Title: Age-related alterations in the genetics and genomics of the male germ line
Abstract: Paternal aging is associated with increased risk of genetic disease transmission to the offspring. The changes associated with aging arise predominantly through formation of single nucleotide variation through DNA replication errors, as well as possibly chronic exposure to environmental toxins and reactive oxygen species exposure. Several age-related reproductive factors are also contributory, including the systemic hormonal milieu, accumulation of environmental toxin exposure, aging germ cells, and accumulation of de novo genetic and genomic abnormalities in germ cells. In this article we review the age-related genetic and genomic changes that occur in the male germ line. Paternal aging is associated with increased risk of genetic disease transmission to the offspring. The changes associated with aging arise predominantly through formation of single nucleotide variation through DNA replication errors, as well as possibly chronic exposure to environmental toxins and reactive oxygen species exposure. Several age-related reproductive factors are also contributory, including the systemic hormonal milieu, accumulation of environmental toxin exposure, aging germ cells, and accumulation of de novo genetic and genomic abnormalities in germ cells. In this article we review the age-related genetic and genomic changes that occur in the male germ line. Discuss: You can discuss this article with its authors and with other ASRM members at https://www.fertstertdialog.com/users/16110-fertility-and-sterility/posts/14151-23433 Discuss: You can discuss this article with its authors and with other ASRM members at https://www.fertstertdialog.com/users/16110-fertility-and-sterility/posts/14151-23433 Over the last 6 decades, couples have delayed marriage and reproduction because of various socioeconomic factors and shifting gender roles in the work force. During this time the median age of fathers at the time of first marriage increased by 25% to the current median age of 29 years (1US Census Bureau. U.S. Census Bureau, dicennial censuses, 1890 to 1940, and current population survey, annual social and economic supplements, 1947 to 2015. Washington, DC: US Census Bureau. Available at: https://www.census.gov/hhes/families/files/graphics/MS-2.pdf. Accessed November 9, 2016.Google Scholar). Similarly, from 1980 to 2010 the number of men fathering children between the age groups of 35–39 and 40–44 years rose by 48% and 51%, respectively (2Martin J.A. Hamilton B.E. Ventura S.J. Osterman M.J. Wilson E.C. Mathews T.J. Births: final data for 2010.Natl Vital Stat Rep. 2012; 61: 1-72Google Scholar). Although the reproductive effects of advancing maternal age are well known, the effects of advancing paternal age are less well studied. However, as increasingly more babies are born to older fathers, interest in studying the implications of advancing paternal age on reproductive outcomes and offspring health has similarly risen. Multiple studies investigating the association between paternal age and reproductive outcomes concluded that increasing age is associated with impaired semen parameters; fivefold longer time to pregnancy; and reduced fertilization rates, embryo quality, implantation rates, pregnancy rate, and live-born deliveries (3Ferreira R.C. Braga D.P. Bonetti T.C. Pasqualotto F.F. Iaconelli Jr., A. Borges Jr., E. Negative influence of paternal age on clinical intracytoplasmic sperm injection cycle outcomes in oligozoospermic patients.Fertil Steril. 2010; 93: 1870-1874Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 4Aboulghar M. Mansour R. Al-Inany H. Abou-Setta A.M. Aboulghar M. Mourad L. et al.Paternal age and outcome of intracytoplasmic sperm injection.Reprod Biomed Online. 2007; 14: 588-592Abstract Full Text PDF PubMed Scopus (57) Google Scholar, 5Bellver J. Garrido N. Remohi J. Pellicer A. Meseguer M. Influence of paternal age on assisted reproduction outcome.Reprod Biomed Online. 2008; 17: 595-604Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 6Spandorfer S.D. Avrech O.M. Colombero L.T. Palermo G.D. Rosenwaks Z. Effect of parental age on fertilization and pregnancy characteristics in couples treated by intracytoplasmic sperm injection.Hum Reprod. 1998; 13: 334-338Crossref PubMed Scopus (151) Google Scholar, 7Frattarelli J.L. Miller K.A. Miller B.T. Elkind-Hirsch K. Scott Jr., R.T. Male age negatively impacts embryo development and reproductive outcome in donor oocyte assisted reproductive technology cycles.Fertil Steril. 