Patent Publication Number: US-2023160013-A1

Title: Methods and kits for identifying advanced paternal age related epigenetic dysregulation

Description:
RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional application No. 63/013,090 titled “Methods and Kits for Identifying Advanced Paternal Age Related Epigenetic Dysregulation” filed Apr. 21, 2020 which is incorporated herein by reference. 
    
    
     TECHNICAL FIELD 
     The present invention relates to an association of advanced paternal age and increased risk of epigenetic events during embryogenesis. The present invention also relates to the identification of a number of genes that act as markers for epigenetic screening associated with adverse outcomes during embryogenesis. 
     BACKGROUND OF THE INVENTION 
     Advanced paternal age (APA) has been associated with adverse outcomes including birth defects and childhood neurodevelopmental diseases, like autism spectrum disorder, schizophrenia, and bipolar disorder. In spite of this, the average age of first time mothers and fathers in the United States is increasing, such that the average mean age of pregnancy for first time mothers has gone from about 25 years old to about 26 years old in the last 25 years. The same trend is seen in first time fathers, as the average age of men having a first child has risen by about 3 years since the 1980s. As such, it can be anticipated that the incidence of adverse outcomes associated with APA will continue to rise. The ability to identify risk factors and to develop diagnostic screens for individuals of APA is of need in the art, such that individuals who choose to start a family at an older age can limit their risk of having a newborn with an adverse outcome. 
     The present invention is directed toward overcoming one or more of the problems discussed above. 
     SUMMARY OF THE INVENTION 
     Disclosed herein are methods and kits useful in the identification, screening and diagnosis of epigenetic dysregulation during embryogenesis, for example epigenetic dysregulation in a blastocyst. 
     Embodiments herein include, for example, methods for identifying a blastocyst, sperm, or sperm population with increased epigenetic dysregulation, and in particular, epigenetic dysregulation associated with adverse outcomes during embryogenesis. The method includes obtaining a blastocyst, sperm, or sperm population of interest, and identifying DNA methylation errors in the blastocyst. An increased risk of adverse outcome is correlated to epigenetic dysregulation in a blastocyst, sperm, or sperm population having global DNA with an overall hypomethylated shift, or correlated with a blastocyst having one or more, or two or more, autism spectrum disorder, schizophrenia, or bipolar disorder candidate genes being hypermethylated. In aspects of the case, the autism spectrum disorder candidate genes include: ABAT, ABCA7, ADSL, ANKRD11, CACNA1C, CACNA1H, CCDC88C, CDH11, CIC, CLSTN3, CNTNAP2, CSMD1, DDX3X, DHCR7, DPP6, EHMT1, FLT1, GLIS1, GNAS, GRIN1, GRM8, HDAC6, ITGB7, KCNQ2, NRP2, NSD1, P4HA2, PACS1, PIK3R2, PXDN, SGSH, SHANK2, SHANK3, SLC38A10, TET2, TT12, ZNF804A, and UBE3B. In some aspects of the case, the schizophrenia candidate genes include TCF3 and ZNF804A. In some aspects of the case, the bipolar disorder candidate genes include COMT, DRD4, GRIN1, MBP, PRKCZ, SHANK2, TRPM2, and ZNF804A. In some aspects of the case, opioid signaling pathway candidate genes include any of the neurodevelopmental disease candidate genes recited above. In some cases, the methods can be used to screen blastocysts, sperm, or sperm population for epigenetic dysregulation, where a blastocyst, sperm, or sperm population having epigenetic dysregulation as described above would not be used for an implantation procedure during in vitro fertilization, or in the case of the sperm, not used to fertilize an egg. In some cases, the methods can be used to screen blastocysts, sperm, or sperm population for epigenetic dysregulation, and blastocysts, sperm, or sperm population can be ranked to allow for the selection of the healthiest embryo for implantation or allow for the healthiest sperm to be used for fertilization. 
     In other embodiments, methods are provided for determining a blastocyst&#39;s epigenetic status. Methods include determining the level of methylation and gene expression in one or more, or two or more, autism spectrum disorder, schizophrenia, or bipolar disorder candidate genes in the blastocyst. In some aspects, the level of methylation and gene expression in the autism spectrum disorder, schizophrenia, or bipolar disorder genes are compared to a known control having a normal, or non-phenotypic, epigenetic status (in some cases, normal or non-phenotypic epigenetic status is associated with the methylation of the same one or more, or two or more, autism spectrum disorder genes found in sperm of younger fathers, typically less than 35 years old). As noted above, the autism spectrum disorder candidate genes can be any one of ABAT, ABCA7, ADSL, ANKRD11, CACNA1C, CACNA1H, CCDC88C, CDH11, CIC, CLSTN3, CNTNAP2, CSMD1, DDX3X, DHCR7, DPP6, EHMT1, FLT1, GLIS1, GNAS, GRIN1, GRM8, HDAC6, ITGB7, KCNQ2, NRP2, NSD1, P4HA2, PACS1, PIK3R2, PXDN, SGSH, SHANK2, SHANK3, SLC38A10, TET2, TT12, ZNF804A, or UBE3B. The schizophrenia candidate genes can be TCF3 and ZNF804A. The bipolar disorder candidate genes can be COMT, DRD4, GRIN1, MBP, PRKCZ, SHANK2, TRPM2, and ZNF804A. Opioid signaling pathway candidate genes can be any of the neurodevelopmental disease candidate genes recited above, for example, CACNA1H, GRIN1, and PRKCZ. 
     In alternative methods for determining a blastocyst&#39;s epigenetic status, the method includes determining the global DNA methylation of the blastocyst, where a hypomethylated or hypermethylated shift in global DNA methylation, as compared to a normal epigenetic status, is considered an abnormal epigenetic status. An abnormal epigenetic status is associated with a higher risk for the blastocyst to result in an adverse outcome as compared to a blastocyst having a normal epigenetic status. 
     Finally, embodiments herein include kits for testing the epigenetic status of one or more cells. In one aspect, the one or more cells are from a blastocyst, and the kit includes means for determining methylation level of the blastocyst, and means for determining gene expression in two or more target genes in the blastocyst. The two or more genes in the blastocyst can be autism spectrum disorder genes. In some kits, instructions including a standard range of values for a normal range of methylation and gene expression for autism spectrum disorder candidate genes is provided. Instructions can also include the methodology for conducting the methylation and gene expression testing. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG.  1 A  and  FIG.  1 B  demonstrate global DNA methylation in young and APA sperm.  FIG.  1 A  illustrates the distribution of the average Young and APA means for significant sperm CpGs visualized using boxplots. The bottom and top of each box represent the 1st and 3rd quantiles of the distributions, and the line between represents the median. The distributions are significantly different according to a Mann-Whitney test (*p=1.33×10-21). 
         FIG.  1 B  shows LINE 1  bisulfite pyrosequencing. CpG sites on the x-axis are followed by the average percent methylation across all CpGs for Young (gray) and APA (black) individuals. Each line represents results for individual sperm samples (n=18 Young and n=18 APA sperm samples). Significant hypermethylation is observed in APA (70%) relative to Young (67%), *p&lt;0.05. 
         FIGS.  2 A through  2 L  provide plots of sperm methylation validation results. Each plot represents pyrosequencing results for selected validation genes at distinct CpG sites within a significant DMR:  FIG.  2 A —CACNA1H;  FIG.  2 B —COMT;  FIG.  2 C —DRD4;  FIG.  2 D —GRIN1;  FIG.  2 E —KCNQ1;  FIG.  2 F —PRKCZ;  FIG.  2 G —SHANK2;  FIG.  2 H —SHANK3;  FIG.  2 I —TCF3;  FIG.  2 J —TRPM2;  FIG.  2 K —CNTNAP2;  FIG.  2 L —MBP;  FIG.  2 M —ZNF804A. CpG sites on the x-axis are followed by the average percent methylation across all CpGs for young (gray) and APA (black) individuals. Each line represents results for individual sperm samples (n=18 young and n=18 APA sperm samples). Significant hypomethylation in APA relative to young is demonstrated in plots (A-J), and significant hypermethylation in APA relative to young is demonstrated in plots (K-M); *p≤0.01, **p≤0.001 
         FIG.  3    provides plots demonstrating Sperm DNA methylation change by paternal age. Linear regression models demonstrate a significant negative association between sperm DNA methylation and paternal age in APA fathers (≥50 years; n=18 APA sperm samples). Models were fitted using the lm( ) function in R with default arguments, and those with p≤0.05 were considered significant. R-squared, slope, and p-values are displayed above each plot, and shaded gray areas around each black regression line represent a 95% confidence interval. 
         FIG.  4 A  and  FIG.  4 B  illustrate sperm and blastocyst DNA methylation validation results for CACNA1H and SHANK2. Bar plots show the average methylation changes within a significant DMR for two selected validation genes, CACNA1H ( FIG.  1 A ) and SHANK2 ( FIG.  1 B ), in sperm (n=12) and blastocyst (n=12) for Young (grey) and APA (black) fathers. Error bars represent one standard deviation. Significant hypomethylation is demonstrated for both sperm and blastocyst in CACNA1H and for sperm in SHANK2. *p≤0.05. 
     
