Female-biased embryonic death from inflammation induced by genomic instability.

Genomic instability can trigger cellular responses that include checkpoint activation, senescence and inflammation1,2. Although genomic instability has been extensively studied in cell culture and cancer paradigms, little is known about its effect during embryonic development, a period of rapid cellular proliferation. Here we report that mutations in the heterohexameric minichromosome maintenance complex-the DNA replicative helicase comprising MCM2 to MCM73,4-that cause genomic instability render female mouse embryos markedly more susceptible than males to embryonic lethality. This bias was not attributable to X chromosome-inactivation defects, differential replication licensing or X versus Y chromosome size, but rather to 'maleness'-XX embryos could be rescued by transgene-mediated sex reversal or testosterone administration. The ability of exogenous or endogenous testosterone to protect embryos was related to its anti-inflammatory properties5. Ibuprofen, a non-steroidal anti-inflammatory drug, rescued female embryos that contained mutations in not only the Mcm genes but also the Fancm gene; similar to MCM mutants, Fancm mutant embryos have increased levels of genomic instability (measured as the number of cells with micronuclei) from compromised replication fork repair6. In addition, deficiency in the anti-inflammatory IL10 receptor was synthetically lethal with the Mcm4Chaos3 helicase mutant. Our experiments indicate that, during development, DNA damage associated with DNA replication induces inflammation that is preferentially lethal to female embryos, because male embryos are protected by high levels of intrinsic testosterone.

Loss of KANSL3 leads to defective inner cell mass and early embryonic lethality.

e-Lysine acetylation is a prominent histone mark found at transcriptionally active loci. Among many lysine acetyl transferases, nonspecific lethal complex (NSL) members are known to mediate the modification of histone H4. In addition to histone modifications, the KAT8 regulatory complex subunit 3 gene (Kansl3), a core member of NSL complex, has been shown to be involved in several other cellular processes such as mitosis and mitochondrial activity. Although functional studies have been performed on NSL complex members, none of the four core proteins, including Kansl3, have been studied during early mouse development. Here we show that homozygous knockout Kansl3 embryos are lethal at peri-implantation stages, failing to hatch out of the zona pellucida. When the zona pellucida is removed in vitro, Kansl3 null embryos form an abnormal outgrowth with significantly disrupted inner cell mass (ICM) morphology. We document lineage-specific defects at the blastocyst stage with significantly reduced ICM cell number but no difference in trophectoderm cell numbers. Both epiblast and primitive endoderm lineages are altered with reduced cell numbers in null mutants. These results show that Kansl3 is indispensable during early mouse embryonic development and with defects in both ICM and trophectoderm lineages.

Placentation defects are highly prevalent in embryonic lethal mouse mutants.

Large-scale phenotyping efforts have demonstrated that approximately 25-30% of mouse gene knockouts cause intrauterine lethality. Analysis of these mutants has largely focused on the embryo and not the placenta, despite the crucial role of this extraembryonic organ for developmental progression. Here we screened 103 embryonic lethal and sub-viable mouse knockout lines from the Deciphering the Mechanisms of Developmental Disorders program for placental phenotypes. We found that 68% of knockout lines that are lethal at or after mid-gestation exhibited placental dysmorphologies. Early lethality (embryonic days 9.5-14.5) is almost always associated with severe placental malformations. Placental defects correlate strongly with abnormal brain, heart and vascular development. Analysis of mutant trophoblast stem cells and conditional knockouts suggests that a considerable number of factors that cause embryonic lethality when ablated have primary gene function in trophoblast cells. Our data highlight the hugely under-appreciated importance of placental defects in contributing to abnormal embryo development and suggest key molecular nodes that govern placenta formation.

Requirement for antiapoptotic MCL-1 during early erythropoiesis.

