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An obituary for periodical editor E. Thayer Gaston is presented.

Expression of the double homeobox 4 ( DUX4 ) transcription factor is highly regulated in early embryogenesis and is subsequently epigenetically silenced. Ectopic expression of DUX4 due to hypomethylation of the D4Z4 repeat array on permissive chromosome 4q35 alleles is associated with facioscapulohumeral muscular dystrophy (FSHD). In peripheral blood samples from 188 healthy individuals, D4Z4 methylation was highly variable, ranging from 19% to 76%, and was not affected by age. In 48 FSHD2 patients, D4Z4 methylation varied from 3% to 30%. Given that DUX4 is one of the earliest transcribed genes after fertilization, the D4Z4 array is expected to be unmethylated in mature germ cells. Deep bisulfite sequencing of 188 mainly normozoospermic sperm samples revealed an average methylation of 2.5% (range 0.3-22%). Overall, the vast majority (78%) of individual sperm cells displayed no methylation at all. In contrast, only 19 (17.5%) of 109 individual germinal vesicle (GV) oocytes displayed D4Z4 methylation <2.5%. However, it is not unexpected that immature GV oocytes which are not usable for assisted reproduction are endowed with D4Z4 (up to 74%) hypermethylation and/or abnormal ( PEG3 and GTL2 ) imprints. Although not significant, it is interesting to note that the pregnancy rate after assisted reproduction was higher for donors of sperm samples and oocytes with <2.5% methylation.

Children from old fathers carry an increased risk for autism spectrum (ASD) and other neurodevelopmental disorders, which may at least partially be mediated by paternal age effects on the sperm epigenome. The brain enriched guanylate kinase associated (BEGAIN) protein is involved in protein-protein interactions at and transmission across synapses. Since several epigenome-wide methylation screens reported a paternal age effect on sperm BEGAIN methylation, here we confirmed a significant negative correlation between BEGAIN promoter methylation and paternal age, using more sensitive bisulfite pyrosequencing and a larger number of sperm samples. Paternal age-associated BEGAIN hypomethylation was also observed in fetal cord blood (FCB) of male but not of female offspring. There was no comparable maternal age effect on FCB methylation. In addition, we found a significant negative correlation between BEGAIN methylation and chronological age (ranging from 1 to 70 years) in peripheral blood samples of male but not of female donors. BEGAIN hypomethylation was more pronounced in male children, adolescents and adults suffering from ASD compared to controls. Both genetic variation (CC genotype of SNP rs7141087) and epigenetic factors may contribute to BEGAIN promoter hypomethylation. The age- and sex-specific BEGAIN methylation trajectories in the male germ line and somatic tissues, in particular the brain, support a role of this gene in ASD development.

