Androgenetic Embryos Research Articles

P-806 Heritable genome editing in haploid mouse embryonic stem cells

Abstract Study question Can we edit a specific gene by CRISPR-Cas9 in haploid mouse embryonic stem cells? Summary answer We successfully carried out heritable genome editing of a coat pigmentation gene in haploid mouse embryonic stem cells via electroporation. What is known already Embryonic stem cells have been widely used as a substrate to carry out genetic studies due to their availability, low cost and ease of handling. CRISPR-Cas9 has been successful in editing genes in the human model. Gene editing occurred by homology directed repair or non-homologous end joining. The utilization of a haploid genome would confirm the feasibility of non-homologous end joining repair mechanism. Study design, size, duration In the past 3 months, diploid as positive control and experimental haploid embryonic stem cells were independently cultured for genome editing experiments. Electroporation was used as a means to introduce CRISPR-Cas9 into cells. The negative control cohort consisted of embryonic stem cells electroporated without delivering any CRISPR solution. CRISPR-Cas9 was designed to knock out the Tyr gene to create an albino phenotype. Participants/materials, setting, methods Diploid embryonic stem cells were derived from C57BL/6 mice. The haploid counterpart instead was obtained from haploid androgenetic embryos using spermatozoa from Oct4-EGFP mice. CRISPR solution was prepared by combining Tyr gRNA and Cas9 protein. For each experiment, one million cells were suspended in CRISPR solution and an electroporation buffer. Then, electric pulses were applied for electroporation with 4 different parameters. Gene modification at the target site was confirmed using T7E1 cleavage assay. Main results and the role of chance A 584 base pairs region around the CRISPR target site was amplified in all the diploid and haploid mouse embryonic stem cells. After performing T7E1 cleavage assay, editing efficiency was calculated following the manufacturer protocol. Precisely, after testing out different electroporation conditions on diploid mESCs, 1350V/20ms/2 pulses together with the combination of three sgRNAs (exon 1 and exon 2) and Cas9 protein, provided the best genome editing efficiency at 36.8% with a viability of 80%. Interestingly, when delivering only one type of gRNA (exon 2) together with Cas9 protein, we saw an efficiency rising to 77.6%. We then used the same condition to edit the Tyr gene in the genome of haploid androgenetic stem cells and confirmed that also in these cells, we were able to obtain a comparable genome editing efficiency at 68.4%. Limitations, reasons for caution While these experimental observations are limited, it is encouraging that it is possible to target and edit a haploid genome with a single allele. Wider implications of the findings The utilization of a haploid cell culture model paves the way to utilize CRISPR-Cas9 for gamete genome editing. This will provide a consistent and reliable method to correct inheritable genetic conditions on gametes allowing uniform gene editing of all cell progeny. Trial registration number Not applicable

P-805 Sperm and embryo genome editing by CRISPR-Cas9 in a mouse model

Abstract Study question We wonder whether it is possible to edit a specific gene in individual mammalian spermatozoa as well as embryo by using CRISPR-Cas9? Summary answer We were able to successfully edit the coat pigment gene on individual spermatozoa as well as embryos by injecting CRISPR-Cas9 during Piezo-ICSI. What is known already Earlier investigations into embryo genome editing with CRISPR-Cas9 were conducted during the S-phase or zygote stage, encountering obstacles such as mosaicism and loss of heterozygosity. To overcome these challenges, performing genome editing at the gamete level is considered more favorable. While it may be achievable in oocytes, sperm genome editing can be ambitious due to the tightly supercoiled DNA around the protamine cores. In initial attempts, simple permeabilization of the sperm membrane allowed CRISPR-Cas9 penetration into the cell; however, the attempts at genomic editing proved to be unsuccessful. Study design, size, duration In the past several months, oocytes were divided into two groups: half of them were used to perform embryo genome editing, whilst the other half was used to edit exclusively the sperm genome through oocyte-mediated sperm decondensation (OMSD), where a single spermatozoon was injected into an enucleated oocyte and used the ooplasmic machinery for DNA decondensation and editing. In both models, CRISPR-Cas9 was used to knock out Tyr gene to create an albino phenotype. Participants/materials, setting, methods B6D2F1 mice were used to retrieve oocytes and spermatozoa. A cohort of oocytes used for the OSMD approach were enucleated, whilst the others were maintained with an intact nucleus. All oocytes, enucleated or not, were injected with a single spermatozoon with Tyr-sgRNA and Cas9 protein. Embryos were cultured until the 8-cell stage and processed for T7E1 cleavage analysis. Genome editing efficiency was calculated following the manufacturer protocol. Main results and the role of chance Of the 40 oocytes used for the study, 20 intact oocytes were used to perform embryo genome editing in the standard fashion, while 20 were enucleated for OMSD experiments. After undergoing Piezo-ICSI with CRISPR-Cas9 solution, 90.0% (18/20) of intact oocytes fertilized. After 48 hours in culture, diploid embryos reached the 8-cell stage at 88.9% (16/18). The 584-bp region of the target site of extracted DNA was amplified and gene modification of the edited diploid embryo cohort was confirmed in 87.5% (14/16). Compared to the OMSD cohort, enucleated oocytes fertilized at 85.0% (17/20) while cleavage rate was lower at 64.7% (11/17) and sperm genome modification at 81.8% (9/11). Both diploid embryos and haploid androgenetic embryos evidence an editing efficiency of 36.8%. Therefore, we verified that genome editing on embryos and sperm achieved the same editing yield. Limitations, reasons for caution While the experimental observations are still limited, it is important to note that these results in the embryo model may be underestimated by the T7E1 cleavage assay that only recognize heterozygous modification. The technique needs further refinement to optimize targeting and editing efficiency. Wider implications of the findings Sperm genome decondensation through an OMSD approach allows CRISPR-Cas9 to edit the sperm genome in order to correct eventual pathogenic mutations present in the gamete. Moreover, these would be the only method to absolutely ensure uniform generation of resulting embryos. Trial registration number not applicable

Allelic reprogramming of chromatin states in human early embryos.