2008; 90: 97-103Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 8Stone B.A. Alex A. Werlin L.B. Marrs R.P. Age thresholds for changes in semen parameters in men.Fertil Steril. 2013; 100: 952-958Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 9Hassan M.A. Killick S.R. Effect of male age on fertility: evidence for the decline in male fertility with increasing age.Fertil Steril. 2003; 79: 1520-1527Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Furthermore, increased paternal age is linked to a broad range of developmental abnormalities (Table 1), such as congenital birth defects and neurologic disorders, and a statistically significant increase in 5-year offspring mortality related to the severity of the congenital malformations, malignancies, and other external causes (11Lian Z.H. Zack M.M. Erickson J.D. Paternal age and the occurrence of birth defects.Am J Hum Genet. 1986; 39: 648-660PubMed Google Scholar, 12Urhoj S.K. Jespersen L.N. Nissen M. Mortensen L.H. Nybo Andersen A.M. Advanced paternal age and mortality of offspring under 5 years of age: a register-based cohort study.Hum Reprod. 2014; 29: 343-350Crossref PubMed Scopus (28) Google Scholar). Several age-related reproductive changes drive these impaired outcomes, including changes to the systemic hormonal milieu, accumulation of environmental toxin exposure, aging germ cells, and accumulation of de novo genetic and genomic abnormalities in germ cells. In this article we review the age-related genetic and genomic changes that occur in the male germ line.Table 1Offspring genetic conditions associated with advanced paternal age.ConditionPaternal age (y)Relative riskPopulation riskAdjusted riskAchondroplasia>507.81/15,0001/1,923Apert syndrome>509.51/50,0001/5,263Pfeiffer syndrome>5061/100,0001/16,666Crouzon syndrome>5081/50,0001/6,250Neurofibromatosis I>503.71/3,000–1/4,0001/810–1/1,080Retinoblastoma>4531/15,000–1/20,0001/5,000–1/6,667Down syndrome40–441.371/1,200aMaternal age 20–29 years.1/876aMaternal age 20–29 years.Klinefelter syndrome>501.61/500 men1/312 menEpilepsy40–451.31/1001/77.0Breast cancer>401.61/8.51/5.3Childhood leukemia>401.141/25,0001/21,930Childhood central nervous system tumor>401.691/36,0001/21,302Note: Adapted with permission from Ramasamy et al. 10Ramasamy R. Chiba K. Butler P. Lamb D.J. Male biological clock: a critical analysis of advanced paternal age.Fertil Steril. 2015; 103: 1402-1406Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar.a Maternal age 20–29 years. Open table in a new tab Note: Adapted with permission from Ramasamy et al. 10Ramasamy R. Chiba K. Butler P. Lamb D.J. Male biological clock: a critical analysis of advanced paternal age.Fertil Steril. 2015; 103: 1402-1406Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar. For fertilization to take place, spermatozoa require a certain amount of reactive oxygen species (ROS), which are the byproducts of oxygen metabolism and consist of reduced oxygen molecules with chemically reactive unpaired electrons, to undergo biologic functions such as capacitation, hyperactivation, acrosome reaction, and oocyte fusion (13de Lamirande E. Gagnon C. A positive role for the superoxide anion in triggering hyperactivation and capacitation of human spermatozoa.Int J Androl. 1993; 16: 21-25Crossref PubMed Scopus (253) Google Scholar). In addition, ROS modulate nuclear maturation and facilitate nuclear condensation in spermatozoa by oxidizing nuclear proteins (14Aitken R.J. Vernet P. Maturation of redox regulatory mechanisms in the epididymis.J Reprod Fertil Suppl. 1998; 53: 109-118PubMed Google Scholar). Although a certain amount of ROS is necessary for normal sperm function, excess ROS can place a detrimental oxidative stress on spermatogenesis and fertilization via damage to sperm DNA and proteins and are associated with poor semen quality and function. Studies comparing ROS levels of healthy fertile men aged <40 years vs. those aged >40 years found significantly lower ROS levels in the seminal ejaculate (15Cocuzza M. Athayde K.S. Agarwal A. Sharma R. Pagani R. Lucon A.M. et al.Age-related increase of reactive oxygen species in neat semen in healthy fertile men.Urology. 2008; 71: 490-494Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Reactive oxygen species harm sperm by entering the nucleus, binding to DNA, and inducing double-strand breaks (16Singh N.P. Muller C.H. Berger R.E. Effects of age on DNA double-strand breaks and apoptosis in human sperm.Fertil Steril. 