Objective: Axial neck pain is a prevalent condition that causes significant morbidity and productivity loss. This study aimed to review the current literature and define the impact of surgical intervention on the management of cervical axial neck pain., Methods: A search was conducted of three databases (Ovid MEDLINE, Embase, and Cochrane) for randomized controlled trials and cohort studies written in the English language with a minimum 6-month follow-up. The analysis was limited to patients with axial neck pain/cervical radiculopathy and preoperative/postoperative Neck Disability Index (NDI) and visual analog scale (VAS) scores. Literature reviews, meta-analyses, systematic reviews, surveys, and case studies were excluded. Two patient groups were analyzed: the arm pain predominant (pAP) cohort and the neck pain predominant (pNP) cohort. The pAP cohort had preoperative VAS neck scores that were lower than the arm scores, whereas the pNP cohort was defined as having preoperative VAS neck scores higher than the arm scores. A 30% reduction in patient-reported outcome measure (PROM) scores from the baseline represented the minimal clinically important difference (MCID)., Results: Five studies met the inclusion criteria, involving a total of 5221 patients. Patients with pAP showed a slightly higher percent reduction in PROM scores from baseline than those with pNP. The NDI reduction in patients with pNP was 41.35% (mean change in NDI score 16.3/mean baseline NDI score 39.42) (p < 0.0001), whereas those with pAP had a reduction of 45.12% (15.86/35.15) (p < 0.0001). Surgical improvement was slightly but similarly greater in pNP patients compared with pAP patients (16.3 vs 15.86 points, respectively; p = 0.3193). Regarding VAS scores, patients with pNP had a greater reduction in neck pain, with a change from baseline of 53.4% (3.60/6.74, p < 0.0001), whereas those with pAP had a change from baseline of 50.3% (2.46/4.89, p < 0.0001). The difference in VAS scores for neck pain improvement was significant (3.6 vs 2.46, p < 0.0134). Similarly, patients with pNP had a 43.6% (1.96/4.5) improvement in VAS scores for arm pain (p < 0.0001), whereas those with pAP had 66.12% (4.43/6.7) improvement (p < 0.0001). The VAS scores for arm pain were significantly greater in patients with pAP (4.43 vs 1.96 points, respectively; p < 0.0051)., Conclusions: Overall, despite significant variations in the existing literature, there is mounting evidence that surgical intervention can lead to clinically meaningful improvements in patients with primary axial neck pain. The studies suggest that patients with pNP tend to have better improvement in neck pain than in arm pain. In both groups, the average improvements exceeded the MCID values and reached substantial clinical benefit in all studies. Further research is necessary to identify which patients and underlying pathologies will benefit most from surgical intervention for axial neck pain because it is a multifaceted condition with many causes.

This study addresses transpiration in a tropical evergreen mountain forest in the Ecuadorian Andes from the leaf to the stand level, with emphasis on nocturnal plant-water relations. The stand level: Evapotranspiration (ET) measured over 12 months with the Eddy-Covariance (ECov) technique proved as the major share (79%) of water received from precipitation. Irrespective of the humid climate, the vegetation transpired day and night. On average, 15.3% of the total daily ET were due to nocturnal transpiration. Short spells of drought increased daily ET, mainly by enhanced nighttime transpiration. Following leaf transpiration rather than air temperature and atmospheric water vapor deficit, ET showed its maximum already in the morning hours. The tree level: Due to the humid climate, the total water consumption of trees was generally low. Nevertheless, xylem sap flux measurements separated the investigated tree species into a group showing relatively high and another one with low sap flux rates. The leaf level: Transpiration rates of Tapirira guianensis, a member of the high-flux-rate group, were more than twice those of Ocotea aciphylla, a representative of the group showing low sap flux rates. Representatives of the Tapirira group operated at a relatively high leaf water potential but with a considerable diurnal amplitude, while the leaves of the Ocotea group showed low water potential and small diurnal fluctuations. Overall, the Tapirira group performed anisohydrically and the Ocotea group isohydrically. Grouping of the tree species by their water relations complied with the extents of the diurnal stem circumference fluctuations. Nighttime transpiration and hydrological type: In contrast to the isohydrically performing trees of the Ocotea group, the anisohydric trees showed considerable water vapour pressure deficit (VPD)-dependent nocturnal transpiration. Therefore, we conclude that nighttime ET at the forest level is mainly sourced by the tree species with anisohydric performance., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Raffelsbauer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Advanced paternal age is associated with increased risks for reproductive and offspring medical problems. Accumulating evidence suggests age-related changes in the sperm epigenome as one underlying mechanism. Using reduced representation bisulfite sequencing on 73 sperm samples of males attending a fertility center, we identified 1,162 (74%) regions which were significantly (FDR-adjusted) hypomethylated and 403 regions (26%) being hypermethylated with age. There were no significant correlations with paternal BMI, semen quality, or ART outcome. The majority (1,152 of 1,565; 74%) of age-related differentially methylated regions (ageDMRs) were located within genic regions, including 1,002 genes with symbols. Hypomethylated ageDMRs were closer to transcription start sites than hypermethylated DMRs, half of which reside in gene-distal regions. In this and conceptually related genome-wide studies, so far 2,355 genes have been reported with significant sperm ageDMRs, however most (90%) of them in only one study. The 241 genes which have been replicated at least once showed significant functional enrichments in 41 biological processes associated with development and the nervous system and in 10 cellular components associated with synapses and neurons. This supports the hypothesis that paternal age effects on the sperm methylome affect offspring behaviour and neurodevelopment. It is interesting to note that sperm ageDMRs were not randomly distributed throughout the human genome; chromosome 19 showed a highly significant twofold enrichment with sperm ageDMRs. Although the high gene density and CpG content have been conserved, the orthologous marmoset chromosome 22 did not appear to exhibit an increased regulatory potential by age-related DNA methylation changes.