Although BCL-xL is critical to the survival of mature erythrocytes, it is still unclear whether other antiapoptotic molecules mediate survival during earlier stages of erythropoiesis. Here, we demonstrate that erythroid-specific Mcl1 deletion results in embryonic lethality beyond embryonic day 13.5 as a result of severe anemia caused by a lack of mature red blood cells (RBCs). Mcl1-deleted embryos exhibit stunted growth, ischemic necrosis, and decreased RBCs in the blood. Furthermore, we demonstrate that MCL-1 is only required during early definitive erythropoiesis; during later stages, developing erythrocytes become MCL-1 independent and upregulate the expression of BCL-xL. Functionally, MCL-1 relies upon its ability to prevent apoptosis to promote erythroid development because codeletion of the proapoptotic effectors Bax and Bak can overcome the requirement for MCL-1 expression. Furthermore, ectopic expression of human BCL2 in erythroid progenitors can compensate for Mcl1 deletion, indicating redundancy between these 2 antiapoptotic family members. These data clearly demonstrate a requirement for MCL-1 in promoting survival of early erythroid progenitors.

Ubiquitous expression of Akt1 p.(E17K) results in vascular defects and embryonic lethality in mice.

Proteus syndrome is a progressive overgrowth disorder with vascular malformations caused by mosaic expression of the AKT1 c.49G > A, p.(E17K) activating variant which was predicted to cause lethality if expressed ubiquitously. To test that hypothesis, we used the ACTB-Cre gene to activate a conditional Akt1 p.(E17K) allele in the mouse. No offspring that was heterozygous for both Cre and the conditional allele (ßA-Akt1WT/flx) was viable. Fewer than expected numbers of ßA-Akt1WT/flx embryos were seen beginning at E11.5, but a few survived until E17.5. The phenotype ranged from mild to severe, but generally ßA-Akt1WT/flx embryos had fewer visible blood vessels and more hemorrhages than their wild-type littermates, which was suggestive of a vascular abnormality. Examination of E13.5 limb skin showed a primitive capillary network with increased branching complexity and abnormal patterning compared with wild-type skin. By E15.5, wild-type skin had undergone angiogenesis and formed a hierarchical network of remodeled vessels, whereas in ßA-Akt1WT/flx embryos, the capillary network failed to remodel. Mural cell coverage of the blood vessels was also reduced in ßA-Akt1WT/flx skin compared with that of wild type. Restricting expression of Akt1E17K to endothelial, cardiac or smooth muscle cells resulted in viable offspring and remodeled vasculature and did not recapitulate the ßA-Akt1WT/flx phenotype. We conclude that ubiquitous expression of Akt1E17K suppresses remodeling and inhibits the formation of a normal skin vasculature. We postulate that this failure prevents proper circulation necessary to support the growing embryo and that it is the result of interactions of multiple cell types with increased AKT signaling.

CDK5RAP3, a UFL1 substrate adaptor, is crucial for liver development.

Protein modification by ubiquitin and ubiquitin-like proteins (UBLs) regulates numerous biological functions. The UFM1 system, a novel UBL conjugation system, is implicated in mouse development and hematopoiesis. However, its broad biological functions and working mechanisms remain largely elusive. CDK5RAP3, a possible ufmylation substrate, is essential for epiboly and gastrulation in zebrafish. Herein, we report a crucial role of CDK5RAP3 in liver development and hepatic functions. Cdk5rap3 knockout mice displayed prenatal lethality with severe liver hypoplasia, as characterized by delayed proliferation and compromised differentiation. Hepatocyte-specific Cdk5rap3 knockout mice suffered post-weaning lethality, owing to serious hypoglycemia and impaired lipid metabolism. Depletion of CDK5RAP3 triggered endoplasmic reticulum stress and activated unfolded protein responses in hepatocytes. We detected the in vivo interaction of CDK5RAP3 with UFL1, the defined E3 ligase in ufmylation. Notably, loss of CDK5RAP3 altered the ufmylation profile in liver cells, suggesting that CDK5RAP3 serves as a novel substrate adaptor for this UBL modification. Collectively, our study identifies CDK5RAP3 as an important regulator of ufmylation and suggests the involvement of ufmylation in mammalian development.