2003; 80: 1420-1430Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). The relationship between age and DNA fragmentation is well established, with higher levels of DNA damage more often found in older men. Even when matched comparisons are made between young and old subjects who are normozoospermic, a statistically significant correlation (P<.001) remains between age and percent sperm DNA fragmentation (17Das M. Al-Hathal N. San-Gabriel M. Phillips S. Kadoch I.J. Bissonnette F. et al.High prevalence of isolated sperm DNA damage in infertile men with advanced paternal age.J Assist Reprod Genet. 2013; 30: 843-848Crossref PubMed Scopus (56) Google Scholar). Using the sperm chromatin structure assay, Das et al. (17Das M. Al-Hathal N. San-Gabriel M. Phillips S. Kadoch I.J. Bissonnette F. et al.High prevalence of isolated sperm DNA damage in infertile men with advanced paternal age.J Assist Reprod Genet. 2013; 30: 843-848Crossref PubMed Scopus (56) Google Scholar) compared 107 normozoospermic young men (<40 years of age) with 41 normozoospermic older men (≥41 years of age) and found higher DNA damage levels in the older cohort (17% ± 13% vs. 12% ± 18%). Moskovtsev et al. (18Moskovtsev S.I. Willis J. Mullen J.B. Age-related decline in sperm deoxyribonucleic acid integrity in patients evaluated for male infertility.Fertil Steril. 2006; 85: 496-499Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) also used sperm chromatin structure assay to compare DNA fragmentation values in a cohort of men ≥45 years of age with a cohort of men <30 years of age and found that damage levels were twice as high in the ≥45 years cohort (32.0% ± 17.1% vs. 15.2% ± 8.4%). These findings were corroborated by a large meta-analysis of 10,220 patients by Johnson et al. (19Johnson L. Evaluation of the human testis and its age-related dysfunction.Prog Clin Biol Res. 1989; 302 (discussion 1–7): 35-60PubMed Google Scholar), who identified a statistically significant age-dependent increase in DNA fragmentation. Although the mechanism for elevated DNA fragmentation in older men remains unclear, several factors may contribute, including a higher incidence and prevalence of varicoceles (the incidence of varicoceles increases by 10% with each decade of life [20Levinger U. Gornish M. Gat Y. Bachar G.N. Is varicocele prevalence increasing with age?.Andrologia. 2007; 39: 77-80Crossref PubMed Scopus (82) Google Scholar]), environmental exposure to pollutants, and comorbidities such as obesity, diabetes, infections, and other lifestyle issues (reviewed by Sabeti et al. [21Sabeti P. Pourmasumi S. Rahiminia T. Akyash F. Talebi A.R. Etiologies of sperm oxidative stress.Int J Reprod Biomed (Yazd). 2016; 14: 231-240Google Scholar]). Coupled with age-related reduction in antioxidant enzymatic activity (reviewed by Aitken and De Iullis [22Aitken R.J. De Iuliis G.N. On the possible origins of DNA damage in human spermatozoa.Mol Hum Reprod. 2010; 16: 3-13Crossref PubMed Scopus (373) Google Scholar]), higher ROS levels in the semen of older men increase the oxidative stress, resulting in impaired sperm DNA integrity via creation of double-strand DNA breaks, mutations of genomic and mitochondrial DNA, perturbation of DNA repair enzymes, and the accumulation of single nucleotide variants with each mitotic and meiotic replication (Fig. 1). Defective DNA repair in turn may result in greater numerical and structural chromosomal abnormalities, increasing the risk of aneuploidy by twofold among fathers aged >50 years compared with fathers aged 25–29 years in one study by McIntosh et al. (23McIntosh G.C. Olshan A.F. Baird P.A. Paternal age and the risk of birth defects in offspring.Epidemiology. 1995; 6: 282-288Crossref PubMed Scopus (140) Google Scholar). This oxidative stress–mediated DNA damage has been linked with IVF/intracytoplasmic sperm injection failure and abnormal offspring development, particularly with learning disorders and impaired cognition (24Shamsi M.B. Kumar R. Dada R. Evaluation of nuclear DNA damage in human spermatozoa in men opting for assisted reproduction.Indian J Med Res. 2008; 127: 115-123PubMed Google Scholar, 25Reichenberg A. Gross R. Weiser M. Bresnahan M. Silverman J. Harlap S. et al.Advancing paternal age and autism.Arch Gen Psychiatry. 