo quality in terms of CLBRs have focused on embryo transfers performed after the fresh transfer, thus excluding the first embryo of the cohort. To be really informative on the possible detrimental effects of LFEP, evidence should be derived from freeze-all cycles where no fresh embryo transfer is performed in the presence of progesterone elevation, and the entire cohort of embryos is cryopreserved. This was a matched case-control, multicentre (three centres), retrospective analysis including all GnRH antagonist ICSI cycles in which a freeze all (FA) policy of embryos on day 3/5/6 of embryonic development was applied between 2012 and 2018. A total of 942 patients (471 cases with elevated P and 471 matched controls with normal P values) were included in the analysis. Each patient was included only once. The sample was divided according to the following P levels on the day of ovulation triggering: <1.50 ng/ml and ≥1.50 ng/ml. The matching of the controls was performed according to age (±1 year) and number of oocytes retrieved (±10%). The main outcome was CLBR defined as a live-born delivery after 24 weeks of gestation. The baseline characteristics of the two groups were similar. Estradiol levels on the day of trigger were significantly higher in the elevated P group. There was no significant difference in terms of fertilisation rate between the two groups. The elevated P group had significantly more cleavage stage frozen embryos compared to the normal P group while the total number of cryopreserved blastocyst stage embryos was the same. The CLBR did not differ between the two study groups (29.3% and 28.2% in the normal versus LFEP respectively, P = 0.773), also following confounder adjustment using multivariable GEE regression analysis (accounting for age at oocyte retrieval, total dose of FSH, progesterone levels on the day of ovulation trigger, day of freezing, at least one top-quality embryo transferred and number of previous IVF cycles, as the independent variables). This is a multicentre observational study based on a retrospective data analysis. Better extrapolation of the results could be validated by performing a prospective analysis. This is the first study demonstrating that LFEP in the fresh cycle does not hinder CLBR of the subsequent frozen cycles in a FA approach. Thus, a FA strategy circumvents the issue of elevated P in the late follicular phase. No funding was received for this study. Throughout the study period and manuscript preparation, authors were supported by departmental funds from: Centre for Reproductive Medicine, Brussels, Belgium; Infertility Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; Centro Scienze Natalità, San Raffaele Scientific Institute, Milan, Italy; and IVI-RMA, Lisbon, Portugal. E.S. has competing interests with Ferring, Merck-Serono, Theramex and Gedeon-Richter outside the submitted work. E.P. reports grants from Ferring, grants and personal fees from Merck-Serono, grants and personal fees from MSD and grants from IBSA outside the submitted work. All the other authors have no conflicts of interest to declare. N/A.

The incidence of ectopic/heterotopic pregnancies after blastocyst-stage frozen-thawed embryo transfers compared with that after cleavage-stage: a Society for Assisted Reproductive Technologies Clinical Outcomes Reporting System study

ObjectiveTo investigate whether there is a difference in the ectopic/heterotopic pregnancy rate of blastocyst-stage frozen-thawed embryo transfers (FETs) compared with that of cleavage-stage FETs.DesignA retrospective cohort study.SettingNot applicable.Patient(s)Women undergoing autologous FETs at either the blastocyst stage (n = 118,572) or the cleavage stage (n = 117,619), as reported to the Society for Assisted Reproductive Technology from 2004 to 2013.Intervention(s)None.Main Outcome Measure(s)Pregnancy outcomes, specifically ectopic pregnancy rates and heterotopic pregnancy rates.Result(s)Among those who became pregnant, there was a significantly lower incidence of ectopic/heterotopic pregnancies in blastocyst-stage FETs versus that in cleavage-stage FETs (0.8% vs. 1.1%). The differences in ectopic/heterotopic pregnancy rates remained statistically significant after controlling for confounders such as tubal factor infertility and number of embryos transferred.Conclusion(s)Blastocyst-stage FET was associated with a lower ectopic/heterotopic pregnancy rate compared with cleavage-stage FET.