Self-organization of the in vitro attached human embryo.

Implantation of the blastocyst is a developmental milestone in mammalian embryonic development. At this time, a coordinated program of lineage diversification, cell-fate specification, and morphogenetic movements establishes the generation of extra-embryonic tissues and the embryo proper, and determines the conditions for successful pregnancy and gastrulation. Despite its basic and clinical importance, this process remains mysterious in humans. Here we report the use of a novel in vitro system to study the post-implantation development of the human embryo. We unveil the self-organizing abilities and autonomy of in vitro attached human embryos. We find human-specific molecular signatures of early cell lineage, timing, and architecture. Embryos display key landmarks of normal development, including epiblast expansion, lineage segregation, bi-laminar disc formation, amniotic and yolk sac cavitation, and trophoblast diversification. Our findings highlight the species-specificity of these developmental events and provide a new understanding of early human embryonic development beyond the blastocyst stage. In addition, our study establishes a new model system relevant to early human pregnancy loss. Finally, our work will also assist in the rational design of differentiation protocols of human embryonic stem cells to specific cell types for disease modelling and cell replacement therapy.

Dysregulation in Multiple Transcriptomic Endometrial Pathways Is Associated with Recurrent Implantation Failure and Recurrent Early Pregnancy Loss.

Overlapping disease aetiologies associated with multiple altered biological processes have been identified that change the endometrial function leading to recurrent implantation failure (RIF) and recurrent early pregnancy loss (REPL). We aimed to provide a detailed insight into the nature of the biological malfunction and related pathways of differentially expressed genes in RIF and REPL. Endometrial biopsies were obtained from 9 women experiencing RIF, REPL and control groups. Affymetrix microarray analysis was performed to measure the gene expression level of the endometrial biopsies. Unsupervised clustering of endometrial samples shows scattered distribution of gene expression between the RIF, REPL and control groups. 2556 and 1174 genes (p value < 0.05, Fold change > 1.2) were significantly altered in the endometria of RIF and REPL patients' group, respectively compared to the control group. Downregulation in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the differentially expressed genes (DEGs) in RIF and REPL including ribosome and oxidative phosphorylation pathways. Gene Ontology (GO) analysis revealed ribosomes and mitochondria inner membrane as the most significantly downregulated cellular component (CC) affected in RIF and REPL. Determination of the dysregulated genes and related biological pathways in RIF and REPL will be key in understanding their molecular pathology and of major importance in addressing diagnosis, prognosis, and treatment issues

The Zα2 domain of ZBP1 is a molecular switch regulating influenza-induced PANoptosis and perinatal lethality during development.

Z-DNA-binding protein 1 (ZBP1) is an innate immune sensor of nucleic acids that regulates host defense responses and development. ZBP1 activation triggers inflammation and pyroptosis, necroptosis, and apoptosis (PANoptosis) by activating receptor-interacting Ser/Thr kinase 3 (RIPK3), caspase-8, and the NLRP3 inflammasome. ZBP1 is unique among innate immune sensors because of its N-terminal Zα1 and Zα2 domains, which bind to nucleic acids in the Z-conformation. However, the specific role of these Zα domains in orchestrating ZBP1 activation and subsequent inflammation and cell death is not clear. Here we generated Zbp1ΔZα2/ΔZα2 mice that express ZBP1 lacking the Zα2 domain and demonstrate that this domain is critical for influenza A virus-induced PANoptosis and underlies perinatal lethality in mice in which the RIP homotypic interaction motif domain of RIPK1 has been mutated (Ripk1mRHIM/mRHIM). Deletion of the Zα2 domain in ZBP1 abolished influenza A virus-induced PANoptosis and NLRP3 inflammasome activation. Furthermore, deletion of the Zα2 domain of ZBP1 was sufficient to rescue Ripk1mRHIM/mRHIM mice from perinatal lethality caused by ZBP1-driven cell death and inflammation. Our findings identify the essential role of the Zα2 domain of ZBP1 in several physiological functions and establish a link between Z-RNA sensing via the Zα2 domain and promotion of influenza-induced PANoptosis and perinatal lethality.