2006; 63: 1026-1032Crossref PubMed Scopus (464) Google Scholar, 26Liebenberg R. van Heerden B. Ehlers R. Du Plessis A.M. Roos J.L. Advancing paternal age at birth is associated with poorer social functioning earlier and later in life of schizophrenia patients in a founder population.Psychiatry Res. 2016; 243: 185-190Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar), although some studies were not performed with the rigor required for evidenced-based medicine. Genomic instability is characterized as a higher frequency of spontaneous genetic and genomic mutations. Genomic instability associated with aging is a multifactorial, complex process that represents the cumulative effects of chronic ROS exposure, telomere shortening, and DNA replication errors, inducing a vicious cycle of chromosome recombination errors, reduced DNA repair efficiency, and further de novo mutations (Fig. 2). Recombination errors can result in chromosomally balanced and unbalanced gametes that ultimately result in aneuploid and often nonviable offspring. Studies assessing the type of chromosomal aneuploidy associated with paternal aging have reported mixed relative risk results for chromosomes 13, 18, 21, and X (reviewed by Sharma et al. [27Sharma R. Agarwal A. Rohra V.K. Assidi M. Abu-Elmagd M. Turki R.F. Effects of increased paternal age on sperm quality, reproductive outcome and associated epigenetic risks to offspring.Reprod Biol Endocrinol. 2015; 13: 35Crossref PubMed Scopus (188) Google Scholar] and Sloter et al. [28Sloter E. Nath J. Eskenazi B. Wyrobek A.J. Effects of male age on the frequencies of germinal and heritable chromosomal abnormalities in humans and rodents.Fertil Steril. 2004; 81: 925-943Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar]). However, a statistically significant positive association between centromeric deletions of chromosome 1 and paternal age was reported by McInnes et al. (29McInnes B. Rademaker A. Martin R. Donor age and the frequency of disomy for chromosomes 1, 13, 21 and structural abnormalities in human spermatozoa using multicolour fluorescence in-situ hybridization.Hum Reprod. 1998; 13: 2489-2494Crossref PubMed Scopus (53) Google Scholar), who performed fluorescent in situ hybridization (FISH) on sperm from 18 men belonging to one of six age groups (20–24, 25–29, 30–34, 35–39, 40–44, and ≥50 years) using probes for chromosomes 13, 21, 1 and a unique probe to detect duplications and deletions of 1p. Historically, reports from the 1950s suggested an association between Down syndrome and advanced paternal age; however, several studies have since emerged, refuting this association (28Sloter E. Nath J. Eskenazi B. Wyrobek A.J. Effects of male age on the frequencies of germinal and heritable chromosomal abnormalities in humans and rodents.Fertil Steril. 2004; 81: 925-943Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Garcia-Ferreyra et al. (30Garcia-Ferreyra J. Luna D. Villegas L. Romero R. Zavala P. Hilario R. et al.High aneuploidy rates observed in embryos derived from donated oocytes are related to male aging and high percentages of sperm DNA fragmentation.Clin Med Insights Reprod Health. 2015; 9: 21-27Crossref PubMed Scopus (27) Google Scholar) recently showed a significantly higher embryo aneuploidy rate associated with fathers aged ≥50 years (73.9% aneuploidy) compared with fathers aged 40–49 (61.1% aneuploidy) and ≤39 years (59.1% aneuploidy). Interestingly, no statistically significant difference was detected in fertilization rates, percentages of zygotes that underwent cleavage, and the quality of embryos at day 3. Although the authors reported that all subjects had normal karyotype, additional cytogenetic testing (such as FISH) was not performed before intracytoplasmic sperm injection, and embryo FISH results were not provided to determine which chromosome(s) was most often affected. The effects of age on chromatin integrity, gene mutations, and aneuploidy in a group of 97 men with an age range of 22–80 years were assessed by Wyrobek et al. (31Wyrobek A.J. Eskenazi B. Young S. Arnheim N. Tiemann-Boege I. Jabs E.W. et al.Advancing age has differential effects on DNA damage, chromatin integrity, gene mutations, and aneuploidies in sperm.Proc Natl Acad Sci U S A. 2006; 103: 9601-9606Crossref PubMed Scopus (309) Google Scholar), who showed no association between X-Y-21 aneuploid sperm per 10,000 sperm vs. age. Because most sperm aneuploidy arises from nondisjunction during meiosis I and meiosis II, the lack of an association between sperm aneuploidy and paternal age but higher rate of embryo aneuploidy implies an error in sperm-related centrosomal function during the embryo cleavage stage. Further studies are necessary to determine the mechanism of aneuploidy transmission from sperm to embryo in men with advanced paternal age. Advanced father's age is also associated with increased incidence of clinical disorders in offspring, ranging from congenital