The reprogramming of parental epigenomes in human early embryos remains elusive. To what extent the characteristics of parental epigenomes are conserved between humans and mice is currently unknown. Here, we mapped parental haploid epigenomes using human parthenogenetic and androgenetic embryos. Human embryos have a larger portion of genome with parentally specific epigenetic states than mouse embryos. The allelic patterns of epigenetic states for orthologous regions are not conserved between humans and mice. Nevertheless, it is conserved that maternal DNA methylation and paternal H3K27me3 are associated with the repression of two alleles in humans and mice. In addition, for DNA-methylation-dependent imprinting, we report 19 novel imprinted genes and their associated germline differentially methylated regions. Unlike in mice, H3K27me3-dependent imprinting is not observed in human early embryos. Collectively, allele-specific epigenomic reprogramming is different in humans and mice.

Haploid androgenetic development of bovine embryos reveals imbalanced WNT signaling and impaired cell fate differentiation†.

Haploid embryos have contributed significantly to our understanding of the role of parental genomes in development and can be applied to important biotechnology for human and animal species. However, development to the blastocyst stage is severely hindered in bovine haploid androgenetic embryos (hAE). To further our understanding of such developmental arrest, we performed a comprehensive comparison of the transcriptomic profile of morula-stage embryos, which were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of transcripts associated with differentiation in haploid and biparental embryos. Among numerous disturbances, results showed that pluripotency pathways, especially the wingless-related integration site (WNT) signaling, were particularly unbalanced in hAE. Moreover, transcript levels of KLF4, NANOG, POU5F1, SOX2, CDX2, CTNNBL1, AXIN2, and GSK3B were noticeably altered in hAE, suggesting disturbance of pluripotency and canonical WNT pathways. To evaluate the role of WNT on hAE competence, we exposed early Day-5 morula stage embryos to the GSK3B inhibitor CHIR99021. Although no alterations were observed in pluripotency and WNT-related transcripts, exposure to CHIR99021 improved their ability to reach the blastocysts stage, confirming the importance of the WNT pathway in the developmental outcome of bovine hAE.

P-723 Pre-fertilization genetic testing to select healthy gametes in a mammalian Marfan syndrome model

Abstract Study question Is it possible to identify disease-free gametes before insemination to prevent vertical transmission of an autosomal dominant disease? Summary answer In a mammalian Marfan syndrome model, we successfully generated and identified healthy androgenotes to be utilized as male gametes to produce unaffected conceptuses. What is known already In patients with autosomal dominant genetic disorders such as Marfan syndrome, germline heterozygosity poses a risk to offspring health. For these patients, preimplantation genetic testing for monogenic disorders (PGT-M) allows us to select unaffected conceptuses for embryo replacement. However, PGT-M is inherently inefficient and can lead to embryo wastage. This has sparked interest in replicating the genome of individual gametes for (1) pre-fertilization genetic testing and (2) subsequent use as genotyped gamete substitutes during IVF. Study design, size, duration For this study, we selected 4 male B6 mice that were heterozygous for the Fbn1tm1Hcd mutation and displayed classic manifestations of Marfan syndrome. Spermatozoa from these mice were used to inseminate enucleated wildtype mouse oocytes and generate haploid androgenetic embryos for (1) Fbn1tm1Hcd genotyping and (2) use as unaffected male gamete substitutes. Participants/materials, setting, methods Using tail clippings and embryo biopsy specimens, a Fbn1tm1Hcd allele-specific PCR primer was developed and validated. Haploid androgenetic embryos were allowed to progress to the 8-cell stage. Subsequently, a single-haploid pseudo-blastomere was isolated for genotyping, while the remainder were individually grafted into activated recipient wildtype oocytes and fused using inactivated Sendai virus to generate biparental conceptuses. Resulting diploid conceptuses were cultured in a time-lapse incubator and then genotyped to confirm the healthy paternal haplotype. Main results and the role of chance A total of 175 wildtype B6 oocytes were enucleated yielding 169 ooplasts (96.6% survival rate). The ooplasts were inseminated with spermatozoa from mutant Fbn1tm1Hcd mice, yielding 144 (85.2%) monopronucleated haploid androgenetic embryos. Of those, 100 (69.4%) progressed to the 8-cell stage. In 47 (47.0%) of them, genetic analysis by PGT-M on an individual pseudo-blastomere characterized the embryo as unaffected because it harbored the wildtype Fbn1 allele. The remaining 7 haploid pseudo-blastomeres from 5 Fbn1 wildtype androgenetic embryos were individually isolated and grafted into 35 recipient oocytes to generate 31 (88.6%) biparental conceptuses with the desired paternal haplotype. These achieved a 61.3% (19/31) blastocyst development rate after 96 hours, displaying normal embryo morphokinesis. A blastocyst biopsy was carried out (n = 19), confirming the unaffected paternal haplotype void of the Fbn1tm1Hcd mutation. Limitations, reasons for caution Although this technique is feasible for pre-fertilization genetic testing, it needs to be reproduced with the same efficiency in humans. A major concern is related to the dynamics and roles of the sperm centrosome in the first mitotic division of human zygotes, which may be missing in the pseudo-gametes. Wider implications of the findings This approach to pre-fertilization genetic testing will benefit patients with heritable genetic conditions. In addition, the inherited decondensation of the male genome by the ooplasm may enable genome editing experiments to be performed. Trial registration number Not applicable