Alternariol exerts embryotoxic and immunotoxic effects on mouse blastocysts through ROS-mediated apoptotic processes.

Alternariol (AOH), a mycotoxin belonging to the genus Alternaria, has been shown to induce cytotoxicity, including apoptosis and cell cycle arrest, in several mammalian cell types. However, its effects on early-stage embryonic development require further investigation. Here, we have shown that AOH exerts embryotoxic effects on mouse blastocyst-stage embryos and long-term adverse effects on immunity in one-day-old newborn mice of the next generation. Significant apoptosis and decrease in total cell number, predominantly through loss of inner cell mass (ICM), and to a minor extent, trophectoderm (TE) cells, were observed in AOH-treated blastocysts. Moreover, AOH exerted detrimental effects on pre- and post-implantation embryo development potential and induced a decrease in fetal weight in in vitro development and embryo transfer assays. Injection of pregnant mice with AOH (1, 3 and 5mg/kg body weight/day) for 4days resulted in apoptosis of blastocyst-stage embryos and injurious effects on embryonic development from the zygote to blastocyst stage or embryo degradation and a further decrease in fetal weight. Furthermore, AOH exerted a long-term impact on the next generation, triggering a significant increase in total oxidative stress content and expression of genes encoding antioxidant proteins. Lower expression of CXCL1, IL-1β and IL-8 related to innate immunity was detected in liver tissue extracts obtained from one-day-old newborns of AOH-injected pregnant mice (5mg/kg body weight/day) relative to their non-treated counterparts. In addition, ROS served as an upstream regulator of AOH-triggered apoptotic processes and impairment of embryonic development. Our collective results highlight the potential of AOH as an embryotoxic and immunotoxic risk factor during embryo and infant development stages in mice.

Paternal preconception phthalate exposure alters sperm methylome and embryonic programming

Preconception environmental conditions have been demonstrated to shape sperm epigenetics and subsequently offspring health and development. Our previous findings in humans showed that urinary anti-androgenic phthalate metabolites in males were associated with altered sperm methylation and blastocyst-stage embryo development. To corroborate this, we examined the effect of preconception exposure to di(2-ethylhexyl) phthalate (DEHP) on genome-wide DNA methylation and gene expression profiles in mice. Eight-week old C57BL/6J male mice were exposed to either a vehicle control, low, or high dose of DEHP (2.5 and 25 mg/kg/weight, respectively) for 67 days (~2 spermatogenic cycles) and were subsequently mated with unexposed females. Reduced representation bisulfite sequencing (RRBS) of epididymal sperm was performed and gastrulation stage embryos were collected for RRBS and transcriptome analyses in both embryonic and extra-embryonic lineages. Male preconception DEHP exposure resulted in 704 differentially methylated regions (DMRs; q-value < 0.05; ≥10% methylation change) in sperm, 1,716 DMRs in embryonic, and 3,181 DMRs in extra-embryonic tissue. Of these, 29 DMRs overlapped between sperm and F1 tissues, half of which showed concordant methylation changes between F0 and F1 generations. F1 transcriptomes at E7.5 were also altered by male preconception DEHP exposure including developmental gene families such as Hox, Gata, and Sox. Additionally, gene ontology analyses of DMRs and differentially expressed genes showed enrichment of multiple developmental processes including embryonic development, pattern specification and morphogenesis. These data indicate that spermatogenesis in adult may represent a sensitive window in which exposure to DEHP alters the sperm methylome as well as DNA methylation and gene expression in the developing embryo.