    
    
     DESCRIPTION 
     The study of epigenetic changes in the genome has resulted in advances in the understanding and treatment of a number of disease states. In particular, epigenetic modifications have now been identified as contributing factors for certain cancers, cognitive dysfunction, autoimmune diseases, and possible reproductive disorders. Of particular relevance herein, the inventors have identified a number of epigenetic events that occur during embryogenesis that correlate with a higher likelihood of an adverse outcome in a newborn, e.g., birth defect, autism spectrum disorder, etc. As disclosed herein, an “epigenetic event” is any process that alters gene activity without changing a DNA sequence. In one aspect, the epigenetic event is DNA methylation. Surprisingly, the inventors have found that these identified epigenetic events are more likely to occur when the father donor is of an APA. 
     Prior to the present disclosure, a causal mechanism underlying paternal age effect had not been elucidated, nor had the mode of inheritance been conclusively established. As described herein, modifiable and inheritable epigenetic information, such as DNA methylation, can be generationally transmitted. A series of epigenetic reprogramming events occur during gametogenesis and immediately after fertilization (Reik, Dean, &amp; Walter, 2001). Erasure and gamete-specific establishment of methylation marks occur during primordial germ cell development. Directly after fertilization, the male pronucleus undergoes a rapid, active demethylation process, while the female pronucleus undergoes passive replication-dependent demethylation, prior to re-establishment in the developing embryo. Imprinted genes, and perhaps other developmentally critical regions, elude the embryonic portion of epigenetic reprogramming, enabling epigenetic generational inheritance (Kobayashi et al., 2012). 
     Disclosed herein, and substantiated by the data provided in the examples, is the finding that compromised DNA methylation profile in aged sperm results in incomplete reprogramming during spermatogenesis. For the first time, the inventors demonstrate a generational correlation in sperm and embryo of an altered human methylation landscape associated with advanced paternal age, contributing to nonequivalent efficiency of methylation re-establishment throughout the human blastocyst genome. Aberrant epigenetic reprogramming is significantly enriched at genes essential for neurological development in both APA sperm and blastocysts, and provides a mechanistic link between the paternal age effect and offspring neurodevelopmental disorders. 
     As disclosed herein, epigenetic events that correlate with a higher likelihood of an adverse outcome include: (1) a hypomethylated shift in global blastocyst DNA; (2) a higher likelihood that any one or more genes in the blastocyst shows both abnormal methylation and abnormal gene expression (increased or decreased); (3) hypermethylation in one or more, or at least two or more, three or more, four or more, etc. of the following genes in a blastocyst: ABAT, ABCA7, ADSL, ANKRD11, CACNA1C, CACNA1H, CCDC88C, CDH11, CIC, CLSTN3, CNTNAP2, CSMD1, DDX3X, DHCR7, DPP6, EHMT1, FLT1, GLIS1, GNAS, GRIN1, GRM8, HDAC6, ITGB7, KCNQ2, NRP2, NSD1, P4HA2, PACS1, PIK3R2, PXDN, SGSH, SHANK2, SHANK3, SLC38A10, TET2, TT12, ZNF804A, or UBE3B (termed the autism spectrum disorder candidate genes herein); TCF3 and ZNF804A (termed schizophrenia candidate genes herein); COMT, DRD4, GRIN1, MBP, PRKCZ, SHANK2, TRPM2, and ZNF804A (termed bipolar candidate genes herein); or (4) hypermethylation and decreased gene expression in any one or more autism spectrum disorder candidate gene, schizophrenia candidate gene, or bipolar disorder candidate gene in the blastocyst. 
     Where one of the above epigenetic events is identified in a blastocyst, it can be used to remove the blastocyst as a possible in vitro fertilization candidate. This is particularly true where the sperm donor for the blastocyst is of APA, or where the sperm donor experienced famine or some other stressful environmental or emotional disruption to epigenetic information. In other aspects, one or more blastocysts can be ranked by the degree or amount of epigenetic events such that the healthiest blastocyst can be chosen for implantation. 
     It is also disclosed herein that the autism spectrum disorder candidate genes, schizophrenia candidate genes, and/or bipolar disorder candidate genes can be used as markers in epigenetic testing, particularly, as markers of potential adverse events during embryogenesis. These genes are associated with altered methylation (either hyper or hypo) and altered expression (either increased or decreased) and are now shown (herein) associated with APA. These findings allow for the development of new screening strategies. 
     Embodiments herein provide methods and kits for the identification and/or screening of blastocysts for epigenetic events, and in particular, for epigenetic events tied to adverse outcomes during reproduction. Embodiments herein provide methods and kits for the identification and/or screening of sperm for epigenetic events, for example, epigenetic events tied to neurodevelopmental disorders in offspring. 
     Embodiments herein include methods for identifying a blastocyst with increased epigenetic dysregulation, and in particular, epigenetic dysregulation associated with adverse outcomes during embryogenesis, i.e. adverse outcomes resulting in offspring affected by neurodevelopmental disorders. The method includes, obtaining a blastocyst of interest and identifying DNA methylation errors in the blastocyst. An increased risk of epigenetic dysregulation is correlated with a blastocyst having global DNA with an overall hypomethylated shift, or correlated with a blastocyst having one or more, or two or more, autism spectrum disorder, schizophrenia, or bipolar disorder candidate genes being hypermethylated. In aspects of the case, the autism spectrum disorder candidate genes include ABAT, ABCA7, ADSL, ANKRD11, CACNA1C, CACNA1H, CCDC88C, CDH11, CIC, CLSTN3, CNTNAP2, CSMD1, DDX3X, DHCR7, DPP6, EHMT1, FLT1, GLIS1, GNAS, GRIN1, GRM8, HDAC6, ITGB7, KCNQ2, NRP2, NSD1, P4HA2, PACS1, PIK3R2, PXDN, SGSH, SHANK2, SHANK3, SLC38A10, TET2, TT12, ZNF804A, or UBE3B. The schizophrenia candidate genes include TCF3 and ZNF804A. The bipolar disorder candidate genes include COMT, DRD4, GRIN1, MBP, PRKCZ, SHANK2, TRPM2, and ZNF804A. Opioid signaling pathway candidate genes can be any of the neurodevelopmental disease candidate genes recited above, for example, CACNA1H, GRIN1, and PRKCZ. 
     In some cases, the methods can be used to screen blastocysts for epigenetic dysregulation, where a blastocyst having epigenetic dysregulation would not be used for an implantation procedure. 
     In other embodiments herein, methods are provided for determining a blastocyst&#39;s epigenetic status. Methods include determining the level of methylation and gene expression in one or more, or two or more, autism spectrum disorder, schizophrenia, or bipolar disorder candidate genes in the blastocyst. In some aspects, the level of methylation and gene expression in the one or more, or two or more, autism spectrum disorder, schizophrenia, or bipolar disorder genes are compared to a known control having a normal or non-phenotypic epigenetic status. As noted above, the autism spectrum disorder candidate genes can be any one of ABAT, ABCA7, ADSL, ANKRD11, CACNA1C, CACNA1H, CCDC88C, CDH11, CIC, CLSTN3, CNTNAP2, CSMD1, DDX3X, DHCR7, DPP6, EHMT1, FLT1, GLIS1, GNAS, GRIN1, GRM8, HDAC6, ITGB7, KCNQ2, NRP2, NSD1, P4HA2, PACS1, PIK3R2, PXDN, SGSH, SHANK2, SHANK3, SLC38A10, TET2, TT12, ZNF804A, or UBE3B. The schizophrenia candidate genes can be TCF3 and ZNF804A. The bipolar disorder candidate genes can be COMT, DRD4, GRIN1, MBP, PRKCZ, SHANK2, TRPM2, and ZNF804A. Opioid signaling pathway candidate genes can be any of the neurodevelopmental disease candidate genes recited above, for example, CACNA1H, GRIN1, and PRKCZ. 
     In alternative methods for determining a blastocyst&#39;s epigenetic status, the method includes determining the global DNA methylation of the blastocyst, where a hypomethylated or hypermethylated shift in the global DNA methylation, as compared to a normal epigenetic status, is considered an abnormal epigenetic status. 
     Finally, embodiments herein include kits for testing the epigenetic status of a blastocyst, which include means for determining methylation level of the blastocyst, and means for determining gene expression in two or more target genes in the blastocyst. The two or more genes in the blastocyst can be autism spectrum disorder genes, schizophrenia genes, or bipolar disorder genes. Standard control values and instructions on how to perform the testing can also be provided. 