P-795 Identification of a specific paternal haplotype in the germline and perpetuating progenies by male gamete replication

Abstract Study question Can we identify and replicate a male germ cell genome to generate progenies carrying a specific genotype? Summary answer We were able to replicate male genome and identify the androgenote with a specific genotype to generate conceptuses with identical paternal haplotype. What is known already For patients with heritable genetic disorders, current treatment relies on preimplantation genetic testing for monogenic disorders (PGT-M) to select unaffected embryos for replacement; however, this can lead to embryo wastage, causing emotional distress. Although genomic editing of gametes is being attempted, CRISPR-based technologies are inaccurate and can lead to loss of heterozygosity in cases where the embryo uses the maternal genome as a template to edit the paternal genome. Therefore, the use of haploid androgenotes may offer a useful tool to select male gametes with genes of interest or a successfully edited genome before the creation of an embryo. Study design, size, duration To generate haploid androgenotes, spermatozoa from hemizygous transgenic mice with GFP expression (B6-EGFP x B6D2F1) were used to inseminate enucleated wildtype oocytes. Constructs were cultured to the 8-cell stage. Two pseudo-blastomeres were isolated for genotyping by DNAseq. A blastomere with GFP expression was used as a male gamete. Experiments were repeated using mice with the Fbn1tm1Hcd mutation as a model for Marfan syndrome. Biparental embryos were created to preserve the mutation as a disease model. Participants/materials, setting, methods To generate haploid androgenotes, metaphase II oocytes from wildtype B6 mice were enucleated and inseminated using spermatozoa from hemizygous GFP transgenic mice. Haploid androgenetic embryos were cultured in a time-lapse incubator up to the 8-cell stage. Biparental embryos were created by fusing a selected androgenote with an activated oocyte from another cohort. Resulting embryos were genotyped to confirm the paternal haplotype. Experiments were reproduced using spermatozoa from heterozygous Fbn1tm1Hcd-mutant mice. Main results and the role of chance In the first set of experiments using spermatozoa from transgenic mice hemizygous of GFP, 200 wildtype oocytes were enucleated and generated 195 (97.5%) ooplasts. Insemination of those ooplasts yielded 168 (86.2%) monopronucleated androgenetic embryos, and subsequently generated 116 (69.0%) 8-cell haploid androgenetic embryos, of which 59 (50.8%) expressed GFP. Up to 2 sibling blastomeres were genotyped individually by DNAseq and confirmed to have an identical paternal haplotype. The remaining blastomeres were used to create biparental embryos and yielded a 97.2% fertilization rate after grafting into 145 oocytes, comparable with the fertilization rate of ICSI controls at 95.6%. Although control zygotes yielded an 88.0% blastulation rate, the experimental embryos yielded a lower blastulation rate of 58.2% (P<0.0001). Albeit with a lower developmental rate, all experimental blastocysts expressed GFP and displayed normal embryo morphokinesis. For the experiment using spermatozoa from Fbn1tm1Hcd mice, 50 8-cell stage haploid androgenetic embryos were generated. Two individual pseudo-blastomeres from 35 haploid embryos were used for genotyping. The remaining 210 haploid blastomeres were used to construct biparental embryos and yielded 119 (56.7%) blastocysts. PCR on embryo biopsies confirmed that the resulting blastocysts inherited the mutant (n = 63) or wildtype (n = 56) paternal haplotype from the original gamete. Limitations, reasons for caution This technique allowed the selection of male gametes with the desired genotype to generate conceptuses with a known haplotype in a reproducible manner. A more prospective experiment is ongoing to simulate clinical application by cryopreserving haploid androgenetic embryos and using only androgenotes with a confirmed genotype to create biparental embryos. Wider implications of the findings This approach to utilize gamete substitutes before fertilization ensures the maintenance of rare and unique genetic materials to generate conceptuses with the desired genotype in transgenic mice in disease models. Indeed, once this technique is implemented in humans, genotyped androgenotes can be used to characterize germline heterozygosity. Trial registration number N/A