CXCR3-Dependent Immune Pathology in Mice following Infection with Toxoplasma gondii during Early Pregnancy.

Toxoplasmosis is a worldwide zoonosis caused by the obligate intracellular parasite Toxoplasma gondii The symptoms of congenital toxoplasmosis range from embryonic death and resorption to subclinical infection, but the mechanism of disease onset remains unclear. C-X-C motif chemokine receptor 3 (CXCR3) is highly expressed in Th1-associated immune cells and plays an important role in the trafficking and activation of immune cells. However, the roles of CXCR3 in T. gondii-induced fetal loss and the molecular mechanism of embryo resorption remain poorly understood. In this study, we investigated the role of CXCR3 in fetal wastage caused by T. gondii infection using CXCR3-deficient (CXCR3-/-) mice. CXCR3-/- and wild-type pregnant mice were inoculated intraperitoneally with T. gondii tachyzoites on day 3.5 of gestation (Gd3.5). Pregnancy rates decreased as the pregnancy progressed in both infected groups; however, infected CXCR3-/- mice showed a significant fetal loss at Gd13.5 compared with that at Gd7.5. All embryos of the infected groups showed necrosis, and embryo resorption was significantly increased in infected CXCR3-/- compared with wild-type mice at Gd13.5. The parasite load of fetoplacental tissues was significantly increased in CXCR3-/- mice at Gd10.5. Moreover, mRNA expression levels of inducible nitric oxide synthase were significantly increased in fetoplacental tissues from infected wild-type mice compared to infected CXCR3-/- mice following the infection. These results suggested that CXCR3-dependent immune responses provide anti-Toxoplasma activity and play an essential role in reducing embryo resorption and fetal loss caused by T. gondii infection during early pregnancy.

Effect of Irisin on LIF and integrin αvß3 in rats of implantation failure.

OBJECTIVE: The aim of this study is to investigate the effect of irisin on leukemia inhibitory factor (LIF) and integrin αvß3 in implantation failure uterus. METHODS: Early pregnant rats were randomly divided into normal group (N), mifepristone treated group (M), irisin group (I) and progestin group (P). The implantation failure model was established using mifepristone. Second, we evaluated the average number of embryos and detected the expression of LIF and integrin αvß3 protein and mRNA in endometrium. RESULTS: Compared with group M, the average number of embryos was significantly higher in group N, P and I, the expression of LIF and integrin αvß3 in endometrium was significantly higher in group N, P and I. CONCLUSION: Irisin could improve the poor receptive state of endometrium by promoting LIF and integrin αvß3 secretion to improve blastocyst implantation in rats of implantation failure.

Identification of Signaling Pathways for Early Embryonic Lethality and Developmental Retardation in Sephs1-/- Mice.

Selenophosphate synthetase 1 (SEPHS1) plays an essential role in cell growth and survival. However, the underlying molecular mechanisms remain unclear. In the present study, the pathways regulated by SEPHS1 during gastrulation were determined by bioinformatical analyses and experimental verification using systemic knockout mice targeting Sephs1. We found that the coagulation system and retinoic acid signaling were most highly affected by SEPHS1 deficiency throughout gastrulation. Gene expression patterns of altered embryo morphogenesis and inhibition of Wnt signaling were predicted with high probability at E6.5. These predictions were verified by structural abnormalities in the dermal layer of Sephs1-/- embryos. At E7.5, organogenesis and activation of prolactin signaling were predicted to be affected by Sephs1 knockout. Delay of head fold formation was observed in the Sephs1-/- embryos. At E8.5, gene expression associated with organ development and insulin-like growth hormone signaling that regulates organ growth during development was altered. Consistent with these observations, various morphological abnormalities of organs and axial rotation failure were observed. We also found that the gene sets related to redox homeostasis and apoptosis were gradually enriched in a time-dependent manner until E8.5. However, DNA damage and apoptosis markers were detected only when the Sephs1-/- embryos aged to E9.5. Our results suggest that SEPHS1 deficiency causes a gradual increase of oxidative stress which changes signaling pathways during gastrulation, and afterwards leads to apoptosis.