malformations to neurodegenerative disorders. Similarly, paternal age is associated with an increased incidence of autosomal dominant disorders such as Apert Syndrome, achrondroplasia, osteogenesis imperfecta, progeria, Marfan syndrome, and Waardenburg syndrome. These disorders are caused by single nucleotide variants, which are more likely to arise in male gametes rather than female gametes owing to the continuing mitotic divisions of spermatogonial stem cells throughout a male's adult life, which create more opportunities for random mutation and mismatch repair failure. In a study of 78 parent–child trios in Iceland, Kong et al. (32Kong A. Frigge M.L. Masson G. Besenbacher S. Sulem P. Magnusson G. et al.Rate of de novo mutations and the importance of father's age to disease risk.Nature. 2012; 488: 471-475Crossref PubMed Scopus (1272) Google Scholar) found that the rate of de novo mutation strongly correlated with paternal age and generated a model predicting that paternal germ-line mutations double every 16.5 years. Francioli et al. (33Francioli L.C. Polak P.P. Koren A. Menelaou A. Chun S. Renkens I. et al.Genome-wide patterns and properties of de novo mutations in humans.Nat Genet. 2015; 47: 822-826Crossref PubMed Scopus (219) Google Scholar) analyzed 11,020 de novo mutations from whole genomes of 250 families and found after accounting for random Poisson distribution that 95% of variation in the global human mutation rate could be attributed to paternal age. They confirmed Kong et al.'s findings that de novo mutations are more common in the children of older fathers and found that, relative to 20-year-old fathers, 40 year olds had twice the rate of mutation and that those mutations were more likely to have functional consequences. A number of single gene mutations are enriched in the setting of advanced paternal age. The fibroblast growth factor receptor 3 (FGFR3) gene codes for a member of the fibroblast growth factor receptor family and is responsible for initiating cell signaling cascades that ultimately result in mitogenesis and differentiation. Mutations of this gene cause skeletal malformations, such as craniosynostosis and achondroplasia (1138G>A). Achondroplasia is inherited in an autosomal dominant pattern and leads among the causes of dwarfism (reviewed by Ramasamy et al. [10Ramasamy R. Chiba K. Butler P. Lamb D.J. Male biological clock: a critical analysis of advanced paternal age.Fertil Steril. 2015; 103: 1402-1406Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar]). An increased risk of achondroplasia associated with advanced paternal age, however, has long been described, with original reports by Penrose in 1955 (34Penrose L.S. Parental age and mutation.Lancet. 1955; 269: 312-313Abstract PubMed Scopus (208) Google Scholar). Corroborating data were provided by Wyrobek et al. (31Wyrobek A.J. Eskenazi B. Young S. Arnheim N. Tiemann-Boege I. Jabs E.W. et al.Advancing age has differential effects on DNA damage, chromatin integrity, gene mutations, and aneuploidies in sperm.Proc Natl Acad Sci U S A. 2006; 103: 9601-9606Crossref PubMed Scopus (309) Google Scholar), who reported a statistically significant and positive correlation between age and the incidence of FGFR3 mutation. The decade-specific incidence of achondroplasia causing FGFR3 mutation increased from 0.55 per 10,000 genomes in subjects aged 20–29 years to 1.85 for men over the age of 60 years or a 3.3% increase per year in the frequency of the mutation. Mutations of other FGFR family members, such as FGFR2, have also been associated with advanced paternal age (35Wilkie A.O. Slaney S.F. Oldridge M. Poole M.D. Ashworth G.J. Hockley A.D. et al.Apert syndrome results from localized mutations of FGFR2 and is allelic with Crouzon syndrome.Nat Genet. 1995; 9: 165-172Crossref PubMed Scopus (740) Google Scholar). Mutations of FGFR2 result in Apert syndrome, which is characterized by acrocephalosyndactyly. Alternate routes exist that allow a father's age to influence offspring. Epigenetics refers to the stable and heritable mechanism of gene expression regulation that does not involve DNA sequence. The major groups of epigenetic changes are DNA methylation, histone modifications, and microRNA expression. An individual can acquire many epigenetic changes throughout his or her life, depending on external stimuli. Although these changes can be stably propagated in somatic cells and regulate cell fate, they do not alter the genetic code. During normal embryonic development the cells undergo a reset of the epigenetic marks, to allow for