O-223 Sperm Genome Editing by CRISPR-Cas9 through an Ooplasm-Mediated Approach

Abstract Study question Can we edit the sperm genome by CRISPR-Cas9 through an ooplasmic-mediated approach? Summary answer The ooplasm-mediated technique induced the decondensation of the male gamete nucleus and allowed CRISPR-Cas9 to access the sperm DNA to carry out successful edits. What is known already Previous studies on genome editing using CRISPR-Cas9 have been performed at the S-phase or zygote stage, but challenges were represented by mosaicism and possible off-target edits. To address these issues, gene editing at the gamete level would be ideal. While this may be feasible in oocytes, it appears ambitious in the spermatozoon due to the DNA hypercoiling around the protamine core and the chromatin compaction. In preliminary experiments, permeabilization of sperm membrane allowed penetration of CRISPR-Cas9 to enter the cell, however, genomic editing was proved unsuccessful. Study design, size, duration In the past 5 months, a total of 128 oocytes were divided into 2 groups. To edit exclusively the male genome through oocyte-mediated sperm decondensation (OMSD), a single spermatozoon was injected into an enucleated oocyte to produce a haploid androgenetic embryo. The control cohort consisted of embryos generated through standard ICSI with the established heritable genome editing (HGE) approach. Both groups were treated with CRISPR-Cas9 aiming to knockout Tyr gene to create an albino phenotype. Participants/materials, setting, methods B6D2F1 mice were used to retrieve oocytes and spermatozoa. A cohort of oocytes used for OSMD approach were enucleated. While the intact oocytes were used for HGE control. All oocytes were injected with a single spermatozoon together with CRISPR-Cas9 solution, containing Tyr gRNA. All embryos were cultured up to the 8-cell stage. Individual blastomeres were isolated and sequenced to proof editing. DNA was extracted and amplified for T7E1 analysis to validate genome editing efficiency. Main results and the role of chance Of the 128 oocytes used for the study, 51 were enucleated for the OMSD experiments, while 77 were used for HGE control. After undergoing ICSI with CRISPR-Cas9 solution, 84.3% (43/51) of the OMSD cohort displayed a single male pronucleus and 79.2% (61/77) control HGE fertilized. In a time-lapse microscopy, 95.3% (41/43) of the OMSD experimental group developed to the 2-cell stage, comparable to the HGE control development at 96.7% (59/61). After 48 hours in culture, the HGE control reached 8-cell development at 88.5% (54/61), while the OMSD cohort cleaved at the lower rate at 60.4% (26/43, P<0.001). A 423-bp region around the CRISPR target site was amplified on DNA extracted by isolated blastomere. Gene modification at the target site was confirmed in 33.7% (70/208) of OMSD cohort and 29.7% (88/296) of the HGE control embryos. Limitations, reasons for caution While the experimental observations are still limited, it is important to note that these results may be underestimated since embryos with completely uniform modifications may go undetected by T7E1 assay. The technique needs further refinement to optimize targeting efficiency and support embryo development. Wider implications of the findings Once the genetic editing method is optimized, individual pseudo-blastomeres generated through this OMSD approach can be utilized as male gametes to fertilize oocyte. The generation of live offspring with the corrected haplotype will serve to prove the safety of genome editing through this technique. Trial registration number N/A

Human zygotic genome activation is initiated from paternal genome

Although parental genomes undergo extensive epigenetic reprogramming to be equalized after fertilization, whether they play different roles in human zygotic genome activation (ZGA) remains unknown. Here, we mapped parental transcriptomes by using human parthenogenetic (PG) and androgenetic (AG) embryos during ZGA. Our data show that human ZGA is launched at the 8-cell stage in AG and bi-parental embryos, but at the morula stage in PG embryos. In contrast, mouse ZGA occurs at the same stage in PG and AG embryos. Mechanistically, primate-specific ZNF675 with AG-specific expression plays a role in human ZGA initiated from paternal genome at the 8-cell stage. AG-specifically expressed LSM1 is also critical for human maternal RNA degradation (MRD) and ZGA. The allelic expressions of ZNF675 and LSM1 are associated with their allelically epigenetic states. Notably, the paternally specific expressions of ZNF675 and LSM1 are also observed in diploid embryos. Collectively, human ZGA is initiated from paternal genome.

P-797 Benefit of Replicating Mammalian Gametes

Abstract Study question Can haploid parthenogenetic and androgenetic embryos generate haploid embryonic stem cells (haESCs) and function as respective oocytes and spermatozoa? Summary answer Haploid embryos generated blastocysts capable of yielding haESCs. Haploid blastomeres of both sexes served as multiple copies of gamete substitutes. What is known already Although creating diploid digyneic or dispermic embryos through nuclear transfer is possible, the conceptuses rarely achieve normal preimplantation development due to unbalanced epigenomes, demonstrating the requirement of both parental epigenomes in the course of embryogenesis. Haploid parthenogenetic embryos can be achieved by inducing calcium oscillations of a metaphase II oocyte, while the paternal counterpart can be generated through the insemination of enucleated oocytes. These embryos may serve to generate haESCs for heterozygosity identification and can be used for reproductive purposes in animal models. Study design, size, duration To generate parthenogenetic embryos, metaphase II oocytes from B6D2F1 mice were artificially activated. Androgenetic embryos were generated from the insemination of enucleated oocytes. Haploid embryos were cultured to blastocysts and seeded on feeder cells (MEF) to derive haESCs. Haploid blastomeres from 8-cell embryos of either sex were isolated and used as artificial gametes; artificial oocytes were generated by grafting a parthenogenetic blastomere to an enucleated recipient oocyte. Androgenetic blastomeres were used as male gametes. Participants/materials, setting, methods Parthenogenetic embryos were generated by exposing metaphase II oocytes to calcium ionophore. Another cohort of B6D2F1 oocytes were enucleated and underwent piezo-ICSI to generate androgenetic embryos. Sendai-virus–mediated nuclear transfer was performed using parthenogenetic blastomeres as donor nucleuses to generate artificial oocytes and subsequently fertilized. Androgenetic blastomeres were used as sperm substitutes by fusion with an intact oocyte to generate zygotes. Control embryos were generated by piezo-actuated ICSI. Developmental characteristics were recorded by time-lapse microscopy. Main results and the role of chance A total of 129 oocytes were parthenogenetically activated by calcium ionophore, and 119 developed a female pronucleus. Androgenetic embryos were generated from the insemination of 375 ooplasts; 317 developed a male pronucleus. The monopronuclear appearance of haploid embryos of both sexes was comparable to the fertilization of control at 90.7%. Haploid embryo development for both sexes up to the 4-cell stage matched the control. However, from the 8-cell stage onward, parthenogenetic and androgenetic embryos cleaved at a lower rate compared to the control at 76.4%, 59.3% and 92.7% (P < 0.0001), respectively. The haploid genome affected the rate of compaction at 73.6%, 55.3%, and 89.2% (P<0.0001), respectively, and the rate of blastulation at 27.8%, 11.2%, and 80.8% (P<0.00001) respectively. Androgenetic embryos showed delayed cleavage from the 4-cell stage onward, while the parthenogenetic counterparts had similar morphokinetics to the control. A total of 20 parthenogenetic and 18 androgenetic blastocysts were plated on MEF, yielding 5 parthenogenetic and 3 androgenetic haESC lines. Both haESCs yielded beating cardiomyocytes on day 10 of differentiation while maintaining haploidy. The utilization of haploid pseudo blastomeres as gametes yielded 93 reconstructed biparental embryos that resulted in 71 blastocysts (76.3%). Time-lapse morphokinetics were comparable to the control. Limitations, reasons for caution Haploid embryos developed to blastocysts at a remarkably lower rate than the control, particularly the androgenotes; however, they can yield pluripotent haESCs capable of differentiation in the mesoderm and subsequent cardiogenesis prone to self-diploidization. Once this system is replicated in humans, the role of the centrosome needs to be investigated. Wider implications of the findings Our study has demonstrated the feasibility of propagating both male and female genomes from single gametes for reproductive applications. Indeed, once a stable culture of haESCs is achieved, genotyped pluripotent haESCs can be used to help identify heterozygosity for inherited disorders. Trial registration number N/A