     Embodiments herein include, methods for identifying sperm or a sperm donor having a predisposition to producing offspring with neurodevelopmental disorders. The method includes obtaining an ejaculate sample from the sperm donor, identifying the degree of DNA methylation errors in the sperm population, and comparing the degree of DNA methylation errors in the donor sperm population to the degree of DNA methylation errors in a control sample. If the degree of DNA methylation errors in the donor sperm population exceeds a threshold indicating a cumulative risk of epigenetic dysregulation, the donor sperm will not be used to fertilize eggs used for in vitro fertilization. In some aspects, the degree of epigenetic changes in a sperm or sperm population allows for a ranking of the sperm or sample to determine the healthiest sperm or sperm sample for use in in vitro fertilization. The cumulative risk is correlated with a sperm population having global DNA with an overall hypomethylated shift, or correlated with a sperm population having a threshold degree of DNA methylation errors in one or more, or two or more, autism spectrum disorder, schizophrenia, or bipolar disorder candidate genes being hypermethylated. In aspects of the case, the autism spectrum disorder candidate genes include ABAT, ABCA7, ADSL, ANKRD11, CACNA1C, CACNA1H, CCDC88C, CDH11, CIC, CLSTN3, CNTNAP2, CSMD1, DDX3X, DHCR7, DPP6, EHMT1, FLT1, GLIS1, GNAS, GRIN1, GRM8, HDAC6, ITGB7, KCNQ2, NRP2, NSD1, P4HA2, PACS1, PIK3R2, PXDN, SGSH, SHANK2, SHANK3, SLC38A10, TET2, TT12, ZNF804A, or UBE3B. The schizophrenia candidate genes can be TCF3 and ZNF804A. The bipolar disorder candidate genes can be COMT, DRD4, GRIN1, MBP, PRKCZ, SHANK2, TRPM2, and ZNF804A. Opioid signaling pathway candidate genes can be any of the neurodevelopmental disease candidate genes recited above, for example, CACNA1H, GRIN1, and PRKCZ. 
     In some cases, the methods can be used to identify sperm exhibiting epigenetic dysregulation, where a sperm having epigenetic dysregulation would not be used for an in vitro fertilization. In some aspects, the methods can be used to rank sperm or a sperm population to permit selection of the healthiest sperm for in vitro fertilization. 
     In other embodiments herein, methods are provided for determining the epigenetic status of an individual sperm, or epigenetic status of a sperm population obtained from a sperm donor. Methods include determining the level of methylation and gene expression in one or more, or two or more, autism spectrum disorder, schizophrenia, and/or bipolar disorder candidate genes in the sperm or sperm population. In some aspects, the level of methylation and gene expression in the one or more, or two or more, autism spectrum disorder schizophrenia, and/or bipolar disorder genes are compared to a known control having a normal or non-phenotypic epigenetic status. As noted above, the autism spectrum disorder candidate genes can be any one of ABAT, ABCA7, ADSL, ANKRD11, CACNA1C, CACNA1H, CCDC88C, CDH11, CIC, CLSTN3, CNTNAP2, CSMD1, DDX3X, DHCR7, DPP6, EHMT1, FLT1, GLIS1, GNAS, GRIN1, GRM8, HDAC6, ITGB7, KCNQ2, NRP2, NSD1, P4HA2, PACS1, PIK3R2, PXDN, SGSH, SHANK2, SHANK3, SLC38A10, TET2, TT12, ZNF804A, or UBE3B. The schizophrenia candidate genes can be TCF3 and ZNF804A. The bipolar disorder candidate genes can be COMT, DRD4, GRIN1, MBP, PRKCZ, SHANK2, TRPM2, and ZNF804A. Opioid signaling pathway candidate genes can be any of the neurodevelopmental disease candidate genes recited above, for example, CACNA1H, GRIN1, and PRKCZ. 
     In alternative methods for determining a sperm or sperm population&#39;s epigenetic status, the method includes determining the global DNA methylation of the sperm or sperm population, where a hypomethylated or hypermethylated shift in the global DNA methylation, as compared to a normal epigenetic status, is considered an abnormal epigenetic status. 
     Finally, embodiments herein include kits for testing the epigenetic status of a sperm or sperm population, which include means for determining methylation level of the sperm population, and means for determining gene expression in one or more, or two or more, target genes in the sperm. The genes in the sperm can be autism spectrum disorder genes, schizophrenia genes, and/or bipolar disorder genes. Standard control values and instructions on how to perform the testing can also be provided. 
     While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims. 
     EXAMPLES 
     The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. Statistical analysis included Student&#39;s t test, Fisher&#39;s exact test, Mann-Whitney U test, Benjamini-Hochberg method, with significance at p&lt;0.05. 
     Example 1: Methylation Plays A Role in Epigenetic Dysregulation of Blastocysts 
     Objective: APA has been associated with adverse outcomes including birth defects and childhood neurodevelopmental diseases, like autism spectrum disorder. Epigenetic dysregulation, such as inherited DNA methylation errors from aged fathers may lead to permanent altered gene expression in offspring, resulting in a clinical phenotype. The aim of this example was to investigate the methylome and subsequent transcriptome of human blastocysts in association with APA. 
     Materials and Methods: Cryopreserved, transferrable quality, human blastocysts were donated with IRB approval and patient consent. Normozoospermic aged fathers (APA; ≥50 years) were compared to normozoospermic younger fathers (Young; ≤35 years) with only donor oocytes to eliminate known female factors. Blastocyst DNA (n=12) was bisulfite converted and sequenced using Methyl-MaxiSeq (Zymo Research), while blastocyst RNA (n=12) underwent small cell number RNA-seq (Illumina). Results were analyzed in conjunction with pathway analysis (DAVID 6.8). Transcription validation (n=24) was performed using qPCR with REST 2009 and methylation validation (n=10) utilized the PyroMark Q24 Advanced system (Qiagen). Statistical analysis included Student&#39;s t-test, ANOVA in R, Fisher&#39;s Exact Test and Pair Wise Fixed Reallocation Randomization Test where appropriate, with significance at p&lt;0.05. 
     Results: An overall shift towards hypomethylation was observed in APA blastocysts (34% APA vs. 40% Young; p&lt;0.05), with 57,286 hypomethylated and 49,709 hypermethylated CpG sites (p&lt;0.05). RNA sequencing revealed 751 genes with significantly decreased, and 195 genes with significantly increased expression (p&lt;0.05). Combining the methylome and transcriptome datasets, 319 genes were significantly and directionally altered in both (p&lt;0.05). Pathway analysis identified “disease mutation” as a highly significant key functional annotation category that includes intellectual disability, tumor suppressor genes, neurodegeneration, and autism spectrum disorder. To date, validation of methylation increase for the autism spectrum candidate genes SHANK2 (45% APA vs. 33% Young; p&lt;0.05) and ANKRD11 (75% APA vs. 43% Young; p&lt;0.05), and the observed decreased effects on their gene expression have been confirmed (p&lt;0.05). 
     Conclusions: This novel study investigated the impact of APA on epigenetic events during embryogenesis. The results reveal an overall hypomethylated shift in blastocysts derived from aged fathers, resulting in significantly altered transcription. Interestingly, autism spectrum candidate genes were primarily hypermethylated with subsequent decreased gene expression, providing a mechanistic link between the advanced paternal age effect and childhood neurodevelopmental disorders. 
     Example 2: Increase of Parental Age Significantly Exacerbated Hypomethylation 
     Six young (≤35 years) and six APA (≥50 years) normozoospermic sperm samples were used for global methylome analysis using a modified reduced representation bisulfite sequence (RRBS; Methyl Mini-Seq platform from Zymo Research) method, and an increase in global methylation was observed in the APA sperm epigenome and confirmed by LINE 1  bisulfite pyrosequencing. See  FIG.  1   . 
     Sperm samples were prepared using two-layer PureSperm density-gradient centrifugation (Nidacon) followed by cell lysis to removed round cell contamination and storage in lysis buffer (Norgen Biotek). Surplus cryopreserved blastocysts were obtained from couples undergoing IVF treatment with informed consent and IRB approval. Similarly, six young and six APA blastocyst samples were analyzed using whole-genome bisulfite sequencing (WGBS; Methyl Maxi-Seq platform from Zymo Research). After identifying 49,722 and 106,995 CpGs statistically significant (p&lt;0.05) between the young and APA groups for sperm and blastocysts, respectively, analysis resulted in 3,405 sperm DMRs and 3,997 blastocyst DMRs. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 APA Sperm and Blastocyst DMRs 
               