Effects of Donor Cell Types on the Development of Bovine Embryos Using Cytoplasm Injection Cloning Technology

Cytoplasm injection cloning technology (CICT) is an efficient technique for evaluating the developmental potential of cloned embryos. In this study, we investigated the effects of donor cell type on the developmental potential and quality of cloned bovine embryos. Adult fibroblasts (AFs) and embryonic cells (ECs) were used as donor cells to clone bovine embryos using CICT. We initially used AF cells to develop cloned embryos and then cultured the cloned day-8 blastocysts for 10 days to obtain ECs as donor cells for second embryo cloning. We found that the bovine blastocysts cloned using AF cells had significantly reduced developmental rates, embryo quality, and ratios of inner cell mass (ICM) to the total number of cells compared to those using ECs as donor cells. Furthermore, there were significant differences in the DNA methyltransferase-, histone deacetylation-, apoptosis-, and development-related genes at the blastocyst stage in embryos cloned from AFs compared to those in embryos cloned from ECs. Our results suggest that using ECs as donor cells for nuclear transfer enhances the quantity and quality of cloned embryos. However, further investigation is required in terms of determining pregnancy rates and developing cloned embryos from different donor cell types.

PGT-A: who and when? Α systematic review and network meta-analysis of RCTs.

Wide controversy is still ongoing regarding efficiency of preimplantation genetic testing for aneuploidy (PGT-A). This systematic review and meta-analysis, aims to identify the patient age group that benefits from PGT-A and the best day to biopsy. A systematic search of the literature was performed on MEDLINE/PubMed, Embase and Cochrane Central Library up to May 2020. Eleven randomized controlled trials employing PGT-A with comprehensive chromosomal screening (CCS) on Day-3 or Day-5 were eligible. PGT-A did not improve live-birth rates (LBR) per patient in the general population (RR:1.11; 95%CI:0.87-1.42; n=1513; I2=75%). However, PGT-A lowered miscarriage rate in the general population (RR:0.45; 95%CI:0.25-0.80; n=912; I2=49%). Interestingly, the cumulative LBR per patient was improved by PGT-A (RR:1.36; 95%CI:1.13-1.64; n=580; I2=12%). When performing an age-subgroup analysis PGT-A improved LBR in women over the age of 35 (RR:1.29; 95%CI:1.05-1.60; n=692; I2=0%), whereas it appeared to be ineffective in younger women (RR:0.92; 95%CI:0.62-1.39; n=666; I2=75%). Regarding optimal timing, only day-5 biopsy practice presented with improved LBR per ET (RR: 1.37; 95% CI: 1.03-1.82; I2=72%). PGT-A did not improve clinical outcomes for the general population, however PGT-A improved live-birth rates strictly when performed on blastocyst stage embryos of women over the 35-year-old mark.

Retinoic acid induces NELFA‐mediated 2C‐like state of mouse embryonic stem cells associates with epigenetic modifications and metabolic processes in chemically defined media

ObjectivesMouse embryonic stem cells (ESCs) are derived from the inner cell mass of blastocyst‐stage embryos and cultured in different culture media with varied pluripotency. Sporadically, a small population of ESCs exhibit 2‐cell stage embryonic features in serum containing medium. However, whether ESCs can transit into 2‐cell embryo‐like (2C‐like) cells in the chemically defined media remains largely unknown.Materials and MethodsWe established a robust in vitro induction system, based on retinoic acid (RA) containing chemically defined media, which can efficiently increase the subpopulation of 2C‐like cells. Further test the pluripotency and 2C features of ESCs cultured in RA. 2C reporter‐positive cells were selected by FACS; the level of protein was detected via immunofluorescence staining and western blot; the level gene expressions were measured by RNA‐seq.ResultsRetinoic acid drives a NELFA (negative elongation factor A)‐mediated 2C‐like state in mouse ESCs, characterized with 2C‐specific transcriptional networks and the ability to contribute trophectoderm (TE) when injected into developing embryos. In addition, RA treatment triggers DNA hypomethylation, active histone modification, suppressed glycolysis metabolism and reduced protein synthesis activity of ESCs.ConclusionsWe showed that RA has a broader role in 2C‐like cells state, not only is one of the upstream regulators of the 2C‐like state in chemically defined media but also illuminates genetic and epigenetic regulations that govern ESCs to 2C‐like transition.