P-798 Utilization of Haploid Androgenetic Blastomeres to Characterize Male Genomic Inheritance and as a Male Gamete Substitute

Abstract Study question Can sperm genomes be replicated by generating androgenetic embryos, and can resulting haploid blastomeres be used as a substitute for male gametes with known genotype? Summary answer Once utilized as male gametes, haploid androgenetic blastomeres can be used for gamete genotyping and can sustain embryo development to live offspring. What is known already Heterozygosity in human spermatozoa may represent a risk to offspring health. This has led to attempts to replicate the gamete genome by generating haploid embryonic stem cells (haESCs) that can be used to typify heterozygosity or as gamete substitutes. Although haESC lines are unstable with a tendency to self-diploidize, utilizing haploid androgenetic cells in the early embryonic stages, such as the blastomere stage, can provide multiple copies of genetically functional pseudo-gametes. Study design, size, duration Haploid androgenetic embryos were generated using spermatozoa from B6 heterozygous mice (B6-EGFP x B6D2F1) and cultured until the 8-cell stage. Individual haploid blastomeres with green fluorescent protein (GFP) expression were fused with activated recipient oocytes. Resulting conceptuses were cultured in a time-lapse system to monitor each stage of preimplantation development. Blastocysts with GFP expression were transferred to pseudo-pregnant surrogates. Pregnancy and offspring reproductive health were evaluated. Piezo-actuated ICSI was performed on unmanipulated oocytes as control. Participants/materials, setting, methods To generate haploid androgenetic embryos, metaphase II oocytes of B6D2F1 mice were treated with cytochalasin B, enucleated under Oosight®, and inseminated by piezo-actuated ICSI. Haploid androgenetic embryos were cultured to the 8-cell stage. Androgenetic blastomeres with GFP expression were coated with inactivated Sendai-virus and grafted to an activated recipient oocyte from another cohort. Cleavage parameters of reconstructed zygotes were compared to controls. Blastocysts were transferred into the uterine cavity of a 2.5-day post-coitus CD-1 surrogate. Main results and the role of chance A total of 421 oocytes were enucleated with a survival rate of 98.6%. The ooplasts underwent ICSI; 351 (84.6%) constructs developed a single male pronucleus 4-6h post-ICSI, and 208 (59.3%) constructs cleaved to the 8-cell stage. Parthenogenic activation by calcium ionophore was successful in 96.4% of the recipient oocytes, confirmed by the appearance of a single female pronucleus. A total of 148 haploid androgenotes with GFP expression were grafted onto corresponding activated oocytes; 145 successfully fused. Up to 2 unused sibling haploid blastomeres per embryo were genotyped using whole exome sequencing by a reference laboratory. A total of 481 untreated oocytes were inseminated, yielding 437 control zygotes. The cleavage of reconstructed embryos into the 2-cell (96.4%), 4-cell (94.3%), and 8-cell stage (91.4%) was comparable to that of control conceptuses (97.5%, 93.8%, and 92.7% respectively). Although morula compaction (80.0%) and blastulation (60.7%) were lower than control (89.2% and 80.8%, P <0.001, respectively), morphokinetic parameters were comparable. A total of 45 blastocysts were transferred into 5 recipient mice; 8 blastocysts implanted (17.8%) in 2 surrogates, yielding 5 live offspring (11.1%) weighing 1.51±0.22g. All live offspring expressed GFP and were confirmed fertile once reaching adulthood. Limitations, reasons for caution Although copies of gamete substitutes were generated that could be genotyped and used to produce live offspring with the desired phenotype in a mouse model, the system needs to be optimized and proven reproducible in humans, specifically in regard to the role of the sperm centrosome. Wider implications of the findings Once our proposed technique is implemented in humans, it may benefit patients to control germline heterozygosity by allowing genotyping of identical copies of the gametes. This technique, when used in conjunction with heritable genomic editing, can obviate the inherently highly condensed sperm genome to provide genome-edited gamete substitutes. Trial registration number N/A

Allele-specific H3K9me3 and DNA methylation co-marked CpG-rich regions serve as potential imprinting control regions in pre-implantation embryo.