            
           
           
               
               
               
               
               
            
               
                   
                 APA sperm 
                   
                 APA blastocyst 
                   
               
               
                   
                 (n = 3,405 
                   
                 (n = 3,997 
                   
               
               
                   
                 DMRs) 
                   
                 DMRs) 
                   
               
               
                   
                 Paternal age: 
                   
                 Paternal age: 
                   
               
               
                   
                 Young (28.5 ± 
                   
                 Young (32.7 ± 
                   
               
               
                   
                 2.1 years) 
                   
                 1.4 years) 
                   
               
               
                   
                 APA (54.5 ± 3.9 
                   
                 APA (52.7 ± 3.0 
                   
               
               
                   
                 years) 
                   
                 years) 
               
               
                   
               
               
                   
                 Hypermethylated 
                 Hypomethylated 
                 Hypermethylated 
                 Hypomethylated 
               
               
                 Total DMRs 
                 1929 
                 1476 
                 1729 
                 2268 
               
               
                 Significant 
                 8729 
                 7262 
                 6217 
                 8244 
               
               
                 CpGs in DMRs 
                   
                   
                   
                   
               
               
                 Average CpGs 
                 4.5 (range 3-35) 
                 4.9 (range 3-40) 
                 3.6 (range 3-25) 
                 3.6 (range 3-14) 
               
               
                 per DMR 
                   
                   
                   
                   
               
               
                 Average DMR 
                 211 bp (range 3- 
                 370 bp (range 3- 
                 550 bp (range 3- 
                 544 (range 4- 
               
               
                 window width 
                 4069) 
                 3792) 
                 5432) 
                 4033) 
               
               
                 Total associated 
                 612 
                 810 
                 1087 
                 1457 
               
               
                 genes 
                   
                   
                   
                   
               
               
                 Average genes 
                 0.32 
                 0.55 
                 0.63 
                 0.64 
               
               
                 per DMR 
                   
                   
                   
                   
               
               
                 Total unique 
                 568 
                 766 
                 803 
                 1173 
               
               
                 associated genes 
                   
                   
                   
                   
               
               
                 DMRs not 
                 1291 (67%) 
                 641 (43%) 
                 428 (25%) 
                 497 (22%) 
               
               
                 associated with 
                   
                   
                   
                   
               
               
                 a gene 
               
               
                   
               
            
           
         
       
     
     Hypomethylated DMRs in APA sperm were significantly enriched at CpG islands (p=1.80E-22), shelves (p=1.16E-175), and shores (p=1.76E-26). CpG island annotations were downloaded from the UCSC genome browser hg19. A similar trend was observed for hypomethylated DMRs in APA blastocysts. 
     Comparing sperm and blastocyst DMR-associated genes yielded a highly significant enrichment of genes between the two methylomes upon paternal aging (p=3.46E-55). Identification was made of 167 hypomethylated genes and 61 hypermethylated genes between the two methylomes with 10 genes exhibiting both hypo and hypermethylated DMRs. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Overlapping DMRs 
               
            
           
           
               
               
            
               
                   
                 APA Sperm-Blastocyst Overlapping DMRs 
               
            
           
           
               
               
               
               
            
               
                   
                 Hypermethylated 
                 Hypomethylated 
                 Any 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Overlapping DMR- 
                 61 
                 167 
                 323 
               
               
                 Associated Genes 
                   
                   
                   
               
               
                 Fold Enrichment 
                 2.36 
                 4.31 
                 3.19 
               
               
                 (odds ratio) 
                   
                   
                   
               
               
                 OR 95% Confidence 
                 1.77-3.10 
                 3.58-5.17 
                 2.79-3.65 
               
               
                 Interval 
                   
                   
                   
               
               
                 p-value 
                 2.25E−08 
                 6.14E−45 
                 3.46E−55 
               
               
                   
               
            
           
         
       
     
     Normozoospermic sperm samples from an additional 12 young and 12 APA were used for methylation validation. See  FIG.  2   . Genes selected for methylation validation included altered DMRs, those identified in significant pathways, genes implicated in neurodevelopmental disorders, those localized to significant cytobands, imprinted genes, and those corresponding to published literature on aging sperm. See Table 3. Using linear regression models, hypomethylation was significantly exacerbated as paternal age increased from 50 years to over 60 years. See  FIG.  3   . 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 DMR-associated genes previously identified in aging human sperm 
               
            
           
           
               
               
               
            
               
                   
                 Methylation 
                   
               
               
                 Gene 
                 Status 
                 Association 
               
               
                   
               
               
                 AGRN 
                 Hypomethylated 
                   
               
               
                 ARC 
                 Hypomethylated 
                   
               
               
                 ATHL1 
                 Hypomethylated 
                   
               
               
                 BEGAIN 
                 Hypomethylated 
                   
               
               
                 C7orf50 
                 Hypomethylated 
                   
               
               
                 CACNA1H 
                 Hypomethylated 
                 Autism Spectrum Disorder, 
               
               
                   
                   
                 Opioid Signaling 
               
               
                 CCDC114 
                 Hypomethylated 
                   
               
               
                 DAPK3 
                 Hypomethylated 
                   
               
               
                 DLGAP2 
                 Hypomethylated 
                 Imprinted 
               
               
                 DRD4 
                 Hypomethylated 
                 Bipolar Disorder 
               
               
                 ELANE 
                 Hypomethylated 
                   
               
               
                 FOXK1 
                 Hypomethylated 
                   
               
               
                 GET4 
                 Hypomethylated 
                 Schizophrenia 
               
               
                 GRIN1 
                 Hypomethylated 
                 Autism Spectrum Disorder, 
               
               
                   
                   
                 Bipolar Disorder, Opioid 
               
               
                   
                   
                 Signaling 
               
               
                 HOXA10 
                 Hypomethylated 
                   
               
               
                 KCNA7 
                 Hypomethylated 
                   
               
               
                 KCNF1 
                 Hypomethylated 
                   
               
               
                 KCNQ1 
                 Hypomethylated 
                 Imprinted 
               
               
                 KDM2B 
                 Hypomethylated 
                   
               
               
                 LDLRAD4 
                 Hypomethylated 
                   
               
               
                 LONP1 
                 Hypomethylated 
                   
               
               
                 MPPED1 
                 Hypomethylated 
                   
               
               
                 NADK 
                 Hypomethylated 
                   
               
               
                 NCOR2 
                 Hypomethylated 
                 Schizophrenia, Frontal Cortex 
               
               
                   
                   
                 of Autistic Brains 
               
               
                 NSMF 
                 Hypomethylated 
                   
               
               
                 PALM 
                 Hypomethylated 
                   
               
               
                 PAX2 
                 Hypomethylated 
                   
               
               
                 PITPNM1 
                 Hypomethylated 
                   
               
               
                 PTPRN2 
                 Hypomethylated 
                 Frontal Cortex of Autistic 
               
               
                   
                   
                 Brains 
               
               
                 PURA 
                 Hypomethylated 
                   
               
               
                 SECTM1 
                 Hypomethylated 
                   
               
               
                 SLC22A18AS 
                 Hypomethylated 
                 Schizophrenia, Imprinted 
               
               
                 SOHLH1 
                 Hypomethylated 
                   
               
               
                 THBS3 
                 Hypomethylated 
                   
               
               
                 UNKL 
                 Hypomethylated 
                 Schizophrenia 
               
               
                 USP36 
                 Hypomethylated 
                   
               
               
                 WFDC1 
                 Hypomethylated 
                   
               
               
                 ZFPM1 
                 Hypomethylated 
                 Schizophrenia 
               
               
                 BCL11A 
                 Hypermethylated 
                 Autism Spectrum Disorder, 
               
               
                   
                   
                 Bipolar Disorder 
               
               
                 CCDC144NL 
                 Hypermethylated 
                   
               
               
                 FAM86C1 
                 Hypermethylated 
               
               
                   
               
            
           
         
       
     
     Twelve blastocysts (derived from young paternal age fathers) and 12 APA blastocysts were selected for blastocyst methylation validation in DMRs from two genes associated with neurodevelopmental disorders. Pyrosequencing data confirmed significant hypomethylation along the amplified CACNA1H DMR region (40% average methylation for young, 25% APA, p, 0.05) while validation of the SHANK2 blastocyst DMR trended towards hypomethylation (young 75%, APA 56%, p&lt;0.1). See  FIG.  4    and Table 4. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Methylome read mapping statistics 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Average 
                 Bisulfite 
                 Gene 
                   
                 CpG 
               
               
                   
                 Total Read 
                 Mapping 
                 Unique 
                 CpG 
                 Conversion 
                 Body 
                 Promoter 
                 Island 
               
               
                 Samples 
                 Number 
                 Efficiency 
                 CpGs 
                 Coverage 
                 Rate 
                 Coverage 
                 Coverage 
                 Coverage 
               
               
                   
               
            
           
           
               
            
               
                 Sperm (Methyl-MiniSeq) 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 YNG 1 
                  30,919,771 
                 47% 
                  9,717,284 
                  8X 
                 99% 
                 85% 
                 74% 
                 77% 
               
               
                 YNG 2 
                  33,860,516 
                 46% 
                  9,864,863 
                  8X 
                 99% 
                 85% 
                 74% 
                 79% 
               
               
                 YNG 3 
                  33,330,722 
                 46% 
                 10,355,505 
                  8X 
                 99% 
                 85% 
                 75% 
                 80% 
               
               
                 YNG 4 
                  32,212,578 
                 42% 
                 10,145,132 
                  7X 
                 99% 
                 85% 
                 74% 
                 78% 
               