Fertilization by ICSI generates a higher number of live births than IVF in a pioneer facility applying >90% single blastocyst-stage embryo transfers

Fertilization by ICSI generates a higher number of live births than IVF in a pioneer facility applying >90% single blastocyst-stage embryo transfers

Fertilization by ICSI generates a higher number of live births than IVF in a pioneer facility applying &gt;90% single blastocyst-stage embryo transfers

Following earlier studies introducing an IVF-ICSI Split model on couples with unexplained infertility to avoid the scenario of unexplained failed or poor fertilization, PIVET has adopted a high ICSI rate approaching 90%, whereas the general rate among Australian facilities is around 60%. This observational study with retrospective data analysis reports on the IVF±ICSI procedures conducted over the period 2011 to 2019 with follow-up of ensuing pregnancies through 2020. Using autologous oocytes, 2343 women had 3434 IVF±ICSI cycles where 84.5% of women had 88.9% of initiated treatment cycles using ICSI and only 5.3% of women had 4.0% of cycles by IVF. The remaining 10.1% of women utilized the IVF-ICSI Split model for the remaining 7.2% of cycles. It was shown that oocyte fertilization rates were significantly higher for ICSI (p&lt;0.0001), but not significant for women &gt;40 years. The utilization rates of the ensuing embryos were ~45% across all ages with no significant differences across the ages, except for those small numbers of women ≥45 years who had a higher rate from IVF-generated embryos (p&lt;0.0002). Pregnancy outcome were higher from ICSI-generated embryos across the age groups, being especially marked among the younger women &lt;40 years (p&lt;0.0001). Miscarriage rates were lowest for the IVF-generated pregnancies (overall 6.7% vs 22.8%, p&lt;0.0001) but nevertheless the final live birth productivity rates per initiated treatment cycle remained higher from the ICSI-generated pregnancies (56.5% vs 46.3%; p&lt;0.0001). Although this study does not meet the highest standards for EBM, it emanates from a pioneer facility with &gt;40 years of published activity and which practices 90% blastocyst transfers in &gt;90% SET cycles. The study supports a high ICSI rate of almost 90% and an IVF-ICSI Split rate of 10%.

Comprehensive Transcriptome Analysis of mRNA Expression Patterns of Early Embryo Development in Goat under Hypoxic and Normoxic Conditions.

Simple SummaryOxygen plays a vital role in the development of early embryos, no matter whether it is too high or low, it will adversely affect the early embryo development, but the mechanisms involved in these effects are still unclear. RNA-seq was performed to compare 8-cell-stage and blastocyst-stage goat embryos under hypoxic and normoxic conditions, the mRNA expression mechanisms of 8-cell- and blastocyst-stage embryos were systematically analyzed under hypoxic and normoxic conditions. Functional enrichment analysis indicated that these differentially expressed genes (DEGs) were mainly related to biological processes and function regulation. In conclusion, we can infer that oxidative stress regulates early embryo development by affecting the expression of zygotic genes and transcription factors, and those stress genes play a potential role in adaptation to normoxic environments in goat embryos.It has been reported that hypoxic environments were more suitable for the in vitro development of mammalian embryos, but the underlying mechanisms were still unclear. In the present study, RNA-seq was performed to compare 8-cell-stage and blastocyst-stage goat embryos under hypoxic and normoxic conditions; zygotes were checked at 72 and 168 h to 8-cell stage (L8C) and blastocyst stage (LM) in hypoxic conditions and 8-cell stage (H8C) and blastocyst stage (HM) in normoxic conditions. In the H8C and L8C groups, 399 DEGs were identified, including 348 up- and 51 down-regulated DEGs. In the HM and LM groups, 1710 DEGs were identified, including 1516 up- and 194 down-regulated DEGs. The expression levels of zygotic genes, transcription factors, and maternal genes, such as WEE2, GDF9, HSP70.1, BTG4, and UBE2S showed significant changes. Functional enrichment analysis indicated that these DEGs were mainly related to biological processes and function regulation. In addition, combined with the pathway–gene interaction network and protein–protein interaction network, twenty-two of the hub genes were identified and they are mainly involved in energy metabolism, immune stress response, cell cycle, receptor binding, and signal transduction pathways. The present study provides comprehensive insights into the effects of oxidative stress on early embryo development in goats.