proper cell fate differentiation (reviewed in references [36Messerschmidt D.M. Knowles B.B. Solter D. DNA methylation dynamics during epigenetic reprogramming in the germline and preimplantation embryos.Genes Dev. 2014; 28: 812-828Crossref PubMed Scopus (437) Google Scholar, 37Zheng H. Huang B. Zhang B. Xiang Y. Du Z. Xu Q. et al.Resetting epigenetic memory by reprogramming of histone modifications in mammals.Mol Cell. 2016; 63: 1066-1079Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar]). Because of these events, it was believed that epigenetic mutations could not be passed down through the germ line. However, this view is being challenged by increasing evidence linking maternal epigenetic states to effects on progeny (38Mitchell E. Klein S.L. Argyropoulos K.V. Sharma A. Chan R.B. Toth J.G. et al.Behavioural traits propagate across generations via segregated iterative-somatic and gametic epigenetic mechanisms.Nat Commun. 2016; 7: 11492Crossref PubMed Scopus (22) Google Scholar). Of particular interest are the analyses linking advanced age to an increased likelihood of diseases such as autism spectrum disorder, schizophrenia, or Down syndrome in the progeny (39Feinberg J.I. Bakulski K.M. Jaffe A.E. Tryggvadottir R. Brown S.C. Goldman L.R. et al.Paternal sperm DNA methylation associated with early signs of autism risk in an autism-enriched cohort.Int J Epidemiol. 2015; 44: 1199-1210Crossref PubMed Scopus (86) Google Scholar). These diseases are associated with epigenetic changes, and it is important to investigate whether the paternal epigenetic state affected these children. There is increasing literature suggesting that some marks can be passed down through maternal nongenetic means to the new generation (38Mitchell E. Klein S.L. Argyropoulos K.V. Sharma A. Chan R.B. Toth J.G. et al.Behavioural traits propagate across generations via segregated iterative-somatic and gametic epigenetic mechanisms.Nat Commun. 2016; 7: 11492Crossref PubMed Scopus (22) Google Scholar); however, a precise mechanism has not been established, and the role of the paternal epigenetic state needs further investigation. Several studies have associated advanced paternal age with a greater burden of DNA methylation changes. Oakes et al. (40Oakes C.C. Smiraglia D.J. Plass C. Trasler J.M. Robaire B. Aging results in hypermethylation of ribosomal DNA in sperm and liver of male rats.Proc Natl Acad Sci U S A. 2003; 100: 1775-1780Crossref PubMed Scopus (148) Google Scholar) showed increased ribosomal DNA methylation in the sperm of older rats, suggesting a mechanism for age-related DNA methylation changes. Restriction landmark genomic scanning, a method used to determine specific methylation patterns of CpG island sequences, allowed Oakes et al. to find a region of the ribosomal DNA locus that is preferentially hypermethylated with age in both spermatozoa and liver. These findings suggest that the maintenance of normal DNA methylation levels in spermatozoa over the course of a lifetime changes and may explain age-related abnormalities in the offspring (40Oakes C.C. Smiraglia D.J. Plass C. Trasler J.M. Robaire B. Aging results in hypermethylation of ribosomal DNA in sperm and liver of male rats.Proc Natl Acad Sci U S A. 2003; 100: 1775-1780Crossref PubMed Scopus (148) Google Scholar). Another study, by Jenkins et al. (41Jenkins T.G. Aston K.I. Cairns B.R. Carrell D.T. Paternal aging and associated intraindividual alterations of global sperm 5-methylcytosine and 5-hydroxymethylcytosine levels.Fertil Steril. 2013; 100: 945-951Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), evaluated the global methylation levels in sperm of fertile men. They discovered that there is a statistically significant increase of global sperm 5-mC and 5-hmC levels by 1.76% and 5%, respectively, per year. These findings underscore the importance of epigenetic aberrations and their contribution to the offspring risks associated with advanced paternal age (reviewed in reference [10Ramasamy R. Chiba K. Butler P. Lamb D.J. Male biological clock: a critical analysis of advanced paternal age.Fertil Steril. 2015; 103: 1402-1406Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar]). Although the evidence arguing that epigenetic changes occur with age and that they are associated with poor outcomes in the progeny is increasing, the factors that lead to transgenerational epigenetic inheritance need further investigation. One such factor is diet. A study in mice by Terashima et al. (42Terashima M. Barbour S. Ren J. Yu W. Han Y. Muegge K. Effect of high fat diet on paternal sperm histone distribution and male offspring liver gene expression.Epigenetics. 