               
                 YNG 5 
                  32,023,879 
                 47% 
                  9,912,019 
                  8X 
                 99% 
                 85% 
                 74% 
                 78% 
               
               
                 YNG 6 
                  32,236,741 
                 48% 
                  9,837,071 
                  8X 
                 99% 
                 85% 
                 75% 
                 79% 
               
               
                 APA 1 
                  31,247,009 
                 47% 
                 10,129,874 
                  8X 
                 99% 
                 85% 
                 75% 
                 79% 
               
               
                 APA 2 
                  32,567,449 
                 45% 
                 10,220,222 
                  8X 
                 99% 
                 85% 
                 75% 
                 79% 
               
               
                 APA 3 
                  32,471,117 
                 42% 
                  9,873,598 
                  7X 
                 99% 
                 85% 
                 74% 
                 77% 
               
               
                 APA 4 
                  30,202,170 
                 43% 
                  9,663,748 
                  7X 
                 99% 
                 85% 
                 73% 
                 77% 
               
               
                 APA 5 
                  31,611,911 
                 45% 
                  9,816,449 
                  8X 
                 99% 
                 85% 
                 74% 
                 77% 
               
               
                 APA 6 
                  35,743,721 
                 48% 
                 10,036,958 
                  9X 
                 99% 
                 85% 
                 76% 
                 81% 
               
            
           
           
               
            
               
                 Blastocysts (Methyl-MaxiSeq) 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 YNG 1 
                 422,906,276 
                 62% 
                 22,896,016 
                 30X 
                 99% 
                 85% 
                 69% 
                 56% 
               
               
                 YNG 2 
                 484,339,064 
                 61% 
                 28,370,405 
                 30X 
                 99% 
                 86% 
                 71% 
                 60% 
               
               
                 YNG 3 
                 462,453,232 
                 61% 
                 43,593,680 
                 17X 
                 99% 
                 89% 
                 84% 
                 75% 
               
               
                 YNG 4 
                 481,752,559 
                 64% 
                 29,478,372 
                 31X 
                 98% 
                 87% 
                 76% 
                 67% 
               
               
                 YNG 5 
                 487,212,124 
                 62% 
                 36,443,768 
                 23X 
                 99% 
                 88% 
                 81% 
                 71% 
               
               
                 YNG 6 
                 489,702,951 
                 63% 
                 36,094,786 
                 24X 
                 98% 
                 88% 
                 77% 
                 68% 
               
               
                 APA 1 
                 502,958,355 
                 61% 
                 42,018,862 
                 21X 
                 99% 
                 89% 
                 83% 
                 78% 
               
               
                 APA 2 
                 507,076,029 
                 63% 
                 22,090,645 
                 40X 
                 99% 
                 86% 
                 71% 
                 58% 
               
               
                 APA 3 
                 510,044,505 
                 62% 
                 38,726,517 
                 23X 
                 98% 
                 88% 
                 76% 
                 66% 
               
               
                 APA 4 
                 466,725,178 
                 61% 
                 38,469,550 
                 20X 
                 98% 
                 89% 
                 81% 
                 70% 
               
               
                 APA 5 
                 448,954,497 
                 60% 
                 38,299,371 
                 19X 
                 99% 
                 88% 
                 80% 
                 68% 
               
               
                 APA 6 
                 495,812,948 
                 63% 
                 37,621,620 
                 25X 
                 99% 
                 87% 
                 74% 
                 64% 
               
               
                   
               
            
           
         
       
     
     Example 3: Specific Chromosomal Regions are More Susceptible to Age-Related Methylation Alterations 
     Chromosomal enrichment for DMR-associated gene density at individual cytobands was analyzed to determine if specific chromosomal regions are more susceptible to age-related methylation alterations. Methylation alterations were not randomly distributed across the genome, but appear clustered at certain chromosomal locations with chromosome 19 having the greatest significance between young and APA samples (sperm p=5.51E-7, blastocyst p=9.01E-13, overlapping p=7.28E-6). Significant enrichment was identified at 5 cytobands in APA sperm, with four of the five independently enriched in APA blastocyst methylome, and 3 significant in the overlapping APA sperm and blastocyst gene list. See Table 5. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Statistically significant cytobands 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 APA 
                   
                   
                 Odds  
               
               
                   
                 Cytoband 
                 Sample 
                 P-value 
                 FDR 
                 Ratio 
               
               
                   
                   
               
               
                   
                 Chr10q26.3 
                 Sperm 
                 1.79E−04 
                 2.91E−02 
                   
               
               
                   
                   
                 Blastocyst 
                 3.93E−05 
                 2.66E−03 
                   
               
               
                   
                   
                 Overlapping 
                 2.25E−02 
                 N.S. 
                 5.29 
               
               
                   
                 Chr11p15.5 
                 Sperm 
                 6.95E−08 
                 2.45E−05 
                   
               
               
                   
                   
                 Blastocyst 
                 4.92E−02 
                 N.S. 
                   
               
               
                   
                   
                 Overlapping 
                 2.98E−04 
                 3.03E−02 
                 5.95 
               
               
                   
                 Chr16p13.3 
                 Sperm 
                 9.02E−08 
                 2.45E−05 
                   
               
               
                   
                   
                 Blastocyst 
                 2.43E−07 
                 3.81E−05 
                   
               
               
                   
                   
                 Overlapping 
                 6.50E−05 
                 1.05E−02 
                 4.20 
               
               
                   
                 Chr17q25.3 
                 Sperm 
                 7.52E−05 
                 1.53E−02 
                   
               
               
                   
                   
                 Blastocyst 
                 2.81E−07 
                 3.81E−05 
                   
               
               
                   
                   
                 Overlapping 
                 6.59E−03 
                 N.S. 
                 3.80 
               
               
                   
                 Chr19p13.3 
                 Sperm 
                 4.32E−19 
                 3.51E−16 
                   
               
               
                   
                   
                 Blastocyst 
                 3.76E−22 
                 3.06E−19 
                   
               
               
                   
                   
                 Overlapping 
                 5.62E−16 
                 4.57E−13 
                 9.89 
               
               
                   
                   
               
            
           
         
       
     
     Greatest enrichment for all three datasets was chr19p13.3 (sperm p=4.32E-19, blastocyst p=3.76E-22, overlapping p=5.62E-16). Analyzation of the colocalization of DMR-associated genes with known regions of nucleosome retention in sperm identified statistically significant enrichment with mononucleosomes for APA sperm DMRs (p=9.36E-29) and with presence of repressive histone mark H3K27me3 (p=7.21E-6). This colocalization with mononucleosomes was enhanced for the directionally overlapping DMRs identified in both sperm and blastocyst datasets (p=2.52E-18). See Table 6. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Co-localization of DMR-associated genes with  
               
               
                 histones and histone modifications 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Fold 
                 OR 95% 
                   
               
               
                   
                 Overlapping 
                 enrichment 
                 confidence 
                   
               
               
                 Histone gene set* 
                 genes 
                 (odds ratio) 
                 interval 
                 P-value 
               
               
                   
               
            
           
           
               
            
               
                 APA Sperm DMRs 
               
            
           
           
               
               
               
               
               
            
               
                 Mononucleosomes 
                 619 
                 1.89 
                 1.69-2.11 
                 9.36E−29 
               
               
                 H3K27me3 
                 275 
                 1.38 
                 1.20-1.59 
                 7.21E−06 
               
               
                 H3K4me3 
                 541 
                 0.94 
                 0.84-1.05 
                 N.S. 
               
            
           
           
               
            
               
                 APA Blastocyst DMRs 
               
            
           
           
               
               
               
               
               
            
               
                 Mononucleosomes 
                 1074 
                 2.19 
                 2.00-2.39 
                 1.50E−66 
               
               
                 H3K27me3 
                 364 
                 1.05 
                 0.93-1.18 
                 N.S. 
               
               
                 H3K4me3 
                 957 
                 1.08 
                 0.98-1.18 
                 N.S. 
               
            
           
           
               
            
               
                 APA Sperm-Blastocyst Directional Overlapping DMRs 
               
            
           
           
               
               
               
               
               
            
               
                 Mononucleosomes 
                 130 
                 3.32 
                 2.51-4.41 
                 2.52E−18 
               
               
                 H3K27me3 
                 31 
                 0.90 
                 0.59-1.32 
                 N.S. 
               
               
                 H3K4me3 
                 79 
                 0.82 
                 0.62-1.10 
                 N.S. 
               
               
                   
               
               
                 *Hammoud, S. S. et al. Distinctive chromatin in human sperm packages genes for embryo development. Nature 460, 473-478, doi:10.1038/nature08162 (2009). 
               