Parental DNA methylation and histone modifications undergo distinct global reprogramming in mammalian pre-implantation embryos, but the landscape of epigenetic crosstalk and its effects on embryogenesis are largely unknown. Here we comprehensively analyse the association between DNA methylation and H3K9me3 reprogramming in mouse pre-implantation embryos and reveal that CpG-rich genomic loci with high H3K9me3 signal and DNA methylation level (CHM) are hotspots of DNA methylation maintenance during pre-implantation embryogenesis. We further profile the allele-specific epigenetic map with unprecedented resolution in gynogenetic and androgenetic embryos, respectively, and identify 1,279 allele-specific CHMs, including 19 known imprinting control regions (ICRs). Our study suggests that 22 ICR-like regions (ICRLRs) may regulate allele-specific transcription similarly to known ICRs, and five of them are confirmed to be important for mouse embryo development. Taken together, our study reveals the widespread existence of allele-specific CHMs and largely extends the scope of allele-specific regulation in mammalian pre-implantation embryos.

P–133 Monopronuclear (1PN) embryos can derive in healthy pregnancies

Abstract Study question Should 1PN embryos be considered suitable for transfer when normal development is observed at day 3 or day 5? Summary answer In IVF/ICSI cycles, 1PN zygotes are encountered in 2.7% of inseminated oocytes. Transfer of 1PN-embryos should be considered in the absence of suitable 2PN embryos. What is known already During in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) zygotes containing only a single pronucleus (monopronuclear, 1PN) are encountered in 1–7.7% of cases, while the display of two pronuclei is expected in a normally fertilised oocyte. A 1PN zygote can be of gynogenetic or androgenetic origin, but it can also be biparental. Gynogenetic and androgenetic 1PN embryos can be haploid or diploid, so a diploid 1PN embryo is not guaranteed to be normally fertilised. Generally, 1PN are discarded, as they have an increased risk for aneuploidy. However, sporadically they can develop into healthyl babies. Study design, size, duration 1PN-zygotes (n = 1287, 2.7% from all inseminated oocytes) from 1–1–2016 up to 15–12–2020 were retrospectively evaluated. The development and fate (discarded/transferred/cryopreserved) of all embryos were recorded. Embryos were evaluated at day 2, 3 or 5 of development. The policy of our unit is that, in absence of 2PN embryos, normal developed 1PN-embryos can be transferred on day 3. Supernumerary 1PN embryos can be cryopreserved at blastocyst stage. Ongoing pregnancies from fresh embryo transfers (ET) were analysed. Participants/materials, setting, methods In 946 IVF/ICSI cycles, at least one 1PN zygote was observed (total 1287 embryos). ICSI with ejaculated, PESA or TESE sperm counted for a total of 795 embryos, IVF cycles for 494 embryos. Embryo evaluation was performed using a home-made numerical algorithm: A (top embryo; 150–200 points), B (regular embryo; 100–149 points) or C (poor embryo; 0–99 points). Monopronuclear embryos always scored lower than equal developed 2PN embryos. Blastocyst evaluation was according to Gardner score. Main results and the role of chance From the 795 ICSI embryos, 49 (6.1%) were used for fresh ET (26 scored quality A or B), and a total of 60 embryos developed to blastocyst and were cryopreserved. From these 49 ICSI transfers, 4 (8.1%) ongoing pregnancies were obtained, all 4 from DET (1PN+2PN embryo), from which one twin pregnancy was confirmed. From the 494 IVF embryos, 41 (8.3%) were used for fresh ET (24 scored A or B), and 62 blastocysts were cryopreserved. A total of 9/41 (22%) ongoing pregnancies were obtained: 5 from SET (1PN) and 4 from DET (1PN+ 2PN embryo). Therefore, in only five IVF cycles a confirmed pregnancy was observed from a 1PN embryo (all A-quality embryos). Considering six ongoing pregnancies with complete certainty of monopronuclear origin from fresh tranfers could be confirmed from our retrospective data, we can conclude that although the live birth rate of these embryos is very low (around 0,5- 1.0%), they should not be discarded when development is normal and no dipronuclear embryos are present. Limitations, reasons for caution Cryo-thawing data is missing as these embryos were not differentially marked at freezing. Therefore, the cumulative pregnancies from monopronuclear embryos could be higher. Embryos were not evaluated in a time lapse system, so asynchronicity of PN formation could explain missing the right moment for evaluation, while normal fertilized. Wider implications of the findings: Notably, IVF monopronuclear embryos display a higher developmental potential than those derived from ICSI. We suggest that, in absence of dipronuclear embryos, culture to blastocyst stage before considering fresh ET or cryopreservation will help differentiate viable 1PN embryos, reducing the higher chance of genetic anomalies and miscarriages. Trial registration number N.A.