A growing number of sperm methylome analyses have identified genomic loci that are susceptible to paternal age effects in a variety of mammalian species, including human, bovine, and mouse. However, there is little overlap between different data sets. Here, we studied whether or not paternal age effects on the sperm epigenome have been conserved in mammalian evolution and compared methylation patterns of orthologous regulatory regions (mainly gene promoters) containing both conserved and non-conserved CpG sites in 94 human, 36 bovine, and 94 mouse sperm samples, using bisulfite pyrosequencing. We discovered three ( NFKB2 , RASGEF1C , and RPL6 ) age-related differentially methylated regions (ageDMRs) in humans, four ( CHD7 , HDAC11 , PAK1 , and PTK2B ) in bovines, and three ( Def6 , Nrxn2 , and Tbx19 ) in mice. Remarkably, the identified sperm ageDMRs were all species-specific. Most ageDMRs were in genomic regions with medium methylation levels and large methylation variation. Orthologous regions in species not showing this age effect were either hypermethylated (>80%) or hypomethylated (<20%). In humans and mice, ageDMRs lost methylation, whereas bovine ageDMRs gained methylation with age. Our results are in line with the hypothesis that sperm ageDMRs are in regions under epigenomic evolution and may be part of an epigenetic mechanism(s) for lineage-specific environmental adaptations and provide a solid basis for studies on downstream effects in the genes analyzed here.

In somatic cells/tissues, methylation of ribosomal DNA (rDNA) increases with age and age-related pathologies, which has a direct impact on the regulation of nucleolar activity and cellular metabolism. Here, we used bisulfite pyrosequencing and show that methylation of the rDNA transcription unit including upstream control element (UCE), core promoter, 18S rDNA, and 28S rDNA in human sperm also significantly increases with donor's age. This positive correlation between sperm rDNA methylation and biological age is evolutionarily conserved among mammals with widely different life spans such as humans, marmoset, bovine, and mouse. Similar to the tandemly repeated rDNA, methylation of human α-satellite and interspersed LINE1 repeats, marmoset α-satellite, bovine alpha- and testis satellite I, mouse minor and major satellite, and LINE1-T repeats increases in the aging male germline, probably related to their sperm histone packaging. Deep bisulfite sequencing of single rDNA molecules in human sperm revealed that methylation does not only depend on donor's age, but also depend on the region and sequence context (A vs. G alleles). Both average rDNA methylation of all analyzed DNA molecules and the number of fully (>50%) methylated alleles, which are thought to be epigenetically silenced, increase with donor's age. All analyzed CpGs in the sperm rDNA transcription unit show comparable age-related methylation changes. Unlike other epigenetic aging markers, the rDNA clock appears to operate in similar ways in germline and soma in different mammalian species. We propose that sperm rDNA methylation, directly or indirectly, influences nucleolar formation and developmental potential in the early embryo., (© 2020 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