Does miRNA Expression in the Spent Media Change During Early Embryo Development?

Distinct miRNA populations have been detected in the spent media of in-vitro culture systems. However, profiling has been limited to media conditioned with blastocyst-stage embryos. Therefore, the aim of the study was to profile extracellular miRNAs throughout the pre-implantation period in bovine embryos. To achieve this, cumulus oocyte complexes were aspirated from ovaries, in-vitro matured, fertilized, and cultured under standard laboratory procedures to the 2-cell, 8-cell, or blastocyst stage of development. At each developmental stage, 25 μl of spent in-vitro culture media was collected, pooled to 300 μl, and processed for total RNA extraction. In-vitro culture media, which never came in contact with any embryos, were additionally processed for total RNA extraction to serve as a negative control. Following hybridization on a GeneChip miRNA 4.0 array, comparative analysis was conducted between spent media and control samples. In total, 111 miRNAs were detected in the spent media samples, with 56 miRNAs identified in blastocyst spent media, 14 miRNAs shared between 8-cell and blastocyst spent media, 7 miRNAs shared between all 3 conditions, and 6 miRNAs exclusive to 2-cell spent media. miRNA-mRNA target prediction analysis revealed that the majority of genes predicted to be regulated by the miRNAs identified in the study have roles in cellular process, metabolic process, and biological regulation. Overall, the study suggest that miRNAs can be detected in the spent media of in-vitro culture system throughout the pre-implantation period and the detected miRNAs may influence genes impacting early embryo development.

When using donor oocytes, does embryo stage matter? An analysis of blastocyst versus cleavage stage embryo transfers using a cryopreserved donor oocyte bank.

Oocyte donor in vitro fertilization (IVF) represents an ideal model to study the effects of embryo stage on reproductive success, as embryos come from young women with high-quality oocytes. Our study aimed to determine if embryo transfer stage affected outcomes in oocyte donor IVF, including the common scenario where only a limited number of quality embryos are available after culture. This retrospective cohort analyzed anonymous vitrified donor oocyte cycles at a single clinic between 2008 and 2015. Overall, 983 recipients underwent 1178 warming cycles resulting in fresh transfer of one-to-two embryos. Our primary outcome was live birth; secondary outcomes included multiple birth, birthweight, and gestational age. Log binomial regression with cluster-weighted generalized estimating equations were used to calculate adjusted risk ratios (aRR) accounting for recipient age, race, and transfer year. Among 132 cleavage and 1046 blastocyst transfer cycles, cleavage transfers were associated with lower probability of live birth (aRR 0.72, 95% CI 0.59-0.88). Subgroup analysis focused on cycles with a limited number of quality embryos 3 days post-fertilization (≤2), as clinically these women were most likely to be considered for cleavage transfers. Among these cycles (120 cleavage, 371 blastocyst), cleavage transfers were still associated with lower live birth rates compared to blastocyst (aRR 0.66, 95% CI 0.51-0.87) CONCLUSIONS: Even in a donor oocyte model with high-quality oocytes, there was a benefit to extended culture and blastocyst transfer, including when only one-to-two quality embryos were available after early culture. This is possibly owed to improved uterine synchronicity or decreased contractility.

Single-embryo transfer: a key strategy to reduce the risk for multiple pregnancy in assisted human reproduction.