P–207 Heterogoneic cell division proven to occur in bovine zygotes

Abstract Study question We hypothesize that the zygote can segregate parental genomes via a non-canonical pathway. We coined this heterogoneic cell division. Can we proof the existence of this new segregational pathway? Summary answer We confirmed the existence of this non-canonical segregation mechanism leading to mixoploidy and provide a catalogue of abnormal zygotic divisions. What is known already Embryos show a high degree of chromosomal instability leading to chromosomal mosaicism. Chromosomal aberrations affect the developmental potential. We developed haplarithmisis which determines the single-cell genome-wide haplotype and copy number and allows to deduce the parental and segregational origin. Analysis of cleavage-stage bovine embryos by haplarithmisis, discovered the presence of uniparental and biparental blastomere lineages in individual embryos. We hypothesized that whole genome segregations can occur via a non-canonical zygotic division, a process termed “heterogoneic” division. Abnormal zygotic division has been observed in bovine and human in vitro produced zygotes using time-lapse, but parental genome segregation has never been disclosed. Study design, size, duration We hypothesized that abnormal cytokinetics and spindle mechanics may underlie the segregation of parental genomes in a separate blastomere line. In vitro produced zygotes were monitored by time-lapse microscopy. Zygotes cleaving 3 or 4 cells were disaggregated, picked and analysed by haplarithmisis. Participants/materials, setting, methods Blastomeres from bovine in vitro produced zygotes cleaving directly into 3 or 4 blastomeres, identified by time-lapse monitoring, were tubed following zona removal and blastomere dissociation and whole-genome amplified. Samples were subsequently hybridized on Illumina Bovine HD BeadChip SNP arrays. Data was analyzed by haplarithmisis, using a the siCHILD-bovine algorithm, to infer the haplotypes and the copy number of the parental genomes. Blastomeres showing failed haplarithmisis plots were low-coverage whole-genome sequenced on a HiSeq4000 sequencer. Main results and the role of chance: We obtained 25 bovine embryos, comprising 82 blastomeres, derived from 12 families (12 cows and 2 bulls) that cleaved directly into 3 or 4 blastomeres. Sixteen, 7 and 2 out of 25 zygotes cleaved respectively in 3 cells, 4 cells and 3 cells and a fragment. In at least 20 embryos, more than one paternal haplotype was identified, showing that a polyspermic fertilization resulted in an abnormal division. All embryos contained a whole-genome abnormality in at least one blastomere, resulting in mixoploid (5), mixed diploid biparental and androgenetic (12), polyploid (4), mixed gynogenetic and androgenetic (2) and androgenetic (2) embryos. Twenty-one embryos had at least one blastomere containing a uniparental signature. Based on the blastomere haplotype profiles we classified the embryos in six segregation categories. In twelve blastomeres haplarithmisis failed. Massive parallel sequencing of the amplified DNA showed the presence of mitochondrial DNA, indicating the blastomere did not contain any genomic DNA. This observation confirms that heterogoneic cell division does occur via different non-canonical zygotic segregations, which result in a variety of chimeric and mixoploid embryos constitutions. Limitations, reasons for caution These findings apply to a small set of bovine in vitro produced abnormally cleaving embryos. Segregation patterns may be incomplete and their true in vitro and in vivo prevalence remains unknown. Based on the haplotypes the non-canonical divisions have been reconstructed. Those patterns can now be evaluated and tested. Wider implications of the findings: This study shows that heterogoneic cell division occurs. We hypothesize this non-canonical division occurs frequently in both in vitro and in vivo, not only in cattle but also in man and hypothesize that persistence of such cell lines might explain the development of androgenetic tumorous outgrowths and mosaic uniparental individuals. Trial registration number Not applicable

Inter-clutch egg differences and androgenesis in rainbow trout (Oncorhynchus mykiss, Walbaum 1792)

Abstract Ionizing radiation (IR) is applied to inactivate the nuclear genome in rainbow trout eggs during induced androgenetic development. However, IR-generated reactive oxygen species (ROS) may affect developmental potential of eggs and reduce the effectiveness of androgenesis. To verify this assumption, androgenetic development of rainbow trout was induced in eggs irradiated with 350 Gy of X-rays. Survival rates, pH of the ovarian fluid and activity of antioxidant enzymes, including SOD, CAT and GPx, were examined in non-irradiated and irradiated eggs originating from four females. Survival rates of androgenetic embryos developing in eggs produced by different females varied from 1% to 57% and these inter-clutch differences were significant. Eggs from female F4, which showed the highest developmental competence for androgenesis, also showed increased activities of SOD, CAT and GPx enzymes. The pH value of the ovarian fluid of each female was over 8 before and after irradiation, therefore it seems that radiation did not affect the ovarian fluid pH. Considering the above-mentioned inter-clutch differences, a strong maternal effect on the effectiveness of androgenesis can be assumed. Eggs with increased activity of antioxidant enzymes before irradiation should be expected to show increased developmental competence for androgenesis.

Dysregulated Gene Expression of Imprinted and X-Linked Genes: A Link to Poor Development of Bovine Haploid Androgenetic Embryos.

Mammalian uniparental embryos are efficient models for genome imprinting research and allow studies on the contribution of the paternal and maternal genomes to early embryonic development. In this study, we analyzed different methods for production of bovine haploid androgenetic embryos (hAE) to elucidate the causes behind their poor developmental potential. Results indicate that hAE can be efficiently generated by using intracytoplasmic sperm injection and oocyte enucleation at telophase II. Although androgenetic haploidy does not disturb early development up to around the 8-cell stage, androgenetic development is disturbed after the time of zygote genome activation and hAE that reach the morula stage are less capable to reach the blastocyst stage of development. Karyotypic comparisons to parthenogenetic- and ICSI-derived embryos excluded chromosomal segregation errors as causes of the developmental constraints of hAE. However, analysis of gene expression indicated abnormal levels of transcripts for key long non-coding RNAs involved in X chromosome inactivation and genomic imprinting of the KCNQ1 locus, suggesting an association with X chromosome and some imprinted loci. Moreover, transcript levels of methyltransferase 3B were significantly downregulated, suggesting potential anomalies in hAE establishing de novo methylation. Finally, the methylation status of imprinted control regions for XIST and KCNQ1OT1 genes remained hypomethylated in hAE at the morula and blastocyst stages, confirming their origin from spermatozoa. Thus, our results exclude micromanipulation and chromosomal abnormalities as major factors disturbing the normal development of bovine haploid androgenotes. In addition, although the cause of the arrest remains unclear, we have shown that the inefficient development of haploid androgenetic bovine embryos to develop to the blastocyst stage is associated with abnormal expression of key factors involved in X chromosome activity and genomic imprinting.