            
           
         
       
     
     Example 4: Effect of Paternal Aging Altering Methylation at Genes Linked to Neurodevelopmental Disorders 
     Neurological signaling pathways were found to be represented among genome regions that were differentially methylated in APA samples. The opioid signaling pathway was the top pathway for the directionally overlapping genes found in both APA sperm and blastocyst datasets. Genes successfully validated from the opioid signaling pathway include CACNA1H, GRIN1 and PRKCZ in APA sperm and CACNA1H in APA blastocysts. The pathway involving nNOS signaling in neurons was highlighted as the top canonical pathway impacted in the APA sperm methylome (p=2.08×10−4), which also comprises similar successfully validated genes (GRIN1 and PRKCZ). Additional pathways impacted by advanced paternal age can be found in Table 7. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Top Canonical Pathways 
               
            
           
           
               
               
               
               
            
               
                   
                 Rank 
                 Pathway 
                 p-value 
               
               
                   
                   
               
            
           
           
               
            
               
                 APA sperm DMRs 
               
            
           
           
               
               
               
               
            
               
                   
                 1 
                 nNOS signaling in neurons 
                 2.08E−04 
               
               
                   
                 2 
                 Sumoylation pathway 
                 2.50E−04 
               
               
                   
                 3 
                 Glutamate receptor signaling 
                 2.54E−04 
               
               
                   
                 4 
                 Opioid signaling pathway 
                 1.24E−03 
               
               
                   
                 5 
                 Dopamine-DARPP32 feedback in cAMP 
                 4.17E−03 
               
               
                   
                   
                 signaling 
                   
               
            
           
           
               
            
               
                 APA blastocyst DMRs 
               
            
           
           
               
               
               
               
            
               
                   
                 1 
                 Opioid signaling pathway 
                 2.45E−07 
               
               
                   
                 2 
                 Netrin signaling 
                 3.58E−07 
               
               
                   
                 3 
                 GPCR-mediated nutrient sensing in 
                 1.69E−06 
               
               
                   
                   
                 enteroendocrine cells 
                   
               
               
                   
                 4 
                 CREB signaling in neurons 
                 4.28E−06 
               
               
                   
                 5 
                 PPARα/RXRα Activation 
                 5.77E−06 
               
            
           
           
               
            
               
                 APA sperm-blastocyst directional overlapping DMRs 
               
            
           
           
               
               
               
               
            
               
                   
                 1 
                 Opioid signaling pathway 
                 2.14E−03 
               
               
                   
                 2 
                 Tight junction signaling 
                 4.84E−03 
               
               
                   
                 3 
                 Wnt/β-catenin signaling 
                 5.42E−03 
               
               
                   
                 4 
                 Role of Wnt/GSK-3β signaling in the 
                 5.71E−03 
               
               
                   
                   
                 pathogenesis of influenza 
                   
               
               
                   
                 5 
                 Phosphatidylethanolamine biosynthesis III 
                 9.26E−03 
               
               
                   
                   
               
            
           
         
       
     
     Publicly available gene lists were compared to the DMRs, and highly significant enrichment for genes was identified in three neurological disorders, specifically, autism spectrum disorder (p=1.94E-4), schizophrenia (p=5.55E-4), and bipolar disorder (p=4.73E-5). This enrichment was identified independently in the APA sperm DMRs and APA blastocyst DMRs. See Table 8. Genes were validated in APA sperm that are associated with autism spectrum disorder (CACNA1H, CNTNAP2, GRIN1, SHANK2, SHANK3, ZNF804A), schizophrenia (TCF3, ZNF804A), bipolar disorder (COMT, DRD4, GRIN1, MBP, PRKCZ, SHANK2, TRPM2, ZNF804A)) and in APA blastocysts (CACNA1H). The opioid signaling pathway is shared by all three candidate neurodevelopmental gene lists. See Table 9. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Neurodevelopmental Disorder Associations 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Fold 
                 OR 95% 
                   
               
               
                   
                 Overlapping 
                 Enrichment 
                 Confidence 
                   
               
               
                 Disease 
                 Genes 
                 (odds ratio) 
                 Interval 
                 p-value 
               
               
                   
               
            
           
           
               
            
               
                 APA Sperm DMRs 
               
            
           
           
               
               
               
               
               
            
               
                 Autism spectrum 
                 35 
                 2.48 
                 1.68-3.57 
                 7.76E−06 
               
               
                 disorder 
                   
                   
                   
                   
               
               
                 Schizophrenia 
                 19 
                 2.31 
                 1.35-3.77 
                 1.57E−03 
               
               
                 Bipolar Disorder 
                 63 
                 1.93 
                 1.45-2.53 
                 8.24E−06 
               
            
           
           
               
            
               
                 APA Blastocyst DMRs 
               
            
           
           
               
               
               
               
               
            
               
                 Autism spectrum 
                 60 
                 2.80 
                 2.06-3.76 
                 2.88E−10 
               
               
                 disorder 
                   
                   
                   
                   
               
               
                 Schizophrenia 
                 40 
                 3.40 
                 2.30-4.94 
                 2.42E−09 
               
               
                 Bipolar Disorder 
                 91 
                 1.71 
                 1.35-2.16 
                 1.17E−05 
               
            
           
           
               
            
               
                 APA Sperm-Blastocyst Directional Overlapping DMRs 
               
            
           
           
               
               
               
               
               
            
               
                 Autism spectrum 
                 10 
                 4.34 
                 2.03-8.28 
                 1.94E−04 
               
               
                 disorder 
                   
                   
                   
                   
               
               
                 Schizophrenia 
                  7 
                 5.30 
                  2.07-11.38 
                 5.55E−04 
               
               
                 Bipolar Disorder 
                 17 
                 3.30 
                 1.87-5.47 
                 4.73E−05 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Top canonical pathways for neurode velopmental disorders 
               
            
           
           
               
               
               
               
               
               
            
               
                 Autism Spectrum 
                   
                   
                   
                   
                   
               
               
                 Disorder 
                 P-value 
                 Schizophrenia 
                 P-value 
                 Bipolar Disorder 
                 P-value 
               
               
                   
               
               
                 GABA Receptor 
                 2.19E−08 
                 Synaptic Long 
                 2.29E−06 
                 Serotonin Receptor 
                 1.26E−23 
               
               
                 Signaling 
                   
                 Term 
                   
                 Signaling 
                   
               
               
                   
                   
                 Depression 
                   
                   
                   
               
               
                 CREB Signaling in 
                 4.90E−07 
                 Synaptic Long 
                 6.03E−06 
                 Circadian Rhythm 
                 2.51E−23 
               
               
                 Neurons 
                   
                 Term 
                   
                 Signaling 
                   
               
               
                   
                   
                 Potentiation 
                   
                   
                   
               
               
                 Glutamate Receptor 
                 5.62E−06 
                 Dopamine- 
                 1.86E−05 
                 CREB Signaling in 
                 2.51E−19 
               
               
                 Signaling 
                   
                 DARPP32 
                   
                 Neurons 
                   
               
               
                   
                   
                 Feedback in 
                   
                   
                   
               
               
                   
                   
                 cAMP Signaling 
                   
                   
                   
               
               
                 Calcium Signaling 
                 4.68E−05 
                 p70S6K 
                 7.76E−05 
                 G-Protein Coupled 
                 6.31E−18 
               
               
                   
                   
                 Signaling 
                   
                 Receptor Signaling 
                   
               
               
                 Opioid Signaling 
                 5.25E−05 
                 CREB Signaling 
                 9.12E−05 
                 GABA Receptor 
                 3.98E−17 
               
               
                 Pathway 
                   
                 in Neurons 
                   
                 Signaling 
                   
               
               
                 Amyotrophic Lateral 
                 6.46E−05 
                 Calcium 
                 1.95E−04 
                 Dopamine 
                 5.01E−17 
               
               
                 Sclerosis Signaling 
                   
                 Signaling 
                   
                 Receptor Signaling 
                   
               
               
                 GPCR-Mediated 
                 6.76E−05 
                 Opioid 
                 1.05E−03 
                 cAMP-mediated 
                 3.16E−15 
               
               
                 Nutrient Sensing in 
                   
                 Signaling 
                   
                 signaling 
                   
               
               
                 Enteroendocrine 
                   
                 Pathway 
                   
                   
                   
               
               
                 Cells 
                   
                   
                   
                   
                   
               
               
                 nNOS Signaling in 
                 1.32E−04 
                 D-myo-inositol- 
                 1.23E−03 
                 Opioid Signaling 
                 3.98E−15 
               
               
                 Skeletal Muscle 
                   
                 5-phosphate 
                   
                 Pathway 
                   
               
               
                 Cells 
                   
                 Metabolism 
                   
                   
                   
               
               
                 Corticotropin 
                 3.02E−04 
                 14-3-3-mediated 
                 1.38E−03 
                 Calcium Signaling 
                 6.31E−15 
               
               
                 Releasing Hormone 
                   
                 Signaling 
                   
                   
                   
               
               
                 Signaling 
                   
                   
                   
                   
                   
               
               
                 Synaptic Long Term 
                 3.72E−04 
                 Role of NFAT in 
                 1.48E−03 
                 Neuroinflammation 
                 3.16E−14 
               
               
                 Depression 
                   
                 Cardiac 
                   
                 Signaling Pathway 
                   
               
               
                   
                   
