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Paternal age and sperm methylation status
REFLECTIONS Paternal age and sperm methylation status The elaboration of knowledge regarding the human genome has simultaneously given rise to a plethora of additional areas of research interest and for which an appreciation of their influence(s) on human genesis has grown. One area, termed epigenetics, focuses on nongenomic influences on gene function and that reside outside the DNA sequence. Put simply, methylation near specific loci (CpG islands) adjacent or even distal (CpG island shores) from gene promotors regulates gene expression. In healthy individuals, CpG islands are, in general, methylation free. CpG areas in the genome not associated with CpG islands are methylated. Methylation is involved in gene silencing. The article by Jenkins et al. (1) used donor semen from healthy normal males to investigate paternal aging and associated alterations (methylation status) in epigenetic regulatory loci, for which changes in regulatory function may have a potential impact on the well-being of offspring in either adolescence or adulthood. It is becoming reasonably well-established that paternal age at offspring birth is associated with an increased risk of not only spontaneous abortion and birth defects but also neurogenic disorders such as autism (2). The causative reason(s) is/are not well-established, but certainly documented candidate suspects include increased aneuploidies, chromatin anomalies, and DNA damage, to name a few (3). Even more obscure is the question of whether there is a paternal agerelated association with epigenetic abnormalities and the subsequent well-being of offspring. Answers to this question have become all the more pressing given the availability of assisted reproductive technologies to help couples of more advanced age procreate. A recent review (3) surveyed the literature database for evidence to link epigenetic alterations and male fertility. It is interesting that abnormalities in methylation, via loss or gain on imprinted genes, histone retention, and transcript deficiencies involved in spermatogenesis appear to all be linked. The consequence(s) of this ‘‘perfect storm’’ may have an impact not only on male fertility but also embryo development. For the former, it was reported that poor-quality spermatozoa demonstrate broad epigenetic abnormalities, as reflected by hypermethylation that extends beyond expected imprinted loci (i.e., global methylation) (4). In regards to paternal influences on embryo development, a recent article (5) provided data using donor sperm that suggests modulation of different regulatory pathways depending on whether sperm are in a hypo- or hypermethylated state. The former condition (hypomethylation) is associated with regulators of cell renewal, and the latter hyper state is suggested to be a mechanism for ‘‘locking in’’ aspects of differentiation associated with the developmental regulators involved in human embryogenesis. With the emerging literature database reflecting that poorquality spermatozoa contain elevated levels of global methylation, the question arises of what the methylation profile is for spermatozoa from normal healthy sperm donors. Jenkins et al. (1) have mined a donor semen depository and analyzed global methylation alterations of regulatory intermediates: 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine 940
(5-hmC). The data show an increase in both 5-mC and 5-hmC levels with advancement in paternal age. Further, 5-mC levels were relatively stable until 55–60 years of age followed by a dramatic hypermethylation at 5-mC residues. Curiously, this seemingly biphasic alteration in methylation mirrors Figure 1A (2), which shows a similar increase in the rate of autism precisely for the paternal age range in question. Specifically, the offspring of men R55 years old have a greater than twofold likelihood for developing autism than the offspring of men %29 years old. A question that arises is whether methylation status in sperm from aged males contributes to neurogenic disorders in offspring such as autism. To validate their initial results, Jenkins et al. (1) hammered down a more scrutinizing level by applying microarray technology to assess methylation in spermatozoa from two representative donors in their donor bank. For both donors, age-associated global hypermethylation was detected, which verified their initial observations. Having validated their experimental method(s), Jenkins et al. evaluated the intraindividual variability in methylation