Manipulation of gametes and embryosin the pig

Summary. Several manipulation techniques including nuclear injections, nuclear transplantation, embryo splitting, chimaera production and sperm injection are discussed with special reference to their application in the pig. The nuclear injection technique is likely to be of greatest use for gene transfer. Gene transfer is feasible for pig embryos. but it is very inefficient. Efficiencies of various steps in the successful production of transgenic offspring as summarized from several published references are as follows: immediate survival of embryos following injection (60%). offspring produced from injected embryos (15%), offspring with an integrated foreign gene (24%) and offspring expressing the integrated foreign gene (60%). The overall efficiency is about 1%. Potential uses for nuclear transplantation are for the production of gynogenetic and androgenetic embryos and for cloning embryos The feasibility of producing androgenetic and gynogenctic embryos will depend on whether in the pig. as in the mouse, the paternal and maternal genomes function differently. Thc feasibility of cloning by nuclear transplantation will depend upon the timing of developmental events in pig embryos and the development of methods for reprogramming nuclei. Pig embryos have been cloned by embryo splitting. Chimaera production and fertilization by sperm injection are likely to be feasible for pig embryos but have not yet found application.

Production of mouse androgenetic embryos using spindle perturbation

To study the functional differences between maternal and paternal genomes in mammalian development, embryos with only one parental genome are often used. Androgenetic embryos are produced by the removal of maternal chromosomes before or after fertilization by techniques that require specialized skills and are associated with high risk of cellular damage. Here, we developed a novel method for producing androgenetic mouse embryos without the invasive enucleation process. We found that during in vitro fertilization in the presence of low-dose nocodazole, a microtubule destabilizing drug, whole oocyte chromosomes were extruded into the second polar body resulting in the production of androgenetic embryos. We further demonstrated that low-dose nocodazole decreased the spindle size and prevented chromosome segregation but did not compromise oocyte meiotic resumption. This led to the formation of a protrusion around the chromosomes, accumulation of protein regulator of cytokinesis 1 (PRC1) to the microtubules around the chromosomes, and assembly of a contractile ring at the neck region of the protrusion. Our method uses the intrinsic cytokinetic mechanism to exclude maternal chromatin from zygotes and may be applicable to other mammals.

Ratio of the zygote cytoplasm to the paternal genome affects the reprogramming and developmental efficiency of androgenetic embryos.

Uniparental embryos have uniparental genomes and are very useful models for studying the specific gene expression of parents or for exploring the biological significance of genomic imprinting in mammals. However, the early developmental efficiency of androgenetic embryos is significantly lower than that of parthenogenetic embryos. In addition, oocytes are able to reprogram sperm nuclei after fertilization to guarantee embryonic development by maternally derived reprogramming factors, which accumulate during oogenesis. However, the importance of maternal material in the efficiency of reprogramming the pronucleus of androgenetic embryos is not known. In this study, androgenetic embryos were constructed artificially by pronucleus transfer (PT) or double sperm injection (DS). Compared with DS embryos, PT embryos that were derived from two zygotes contained more maternal material, like 10-11 translocation methylcytosine deoxygenase 3 (Tet3) and histone variant 3.3 (H3.3). Our experiments confirmed the better developmental potential of PT embryos, which had higher blastocyst rates, a stronger expression of pluripotent genes, a lower expression of apoptotic genes, and superior blastocyst quality. Our findings indicate that the aggregation of more maternal materials in the paternal pronucleus facilitate the reprogramming of the paternal genome, improving embryonic development in PT androgenesis.

From Mother or Father: Uniparental Embryos Uncover Parent-of-Origin Effects in Humans.

In mammals, both parents make unique contributions to the offspring and maternal and paternal genomes are required for development. Two recent papers in Cell Stem Cell (Leng etal., 2019; Sagi etal., 2019) study uniparental embryos and uniparental embryonic stem cells to interrogate parent-of-origin effects in human embryogenesis.

Establishment of mouse androgenetic embryonic stem cells by double sperm injection and differentiation into beating embryoid body.

Androgenetic embryonic stem (AgES) cells offer a possible tool for patient-specific pluripotent stem cells that will benefit genomic imprinting studies and clinic applications. However, the difficulty in producing androgenetic embryos and the unbalanced expression of imprinted genes make the therapeutic applicability of AgES cells uncertain. In this study, we produced androgenetic embryos by injecting two sperm into an enucleated metaphase II (MII) oocyte. By this method, 88.48% of oocytes survived after injection, and 20.24% of these developed to the blastocyst stage. We successfully generated AgES cell lines from the androgenetic embryos and assayed the expression of imprinted genes in the cell lines. We found that the morphological characteristics of AgES cells were similar to that of fertilized embryonic stem cells (fES), such as expression of key pluripotent markers, and generation of cell derivatives representing all three germ layers following in vivo and in vitro differentiation. Furthermore, activation of paternal imprinted genes was detected, H19, ASC12 and Tss3 in AgES cell activation levels were lower while other examined genes showed no significant difference to that of fES cells. Interestingly, among examined maternal imprinted genes, only Mest and Igf2 were significantly increased, while levels of other detected genes were no different to that of fES cells. These results demonstrated that activation of some paternal imprinted genes, as well as recovery of maternal imprinted genes, was present in AgES cells. We differentiated AgES cells into a beating embryoid body in vitro, and discovered that the AgES cells did not show significant higher efficiency in myocardial differentiation potential.