                 Hypertrophy 
               
               
                   
               
            
           
         
       
     
     Using publicly available methylation profiling arrays, two brain tissues from autistic and nonautistic samples were analyzed; 141 (autistic) and 62 (nonautistic) DMRs were identified. The genes within these DMRs significantly overlapped APA sperm (frontal cortex p=5.78E-9, cingulate cortex p=1.62E-4) and APA blastocyst (frontal cortex p=1.94E-16, cingulate cortex p=3.24E-5) DMR-associated gene lists. The frontal cortex was determined to be significantly enriched for opioid signaling (p=2.14E-3) using pathway analysis of the genes associated with the DMRs. See Table 10. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Pathway analysis for DMR-associated genes in autistic brains  
               
               
                 compared with APA sperm and APA blastocyst 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Canonical Pathway  
                 Autistic Frontal 
                 APA  
                 APA  
               
               
                   
                 (p ≤ 0.01) 
                 Cortex 
                 Sperm 
                 Blastocyst 
               
               
                   
                   
               
               
                   
                 Opioid Signaling 
                 2.14E−03 
                 1.23E−03 
                 2.45E−07 
               
               
                   
                 Pathway 
                   
                   
                   
               
               
                   
                 Melatonin Signaling 
                 2.24E−03 
                 6.17E−03 
                 3.72E−04 
               
               
                   
                 Dopamine-DARPP32 
                 2.88E−03 
                 4.17E−03 
                 1.10E−05 
               
               
                   
                 Feedback in cAMP 
                   
                   
                   
               
               
                   
                 Signaling 
                   
                   
                   
               
               
                   
                 Neuregulin Signaling 
                 5.13E−03 
                 5.25E−03 
                 N.S. 
               
               
                   
                   
               
            
           
         
       
     
     The APA datasets were further compared to the current list of known and putative human-imprinted genes. APA sperm had 19 hypomethylated and 7 hypermethylated significant DMRs associated with imprinted genes. APA blastocysts had 22 hypomethylated and 10 hypermethylated significant DMRs associated with imprinted genes, with 6 imprinted genes overlapped in both datasets (p&lt;0.05). See Table 11. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 DMR-associated imprinted genes 
               
            
           
           
               
               
               
               
               
               
            
               
                 APA 
                   
                   
                   
                 Imprinting 
                 Expressed 
               
               
                 Sample 
                 DMR 
                 Chr 
                 Imprinted Gene 
                 Status 
                 Allele 
               
               
                   
               
               
                 Sperm 
                 Hypomethylated 
                 chr1 
                 DVL1 
                 Predicted 
                 Maternal 
               
               
                 Blastocyst 
                 Hypomethylated/ 
                 chr1 
                 OBSCN 
                 Predicted 
                 Paternal 
               
               
                   
                 Hypermethylated 
                   
                   
                   
                   
               
               
                 Blastocyst 
                 Hypermethylated 
                 chr1 
                 PEX10 
                 Predicted 
                 Maternal 
               
               
                 Sperm 
                 Hypomethylated/ 
                 chr1 
                 PRDM16 
                 Predicted 
                 Paternal 
               
               
                   
                 Hypermethylated 
                   
                   
                   
                   
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr1 
                 PRDM16 
                 Predicted 
                 Paternal 
               
               
                 Sperm 
                 Hypermethylated 
                 chr1 
                 PTPN14 
                 Predicted 
                 Maternal 
               
               
                 Sperm 
                 Hypomethylated 
                 chr1 
                 TP73 
                 Imprinted 
                 Maternal 
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr2 
                 GPR1 
                 Imprinted 
                 Paternal 
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr2 
                 MYEOV2 
                 Predicted 
                 Paternal 
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr3 
                 ALDHIL1 
                 Predicted 
                 Maternal 
               
               
                 Blastocyst 
                 Hypermethylated 
                 chr6 
                 IGF2R 
                 Conflicting 
                 Biallelic 
               
               
                   
                   
                   
                   
                 Data 
                   
               
               
                 Blastocyst 
                 Hypomethylated/ 
                 chr6 
                 PRIM2 
                 Conflicting 
                 Biallelic 
               
               
                   
                 Hypermethylated 
                   
                   
                 Data 
                   
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr6 
                 ADTRP 
                 Imprinted 
                 Maternal 
               
               
                 Sperm 
                 Hypomethylated 
                 chr7 
                 DDC 
                 Imprinted 
                 Isoform 
               
               
                   
                   
                   
                   
                   
                 Dependent 
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr7 
                 HOXA3 
                 Predicted 
                 Maternal 
               
               
                 Sperm 
                 Hypermethylated 
                 chr7 
                 MAGI2 
                 Imprinted 
                 Maternal 
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr7 
                 SLC4A2 
                 Predicted 
                 Maternal 
               
               
                 Sperm 
                 Hypomethylated 
                 chr8 
                 DLGAP2 
                 Imprinted 
                 Paternal 
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr8 
                 DLGAP2 
                 Imprinted 
                 Paternal 
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr8 
                 ZFAT 
                 Imprinted 
                 Paternal 
               
               
                 Sperm 
                 Hypomethylated 
                 chr9 
                 EGFL7 
                 Predicted 
                 Paternal 
               
               
                 Sperm 
                 Hypomethylated 
                 chr10 
                 VENTX 
                 Predicted 
                 Maternal 
               
               
                 Sperm 
                 Hypomethylated 
                 chr11 
                 ANO1 
                 Imprinted 
                 Maternal 
               
               
                 Sperm 
                 Hypomethylated 
                 chr11 
                 B4GALNT4 
                 Predicted 
                 Maternal 
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr11 
                 B4GALNT4 
                 Predicted 
                 Maternal 
               
               
                 Sperm 
                 Hypomethylated 
                 chr11 
                 H19 
                 Imprinted 
                 Maternal 
               
               
                 Sperm 
                 Hypomethylated 
                 chr11 
                 IGF2;INS-IGF2 
                 Imprinted 
                 Paternal 
               
               
                 Sperm 
                 Hypomethylated 
                 chr11 
                 KCNQ1 
                 Imprinted 
                 Maternal 
               
               
                 Blastocyst 
                 Hypomethylated/ 
                 chr11 
                 KCNQ1 
                 Imprinted 
                 Maternal 
               
               
                   
                 Hypermethylated 
                   
                   
                   
                   
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr11 
                 NAP1L4 
                 Unknown 
                 Unknown 
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr11 
                 NTM 
                 Imprinted 
                 Maternal 
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr11 
                 OSBPL5 
                 Imprinted 
                 Maternal 
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr11 
                 RAB1B 
                 Predicted 
                 Maternal 
               
               
                 Sperm 
                 Hypomethylated 
                 chr11 
                 SLC22A18AS 
                 Provisional 
                 Maternal 
               
               
                   
                   
                   
                   
                 Data 
                   
               
               
                 Sperm 
                 Hypermethylated 
                 chr11 
                 WT1 
                 Imprinted 
                 Paternal 
               
               
                 Sperm 
                 Hypomethylated 
                 chr12 
                 FBRSL1 
                 Predicted 
                 Maternal 
               
               
                 Blastocyst 
                 Hypomethylated/ 
                 chr12 
                 FBRSL1 
                 Predicted 
                 Maternal 
               
               
                   
                 Hypermethylated 
                   
                   
                   
                   
               
               
                 Sperm 
                 Hypermethylated 
                 chr12 
                 LRP1 
                 Imprinted 
                 Unknown 
               
               
                 Sperm 
                 Hypomethylated 
                 chr14 
                 DLK1 
                 Imprinted 
                 Paternal 
               
               
                 Sperm 
                 Hypomethylated 
                 chr14 
                 RTL1 
                 Imprinted 
                 Paternal 
               
               
                 Sperm 
                 Hypermethylated 
                 chr15 
                 GABRG3 
                 Conflicting 
                 Paternal 
               
               
                   
                   
                   
                   
                 Data 
                   
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr15 
                 SNRPN 
                 Imprinted 
                 Paternal 
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr16 
                 NAA60 
                 Imprinted 
                 Maternal 
               
               
                 Sperm 
                 Hypomethylated 
                 chr16 
                 SOX8 
                 Predicted 
                 Paternal 
               
               
                 Blastocyst 
                 Hypermethylated 
                 chr19 
                 DNMT1 
                 Imprinted 
                 Paternal 
               
               
                 Sperm 
                 Hypermethylated 
                 chr19 
                 LILRB4 
                 Predicted 
                 Maternal 
               
               
                 Blastocyst 
                 Hypermethylated 
                 chr19 
                 NLRP2 
                 Imprinted 
                 Maternal 
               
               
                 Blastocyst 
                 Hypermethylated 
                 chr19 
                 PEG3;ZIM2; 
                 Imprinted 
                 Paternal 
               
               
                   
                   
                   
                 MIMT1 
                   
                   
               
               
                 Sperm 
                 Hypomethylated 
                 chr19 
                 PPAP2C 
                 Predicted 
                 Maternal 
               