2015; 10: 861-871Crossref PubMed Scopus (72) Google Scholar) found that hepatic messenger RNA levels in seven imprinted genes were significantly altered in the offspring of male mice given a high-fat diet (HFD). Although they did not detect DNA methylation changes in the fathers' spermatozoa, they found differential histone H3 occupancy at genes involved in the regulation of embryogenesis and differential H3K4me1 enrichment at transcription regulatory genes in HFD fathers. Another study, by Barbosa et al. (43de Castro Barbosa T. Ingerslev L.R. Alm P.S. Versteyhe S. Massart J. Rasmussen M. et al.High-fat diet reprograms the epigenome of rat spermatozoa and transgenerationally affects metabolism of the offspring.Mol Metab. 2016; 5: 184-197Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), evaluated HFD effects on rat offspring. They found altered expression of the microRNA let-7c in the sperm of F0 rats and their F1 offspring, thus showing a transgenerational epigenetic inheritance that alters the metabolic tissues in the offspring. An earlier study by Anderson et al. (44Anderson L.M. Riffle L. Wilson R. Travlos G.S. Lubomirski M.S. Alvord W.G. Preconceptional fasting of fathers alters serum glucose in offspring of mice.Nutrition. 2006; 22: 327-331Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) suggested that paternal food deprivation results in a consistent decrease in average serum glucose in male and female offspring in mice, which was also accompanied by significant changes in corticosterone and insulin-like growth factor-1. Human studies also suggest a link between parental obesity and epigenetic changes in the offspring. Soubry et al. (45Soubry A. Schildkraut J.M. Murtha A. Wang F. Huang Z. Bernal A. et al.Paternal obesity is associated with IGF2 hypomethylation in newborns: results from a Newborn Epigenetics Study (NEST) cohort.BMC Med. 2013; 11: 29Crossref PubMed Scopus (232) Google Scholar) showed in 79 newborns that differential changes in DNA methylation in multiple human imprinted genes (such as IGF2) associate with both paternal and maternal obesity. A father's obesity was significantly correlated with hypomethylation of the differentially methylated regions of the IGF2 gene. Another study, by Pembrey et al. (46Pembrey M.E. Bygren L.O. Kaati G. Edvinsson S. Northstone K. Sjostrom M. et al.Sex-specific, male-line transgenerational responses in humans.Eur J Hum Genet. 2006; 14: 159-166Crossref PubMed Scopus (847) Google Scholar), investigated paternal ancestors' food supply and effects of smoking on the progeny's health. They found that the paternal grandfather's food supply was linked to the mortality risk ratios only of the grandsons, suggesting a sex-linked inheritance of these traits. All of these studies provide compelling evidence that the paternal contribution to the development of the progeny goes beyond the DNA sequence. The influence of a father's poor nutrition habits, such as overeating, may extend across multiple generations. A large-scale epidemiologic study by Kaati et al. (47Kaati G. Bygren L.O. Edvinsson S. Cardiovascular and diabetes mortality determined by nutrition during parents' and grandparents' slow growth period.Eur J Hum Genet. 2002; 10: 682-688Crossref PubMed Scopus (606) Google Scholar) showed the transgenerational metabolic effects of a father's overfeeding, with elevated cardiovascular disease risk and diabetes in his grandchildren. Further research is needed to elucidate how the paternal epigenetic state is transferred to the progeny, what factors affect the epigenetic state, and why some marks are inherited whereas others are not. Most importantly, researchers need to address the question of what external factors, such as parental age and lifestyle choices, affect future generations the most. In conclusion, paternal aging is associated with increased risk of genetic disease transmission to the offspring. The changes associated with aging arise predominantly through DNA replication errors, as well as possibly chronic exposure to environmental exposure to toxins and ROS exposure. More importantly, age-related reproductive problems are an inevitable complication of an aging population. Recognition and thorough understanding of male reproductive disorders associated with aging will continue to gain in importance because of the medical and societal burden of developmental disorders in offspring. Guidelines on proper evaluation and clinical counseling are lacking.