Decreased ANGPTL4 impairs endometrial angiogenesis during peri-implantation period in patients with recurrent implantation failure.

Insufficient endometrial angiogenesis during peri-implantation impairs endometrial receptivity (ER), which contributes to recurrent implantation failure (RIF) during in vitro fertilization and embryo transfer (IVF-ET). Angiopoietin-like protein 4 (ANGPTL4) acts as a multifunctional secretory protein and is involved in the regulation of lipid metabolism and angiogenesis in various tissues including the endometrium. Herein, we found decreased ANGPTL4 expression in endometrial tissue and serum during peri-implantation period in 18 RIF-affected women with elevated uterine arterial impedance (UAI) compared with the pregnancy controls. ANGPTL4 and peroxisome proliferator-activated receptor gamma (PPARγ) expression were up-regulated upon decidualization on human endometrial stromal cells (HESCs). Rosiglitazone promoted the expression of ANGPTL4 in HESCs and human umbilical vein endothelial cells (HUVECs) via PPARγ. ANGPTL4 promoted the proliferation, migration and angiogenesis of HUVECs in vitro. Our results suggest that decreased abundance of ANGPTL4 in endometrial tissues impairs the endometrial receptivity via restraining endometrial angiogenesis during decidualization; while rosiglitazone-induced ANGPTL4 up-regulation in hESCs and HUVECs through PPARγ. Therefore, ANGPTL4 could be a potential therapeutic approach for some RIF-affected women with elevated UAI.

Roles of the endoplasmic reticulum-resident, collagen-specific molecular chaperone Hsp47 in vertebrate cells and human disease.

Heat shock protein 47 (Hsp47) is an endoplasmic reticulum (ER)-resident molecular chaperone essential for correct folding of procollagen in mammalian cells. In this Review, we discuss the role and function of Hsp47 in vertebrate cells and its role in connective tissue disorders. Hsp47 binds to collagenous (Gly-Xaa-Arg) repeats within triple-helical procollagen in the ER and can prevent its local unfolding or aggregate formation, resulting in accelerating triple-helix formation of procollagen. Hsp47 pH-dependently dissociates from procollagen in the cis-Golgi or ER-Golgi intermediate compartment and is then transported back to the ER. Although Hsp47 belongs to the serine protease inhibitor (serpin) superfamily, it does not possess serine protease inhibitory activity. Whereas general molecular chaperones such as Hsp70 and Hsp90 exhibit broad substrate specificity, Hsp47 has narrower specificity mainly for procollagens. However, other Hsp47-interacting proteins have been recently reported, suggesting a much broader role for Hsp47 in the cell that warrants further investigation. Other ER-resident stress proteins, such as binding immunoglobulin protein (BiP), are induced by ER stress, whereas Hsp47 is induced only by heat shock. Constitutive expression of Hsp47 is always correlated with expression of various collagen types, and disruption of the Hsp47 gene in mice causes embryonic lethality due to impaired basement membrane and collagen fibril formation. Increased Hsp47 expression is associated with collagen-related disorders such as fibrosis, characterized by abnormal collagen accumulation, highlighting Hsp47's potential as a clinically relevant therapeutic target.