relative to aging. For the first experimental set, spermatozoa from semen samples collected over a period of 9 to 21 years showed a high coefficient of variation (CV) relative to samples collected within 14 days of each other, 32% versus 12%, respectively. Thus, spermatozoa from semen samples collected years apart demonstrated significant variability in methylation relative to spermatozoa from semen samples collected within the same spermatogenic cycle. Paradoxically, 5-hmC showed a high CV regardless of interval duration. This latter difference is difficult to explain. In their final experimental set, Jenkins et al. (1) compared the methylation of 5-hmC from sperm versus blood DNA. Blood DNA is known to express low levels of 5-hmC relative to other tissues analyzed. Low 5-hmC levels are thought, but are not proven, to be associated with transcriptionally inactive cells. It was thus reasoned that the transcriptionally quiet spermatozoon should possess low 5-hmC levels, and indeed this was their finding. In fact, the levels bordered the lower detectable limit of the assay that was used. In summary, Jenkins et al. (1) executed rational methods to test their hypothesis of whether global methylation status changes with advancing paternal age. Their interpretation of results is well balanced and not overreaching. Results from the frontier of research most often stimulate rather than quiet questions, and the same can be said for their present work. For example, what might the outcome(s) be for intraindividual variability for 5-mC and even 5-hmC if the interval duration monitored was successive spermatogenic cycles rather than within the same spermatogenic cycle? Also, the investigators indicate that they evaluated nonselected sperm from the semen samples. Clearly not all sperm are capable of being able to transit through cervical mucus or a density gradient column and subsequently serve as the in vivo or in vitro ‘‘fertilizing spermatozoa’’ candidates. Thus, do the methylation levels and patterns differ between naturally or artificially selected and deselected sperm? The investigators acknowledge in their conclusions that ‘‘a large portion of (methylome) changes have occurred VOL. 100 NO. 4 / OCTOBER 2013
Fertility and Sterility® outside CpG islands or other regulatory regions. Thus, these changes may be occurring at . non-regulatory regions.’’ And further they posit: ‘‘DNA methylation alterations in these outlying regions may be limited in their ability to produce biologically relevant changes.’’ If so, then what might the significance of the changes be? Are they merely a reflection of an overall systemic aging process that is also evidenced in the male gamete in the form of chromosomal abnormalities, DNA fragmentation, chromatin anomalies, and so on? In pursuing the gametic changes that come with advancing paternal age, might genomic and nongenomic changes be found to culminate in or rather contribute to a reproductive ‘‘silencing’’ in the aging male, analogous to the cessation of ovarian function in women of advanced age? In this new age of delayed parenthood, which has come in part from the resource of assisted reproductive technologies, is there cause for concern as gametes from not only older women but also older men are being used for procreative purposes? Christopher J. De Jonge, Ph.D. Department of Obstetrics, Gynecology, and Women's Health, University of Minnesota, Minneapolis, Minnesota
http://fertstertforum.com/dejongec-paternal-age-spermmethylation/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
REFERENCES 1.
2.
3. 4.
http://dx.doi.org/10.1016/j.fertnstert.2013.06.024 5.
You can discuss this article with its authors and with other ASRM members at
VOL. 100 NO. 4 / OCTOBER 2013
Jenkins TG, Aston KI, Cairns BR, Carrell DT. Paternal aging and associated intra-individual alterations of global sperm 5-methylcytosine and 5-hydroxymethylcytosine levels. Fertil Steril 2013;100:945–51. Hultman CM, Sandin S, Levine SZ, Lichtenstein P, Reichenberg A. Advancing paternal age and risk of autism: new evidence from a population-based study and a meta-analysis of epidemiological studies. Mol Psychiatry 2011;16: 1203–12. Boissonnas CC, Jouannet P, Jammes H. Epigenetic disorders and male subfertility. Fertil Steril 2013;99:624–31. Houshdaran S, Cortessis VK, Siegmund K, Yang A, Laird PW, Sokol RZ. Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm. PLoS ONE 2007;2:e1289. Hammoud SS, Nix DA, Zhang H, Purwar J, Carrell DT, Cairns BR. Distinctive chromatin in human sperm packages genes for embryo development. Nature 2009;460:473–8.