In the early days of assisted reproductive technology (ART), the main target was achieving gestation. Success rates were low, and multiple embryo transfers became common practice, with multiple pregnancies being 20 times higher than in natural conception. Multiple pregnancy is associated with a higher risk of complications for the mother and the baby than a singleton pregnancy. Added to healthcare costs, multiple pregnancy also involves other costs and psychosocial risks, with a high social and health costs. At present, success rates of assisted human reproduction (AHR) have improved dramatically, partially due to advances in laboratory techniques such as culture of blastocyst-stage embryos and vitrification. Additionally, there is a wide range of counseling, health and economic policies that have demonstrated being effective in increasing single-embryo transfer (SET) practices and reducing multiple pregnancies, which ensures satisfactory success rates. Therefore, single-embryo transfer emerges as the approach of choice for AHR to result in a full-term healthy newborn.

Embryo blastulation and quality between days 5 and 6 of extended embryo culture.

This study aims to know what proportion of culture day 5 pre-blastocyst-stage embryos develop into blastocysts by culture day 6 and what patient and cycle characteristics are associated with delayed blastocyst formation. A retrospective observational cohort analysis was performed including a total of 9886 embryos from 1008 IVF cycles in 835 patients, who underwent treatment between January 1, 2016, and December 31, 2018. Autologous fresh in vitro fertilization (IVF) cycles at a single academic center were included in the analysis. Embryos were group-cultured using single-step culture media. Blastulation was defined as the presence of a new blastocyst. Usable blastulation was defined as the presence of a new good or excellent quality, expanded, hatching, or hatched blastocysts. The mean blastulation rate between days 5 and 6 of extended embryo culture was 30.9%. The mean percentage of embryos developing into usable blastocyst-stage embryos was 19.8%. The factors associated with blastulation on day 6 included the total number of embryos and the number of pre-blastocysts on day 5, as well as the use of ICSI. Age, the number of total embryos, those remained in culture and pre-blastocysts, as well as the blastulation rate on day 5 were associated with usable blastulation. It is important to know the usable blastocyst development rate between culture days 5 and 6 in order to adequately counsel patients debating whether to proceed with fresh ET on day 5 or forego ET with the expectation that embryos will be biopsied for PGT and/or cryopreserved on culture day 6. Our findings provide evidence to help guide patients in this difficult decision.

Association between estradiol levels and clinical outcomes of IVF cycles with single blastocyst embryo transfer

Objective: To determine the correlation of different serum estradiol levels on the trigger day with the clinical and laboratory outcomes of in-vitro fertilization (IVF) cycles comprising a single fresh top-quality blastocyst transfer. Methods: This was a retrospective observational study performed in Morula IVF Clinic Jakarta. Five hundred forty-two women were recruited and grouped according to their serum estradiol levels on the trigger day of follicular maturation as follows: &lt;2 000 pg/mL, 2 000-2 999 pg/mL, 3 000-3 999 pg/mL, and ≥ 4 000 pg/mL. Clinical pregnancy and miscarriage rates were evaluated as the primary outcomes and embryology laboratory results as the secondary outcomes which consisted of the number of retrieved, mature, and fertilized oocytes, the total sum of derived embryos, and top-quality embryos at cleavage and blastocyst stage. Results: Clinical pregnancy and miscarriage rates did not differ among the groups (P&gt;0.05). Nonetheless, the study demonstrated a positive correlation of the serum estradiol levels with the overall laboratory outcomes including the number of retrieved, mature, and fertilized oocytes, the total sum of derived embryos, and top-quality embryos at cleavage and blastocyst stage (P&lt;0.001). The subject group with estradiol level of ≥4 000 pg/mL was superior to the other groups in its respective median number of retrieved, mature, fertilized oocytes, total derived embryos, and top-quality cleavage- and blastocyst-stage embryos. Conclusions: Although an apparent positive correlation is observed between estradiol levels and laboratory outcomes, serum estradiol level on hCG trigger day is not associated with the clinical outcomes of IVF.