Application of Copy Number Variation Sequencing in Genetic Analysis of Miscarriages in Early and Middle Pregnancy.

High-throughput sequencing based on copy number variation (CNV-seq) is commonly used to detect chromosomal abnormalities. This study identifies chromosomal abnormalities in aborted embryos/fetuses in early and middle pregnancy and explores the application value of CNV-seq in determining the causes of pregnancy termination. High-throughput sequencing was used to detect chromosome copy number variations (CNVs) in 116 aborted embryos in early and middle pregnancy. The detection data were compared with the Database of Genomic Variants (DGV), the Database of Chromosomal Imbalance and Phenotype in Humans using Ensemble Resources (DECIPHER), and the Online Mendelian Inheritance in Man (OMIM) database to determine the CNV type and the clinical significance. High-throughput sequencing results were successfully obtained in 109 out of 116 specimens, with a detection success rate of 93.97%. In brief, there were 64 cases with abnormal chromosome numbers and 23 cases with CNVs, in which 10 were pathogenic mutations and 13 were variants of uncertain significance. An abnormal chromosome number is the most important reason for embryo termination in early and middle pregnancy, followed by pathogenic chromosome CNVs. CNV-seq can quickly and accurately detect chromosome abnormalities and identify microdeletion and microduplication CNVs that cannot be detected by conventional chromosome analysis, which is convenient and efficient for genetic etiology diagnosis in miscarriage.

Selenoprotein I is essential for murine embryogenesis.

Selenoprotein I (SELENOI) is an ethanolamine phosphotransferase that catalyzes the third reaction of the Kennedy pathway for the synthesis of phosphatidylethanolamine. Since the role of SELENOI in murine embryogenesis has not been investigated, SELENOI-/+ mating pairs were used to generate global KO offspring. Of 323 weanling pups, no homozygous KO genotypes were found. E6.5-E18.5 embryos (165 total) were genotyped, and only two E18.5 KO embryos were detected with no discernable anatomical defects. To screen embryos prior to uterine implantation that occurs ~ E6, blastocyst embryos (E3.5-E4.4) were flushed from uteruses of pregnant females and analyzed for morphology and genotype. KO embryos were detected in 5 of 6 pregnant females, and 7 of the 32 genotyped embryos were found to be SELENOI KO that exhibited no overt pathological features. Overall, these results demonstrate that, except for rare cases (2/490 = 0.4%), global SELENOI deletion leads to early embryonic lethality.

Effect of single-dose depot leuprolide acetate on embryonal implantation: an experimental rat model.

The objective of this article is to investigate the effect of single-dose depot leuprolide acetate in rat embryonal implantation and its association with glycodelin A, mucin-1 and leukemia inhibitory factor expression. Thirty-two pregnant Wistar Albino rats were divided into four equal groups: untreated control rats in group I (n = 8) and untreated pregnant rats in group II (n = 8) were injected intraperitoneally with single dose of normal saline, treated rats in group III (n = 8) and treated pregnant rats in group IV (n = 8) were given single 1 mg/kg subcutaneous injection of leuprolide acetate at day 8 of pregnancy. The dams were sacrificed on the 15th day of gestation, uterine horn samples were removed. Immunohistochemical examination of the tissue samples prepared from the control and experimental groups, a statistically significant difference was observed between the groups in the luminal-glandular-decidualized epithelium of the uterus with glycodelin A, mucin-1 and leukemia inhibitory factor. A statistically significant difference was observed between the groups for the concentration of glycodelin A but no statistically significant difference was found for the other two molecules. In light of our findings, leuprolide acetate adversely affected expression and concentration of all three molecules in embryonal implantation model.

Mid-Gestation lethality of Atxn2l-Ablated Mice.