               
                 Blastocyst 
                 Hypermethylated 
                 chr19 
                 ZNF229 
                 Predicted 
                 Maternal 
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr20 
                 GNAS 
                 Imprinted 
                 Isoform 
               
               
                   
                   
                   
                   
                   
                 Dependent 
               
               
                 Sperm 
                 Hypomethylated 
                 chr20 
                 HM13 
                 Unknown 
                 Unknown 
               
               
                 Blastocyst 
                 Hypomethylated 
                 chr20 
                 HM13 
                 Unknown 
                 Unknown 
               
               
                   
               
            
           
         
       
     
     Conclusion 
     The above studies demonstrate for the first time the generational inheritance of epigenetic dysregulation from human sperm to the preimplantation embryo. The results demonstrate a mechanism for the paternal age effect from sperm to offspring, with confirmed significant susceptibility at neurodevelopmental genes associated with autism spectrum disorder, schizophrenia, and bipolar disorder. Furthermore, the genes identified in our study significantly overlapped those that were also differentially methylated in the frontal cortex and cingulate cortex of autistic brains (Nardone et al., 2014). An increase in global methylation was observed in the APA sperm epigenome; however, DMR analysis identified a comparable number of hypermethylated and hypomethylated regions. Surprisingly, among individuals, these methylation changes occurred with tight consistency at each CpG site of validated genes, seen in both the original and independent sperm cohorts. Additionally, linear regression analysis clearly illustrated significant progressive methylation alterations as paternal age increased. Interestingly, while considerable DNA methylation changes were identified between the young and APA sperm samples, differential expression of small RNAs was not observed (data not shown), suggesting that miRNA regulation is not an epigenetic mechanism affected by advancing paternal age. 
     There was a highly significant overlap of DMR-associated genes between the sperm and blastocyst methylomes upon paternal aging, suggesting that the same genomic regions are affected by methylation dysregulation. Methylation alterations in our datasets were not randomly distributed across the genome, but appear clustered at certain chromosomal locations. Subtelomeric regions were highly enriched for methylation alterations, particularly cytoband 19p13.3, for both sperm and blastocyst. This cytoband harbors a large number of genes (second only to cytoband 16p13.3, also significant in all three groups). Access to these genes may require a looser chromatin conformation, generating vulnerability to epigenetic alterations. Methylation in subtelomeric regions like these may escape the large-scale epigenetic reprogramming events and therefore have potential susceptibility to disruption and transmission to offspring. 
     As methylation alterations were not randomly distributed, it suggests that particular features of chromatin packaging in APA sperm are vulnerable to epigenetic errors, giving rise to aberrant reprogramming within the same regions in blastocysts. The inventors postulated that the DMRs identified in APA sperm colocalized with regions of retained histones. During spermiogenesis, the majority of histones are exchanged for protamines, but as many as 15% of nucleosomes are retained in regions of high CpG density and enriched at loci of developmental importance, including developmental gene promoters, imprinted loci, and genes encoding miRNAs (de Vries et al., 2013; Hammoud et al., 2009). By comparing the data provided above to those putatively bound to retained histones, previously identified by ChIP data from Hammoud et al., 2009, a statistically significant enrichment of APA sperm DMR-associated genes correlated with nucleosome retention. A comparable enrichment was observed for the directional overlapping DMRs identified in both sperm and blastocyst datasets. Thus, paternal germline methylation alterations induced by advancing age is transmitted in a chromatin context. In particular, hypomethylated DMRs were more likely to be associated with a gene, to be associated with CpG islands and flanking regions, and more likely to colocalize with mononucleosomes. Surprisingly, while the DMR-associated genes significantly colocalized with retained histones, only a handful of DMRs were situated near genes that encode miRNAs, with no significant impact to miRNA expression in APA sperm. 
     Both APA sperm and blastocyst DMRs exhibited significant enrichment for neurodevelopmental genes associated with autism spectrum disorder, schizophrenia, and bipolar disorder. The incidence of these neuropsychiatric conditions is known to increase progressively with increasing paternal age (de Kluiver et al., 2017), likewise DNA methylation alterations have been associated with these disorders (Rutten &amp; Mill, 2009; Wockner et al., 2014; Wong et al., 2014). Genomic imprinting is an epigenetic phenomenon that utilizes DNA methylation to restrict gene expression to only one inherited allele. Imprinted genes play a critical role in brain development and therefore may contribute to the etiologies of neurodevelopmental conditions (Crespi, 2008). Many imprinting disorders present with autistic-like characteristics, including Beckwith-Wiedemann syndrome (chr11p15.5), Angelman syndrome, and Prader-Willi syndrome (chr15q11-13) (Crespi, 2008). Given that imprinting is established in the germline and persists through offspring by escaping widespread epigenetic reprogramming, it is reasonable to consider its involvement in mediating APA effects. In fact, advanced paternal age was associated with methylation differences in brain-expressed imprinted loci in a mouse model, with concurrent behavioral changes (Smith et al., 2013). Both APA datasets demonstrated various human-imprinted genes. DLGAP2, which has been implicated in the development of autism (Nardone et al., 2014; Rasmussen, Rasmussen, &amp; Silahtaroglu, 2017; Soler et al., 2018), was significantly hypomethylated in both APA sperm and blastocyst groups. In addition, the cytoband classically responsible for Beckwith-Wiedemann syndrome (chr11p15.5) was significantly impacted in the APA sperm as well as the overlapping group of DMR associated genes. Hypomethylation at KCNQ1 was statistically significant in APA sperm and blastocysts and was validated in our APA sperm samples, in accordance with previously observed hypomethylation in aged human sperm (Jenkins et al., 2014). 
     Neurodevelopmental disorders in offspring may also manifest as epigenetic errors at genes associated with neurological development and function, including the opioid signaling pathway, in APA sperm. Opioid signaling was identified as a primary pathway affected in APA sperm and blastocysts, as well as in the combined directional overlapping DMRs, and shared by all three candidate gene lists for autism spectrum disorder, schizophrenia, and bipolar disorder. The frontal cortex in autistic brains was also significantly enriched for genes in the opioid signaling pathway and significantly overlapped our APA sperm and blastocyst DMR-associated gene lists (Nardone et al., 2014). The opioid signaling pathway in the brain is a neurotransmitter system involved in mood regulation, and abnormal brain opioid activity likely plays a role in the genesis of neurodevelopmental disorders. Increased opioid receptors are reported in autism and schizophrenia cases (Pellissier, Gandia, Laboute, Becker, &amp; Le Merrer, 2018; Volk, Radchenkova, Walker, Sengupta, &amp; Lewis, 2012), and there is a strong similarity between symptoms of opiate addiction and autistic symptoms (Kalat, 1978). Conversely, autistic-like symptoms have shown to be attenuated by opioid blocking in the severely mentally disabled (Sandman et al., 1983). These contrary reactions suggest that a delicate balance is required in the opioid signaling pathway, where either excess or deficiency may produce autistic-like symptoms (Pellissier et al., 2018). 
     CACNA1H is one gene identified in the opioid signaling pathway. It encodes CaV3.2, a T-type calcium channel abundantly expressed in the brain and implicated in neuronal function and brain development. It interacts with opioid receptors in the opioid signaling pathway to mediate analgesia (Altier &amp; Zamponi, 2008), and missense mutations were identified in individuals with autism spectrum disorder (Splawski et al., 2006). Hypomethylation at CACNA1H was previously observed in aged human sperm compared with the same individual when he was young (Jenkins et al., 2014). Not only was aberrant DMR hypomethylation confirmed for this same gene in the APA sperm methylome and validation data, DMR hypomethylation in the preimplantation blastocyst methylome was assessed and validated in embryos derived from APA fathers. Evidently some level of methylation retention is required for CACNA1H in blastocysts, which is then lost in those derived from APA fathers. Altered CaV3.2 calcium channel activity could ultimately lead to affected neuronal function and brain development in these offspring. 
     DNA methylation during spermatogenesis is susceptible to errors that can be propagated to the subsequent generation. While a large proportion of the sperm ejaculate can be analyzed for epigenetic alterations, only one methylation pattern from a single sperm utilized in fertilization has potential inheritance into the blastocyst. 
     APA is a subtle and varying effect on the human population, and likewise, neurodevelopmental disorders exist on a spectrum due to substantial ranges of symptom severity. Not every child that is born to an APA father is autistic. As such, it would be unreasonable to expect a drastic alteration, similar to a gene mutation or knockout, in sperm DNA methylation in all APA fathers or derived APA blastocysts. 
     A threshold for cumulative risk exists in terms of aberrant epigenetic alterations in sperm that, if surpassed, culminates in a predisposition to disease and ultimately an observed phenotype in offspring. The above data substantiate an increasingly compromised DNA methylation profile as sperm ages, and demonstrates a generational correlation in sperm and embryo of an altered human methylation landscape associated with APA, with significant susceptibility at genes associated with neurodevelopmental disorders.