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(5-hmC). The data show an increase in both 5-mC and 5-hmC levels with advancement in paternal age. Further, 5-mC levels were relatively stable until 55–60 years of age followed by a dramatic hypermethylation at 5-mC residues. Curiously, this seemingly biphasic alteration in methylation mirrors Figure 1A (2), which shows a similar increase in the rate of autism precisely for the paternal age range in question. Specifically, the offspring of men R55 years old have a greater than twofold likelihood for developing autism than the offspring of men %29 years old. A question that arises is whether methylation status in sperm from aged males contributes to neurogenic disorders in offspring such as autism. To validate their initial results, Jenkins et al. (1) hammered down a more scrutinizing level by applying microarray technology to assess methylation in spermatozoa from two representative donors in their donor bank. For both donors, age-associated global hypermethylation was detected, which verified their initial observations. Having validated their experimental method(s), Jenkins et al. evaluated the intraindividual variability in methylation relative to aging. For the first experimental set, spermatozoa from semen samples collected over a period of 9 to 21 years showed a high coefficient of variation (CV) relative to samples collected within 14 days of each other, 32% versus 12%, respectively. Thus, spermatozoa from semen samples collected years apart demonstrated significant variability in methylation relative to spermatozoa from semen samples collected within the same spermatogenic cycle. Paradoxically, 5-hmC showed a high CV regardless of interval duration. This latter difference is difficult to explain. In their final experimental set, Jenkins et al. (1) compared the methylation of 5-hmC from sperm versus blood DNA. Blood DNA is known to express low levels of 5-hmC relative to other tissues analyzed. Low 5-hmC levels are thought, but are not proven, to be associated with transcriptionally inactive cells. It was thus reasoned that the transcriptionally quiet spermatozoon should possess low 5-hmC levels, and indeed this was their finding. In fact, the levels bordered the lower detectable limit of the assay that was used. In summary, Jenkins et al. (1) executed rational methods to test their hypothesis of whether global methylation status changes with advancing paternal age. Their interpretation of results is well balanced and not overreaching. Results from the frontier of research most often stimulate rather than quiet questions, and the same can be said for their present work. For example, what might the outcome(s) be for intraindividual variability for 5-mC and even 5-hmC if the interval duration monitored was successive spermatogenic cycles rather than within the same spermatogenic cycle? Also, the investigators indicate that they evaluated nonselected sperm from the semen samples. Clearly not all sperm are capable of being able to transit through cervical mucus or a density gradient column and subsequently serve as the in vivo or in vitro ‘‘fertilizing spermatozoa’’ candidates. Thus, do the methylation levels and patterns differ between naturally or artificially selected and deselected sperm? The investigators acknowledge in their conclusions that ‘‘a large portion of (methylome) changes have occurred VOL. 100 NO. 4 / OCTOBER 2013
Fertility and Sterility® outside CpG islands or other regulatory regions. Thus, these changes may be occurring at . non-regulatory regions.’’ And further they posit: ‘‘DNA methylation alterations in these outlying regions may be limited in their ability to produce biologically relevant changes.’’ If so, then what might the significance of the changes be? Are they merely a reflection of an overall systemic aging process that is also evidenced in the male gamete in the form of chromosomal abnormalities, DNA fragmentation, chromatin anomalies, and so on? In pursuing the gametic changes that come with advancing paternal age, might genomic and nongenomic changes be found to culminate in or rather contribute to a reproductive ‘‘silencing’’ in the aging male, analogous to the cessation of ovarian function in women of advanced age? In this new age of delayed parenthood, which has come in part from the resource of assisted reproductive technologies, is there cause for concern as gametes from not only older women but also older men are being used for procreative purposes? Christopher J. De Jonge, Ph.D. Department of Obstetrics, Gynecology, and Women's Health, University of Minnesota, Minneapolis, Minnesota
http://fertstertforum.com/dejongec-paternal-age-spermmethylation/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
REFERENCES 1.
2.
3. 4.
http://dx.doi.org/10.1016/j.fertnstert.2013.06.024 5.
You can discuss this article with its authors and with other ASRM members at
VOL. 100 NO. 4 / OCTOBER 2013
Jenkins TG, Aston KI, Cairns BR, Carrell DT. Paternal aging and associated intra-individual alterations of global sperm 5-methylcytosine and 5-hydroxymethylcytosine levels. Fertil Steril 2013;100:945–51. Hultman CM, Sandin S, Levine SZ, Lichtenstein P, Reichenberg A. Advancing paternal age and risk of autism: new evidence from a population-based study and a meta-analysis of epidemiological studies. Mol Psychiatry 2011;16: 1203–12. Boissonnas CC, Jouannet P, Jammes H. Epigenetic disorders and male subfertility. Fertil Steril 2013;99:624–31. Houshdaran S, Cortessis VK, Siegmund K, Yang A, Laird PW, Sokol RZ. Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm. PLoS ONE 2007;2:e1289. Hammoud SS, Nix DA, Zhang H, Purwar J, Carrell DT, Cairns BR. Distinctive chromatin in human sperm packages genes for embryo development. Nature 2009;460:473–8.
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