Depletion of yeast/fly Ataxin-2 rescues TDP-43 overexpression toxicity. In mouse models of Amyotrophic Lateral Sclerosis via TDP-43 overexpression, depletion of its ortholog ATXN2 mitigated motor neuron degeneration and extended lifespan from 25 days to >300 days. There is another ortholog in mammals, named ATXN2L (Ataxin-2-like), which is almost uncharacterized but also functions in RNA surveillance at stress granules. We generated mice with Crispr/Cas9-mediated deletion of Atxn2l exons 5-8, studying homozygotes prenatally and heterozygotes during aging. Our novel findings indicate that ATXN2L absence triggers mid-gestational embryonic lethality, affecting female animals more strongly. Weight and development stages of homozygous mutants were reduced. Placenta phenotypes were not apparent, but brain histology showed lamination defects and apoptosis. Aged heterozygotes showed no locomotor deficits or weight loss over 12 months. Null mutants in vivo displayed compensatory efforts to maximize Atxn2l expression, which were prevented upon nutrient abundance in vitro. Mouse embryonal fibroblast cells revealed more multinucleated giant cells upon ATXN2L deficiency. In addition, in human neural cells, transcript levels of ATXN2L were induced upon starvation and glucose and amino acids exposure, but this induction was partially prevented by serum or low cholesterol administration. Neither ATXN2L depletion triggered dysregulation of ATXN2, nor a converse effect was observed. Overall, this essential role of ATXN2L for embryogenesis raises questions about its role in neurodegenerative diseases and neuroprotective therapies.

Proliferative Activity of Mouse Splenocytes in Physiological Pregnancy and in Models of Spontaneous and Muramylpeptide-Dependent Abortions.

Spontaneous proliferative activity of splenocytes in female CBA mice and the response of these cells to antigens of allogeneic male BALB/c and DBA/2 mice in a mixed splenocyte culture were evaluated by 3H-thymidine incorporation in different pregnancy models. ♀CBA×♂BALB/c mating was used for modeling physiological pregnancy. Spontaneous abortions were reproduced by abortion-prone ♀CBA×♂DBA/2 mating. In order to simulate immunostimulant-induced and immunostimulant-potentiated abortions, 0.83 mg/kg muramyl dipeptide ß-heptylglycoside was intraperitoneally injected to CBA females mated with BALB/c or DBA/2 males, respectively, on gestation days 5 and 7. The increase in the rate of embryo resorption in the models of spontaneous, induced, and potentiated abortions occurred against the background of an increase in the level of spontaneous proliferation of splenocytes and a decrease in their reactivity to paternal antigens on gestation day 9.

Mutations in PADI6 Cause Female Infertility Characterized by Early Embryonic Arrest.

Early embryonic arrest is one of the major causes of female infertility. However, because of difficulties in phenotypic evaluation, genetic determinants of human early embryonic arrest are largely unknown. With the development of assisted reproductive technology, the phenotype of early human embryonic arrest can now be carefully evaluated. Here, we describe a consanguineous family with a recessive inheritance pattern of female infertility characterized by recurrent early embryonic arrest in cycles of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). We have identified a homozygous PADI6 nonsense mutation (c.1141C>T [p.Gln381(∗)]) that is responsible for the phenotype. Mutational analysis of PADI6 in a cohort of 36 individuals whose embryos displayed developmental arrest identified two affected individuals with compound-heterozygous mutations (c.2009_2010del [p.Glu670Glyfs(∗)48] and c.633T>A [p.His211Gln]; c.1618G>A [p.Gly540Arg] and c.970C>T [p.Gln324(∗)]). Immunostaining indicated a lack of PADI6 in affected individuals' oocytes. In addition, the amount of phosphorylated RNA polymerase II and expression levels of seven genes involved in zygotic genome activation were reduced in the affected individuals' embryos. This phenotype is consistent with Padi6 knockout mice. These findings deepen our understanding of the genetic basis of human early embryonic arrest, which has been a largely ignored Mendelian phenotype. Our findings lay the foundation for uncovering other genetic causes of infertility resulting from early embryonic arrest.