In the vineyards of Rhineland-Palatinate (Germany), two different types of Shepherd's Purse ( Capsella bursa-pastoris ) coexist: (1) the common type called 'wild type', and (2) the decandric type called Capsella apetala or ' Spe' with four stamens in place of the four petals. In this study, we compare the anatomical and physiological characters of rosette leaves of the respective types. Progeny of individual plants was cultivated in growth chambers under low- and high-light conditions. Under low-light conditions, the stomata densities of the adaxial and abaxial epidermis did not differ between the two types. When grown under high-light conditions, wild type and Spe , both exhibited increased stomata densities compared to low-light conditions, but Spe to a lesser extent than the wild type. The maximal photosynthetic capacity of Spe was lower in both, low-light and high-light conditions compared to wild-type plants. Under all CO 2 concentrations, Spe seemed to be less productive. The less effective CO 2 assimilation of the Spe mutant C. apetala was accompanied by later flowering . This fact prolonged the vegetative phase of Spe by about two weeks and was sufficient for the maintenance of both populations stably over years., Competing Interests: Renate Scheibe is an Academic Editor for PeerJ., (©2020 Neuffer et al.)

The prevalence of metabolic disorders, in particular obesity has dramatically increased worldwide. Genetic variants explain only a minor part of the obesity epidemic induced by physical inactivity and over-nutrition. Epidemiological studies in humans and animal models indicate that epigenetic changes associated with adverse parental and/or intrauterine factors may contribute to the missing heritability of metabolic disorders. Possible adverse paternal effects are likely transmitted by sperm to the next-generation. To investigate this hypothesis, we have systematically analyzed the effects of male body mass index (BMI) on sperm epigenome and its association with next-generation fetal cord blood (FCB) DNA methylation. Methylation levels of maternally imprinted (PEG1, PEG4, PEG5, and PEG10), paternally imprinted (H19-IG DMR, IGF2-DMR0, and MEG3-IG DMR) regions, and obesity-related non-imprinted HIF3A gene were quantified by bisulphite pyrosequencing in sperm samples of 294 human donors undergoing in vitro fertilization or intracytoplasmic sperm injection, and in 113 FCBs of the resulting offspring. Multivariable regression analyses revealed that MEG3 intergenic differentially methylated region (IG DMR) showed positive correlation between sperm methylation and donor's BMI. A gender-specific correlation between paternal BMI and FCB methylation was observed for MEG3-IG DMR, HIF3A, and IGF2-DMR0. The former two genes displayed same directional nominal association (as sperm) between paternal BMI and FCB methylation in male offspring. Hypomethylation of IGF2-DMR0 with increased paternal BMI was observed in FCBs from female offsprings. Our results suggest that male obesity is nominally associated with modification of sperm DNA methylome in humans, which may affect the epigenome of the next-generation. Nevertheless, it is important to note that none of the associated p-values survived multiple testing adjustments. Future work should test the effect of associated methylation aberrations in the offspring as DNA methylation was shown to control expression and/or imprint establishment across the studied genes., Competing Interests: The authors have declared that no competing interests exist.

Introduction For patients considering undergoing assisted reproductive techniques (ART), many concerns arise when persistent ovarian cysts are found. This large study aimed to determine how ovarian cyst removal affects success rates of IVF/ICSI therapies. Methods 550 patients who underwent an IVF/ICSI treatment between 2002 and 2011 with a persistent ovarian cyst ≤ 5 cm before treatment were analyzed retrospectively. 328 patients' preference was to undergo a laparoscopic cystectomy and 222 patients opted for a conservative management. Control subjects included 13 552 patients undergoing IVF/ICSI at the same period of time without an ovarian cyst. Results After adjusting for age, patients with ovarian cysts without surgery needed a significant higher stimulation dose than the control group (2576.4 vs. 2207.5 IU, p < 0.001). However, on average, they had 1.13 (- 0.25 - 2.01) higher oocyte number retrieved compared to the operated patients (9.0 ± 5.5 vs. 8.2 ± 5.0) (p = 0.012). Patients after surgical cyst removal had a significant lower number of oocytes retrieved (MNOR) in comparison to the control group (8.2 ± 5.0 vs. 9.5 ± 5.4) (p = 0.00). Compared to controls, operated patients had similar clinical pregnancy rate (CPR) (34.2 vs. 33.5%) OR 1.031 (95% CI 0.817 - 1.302) (p = 0.815). Compared to controls, patients without surgery showed significant lower pregnancy rate (34.2 vs. 25,7%) OR 1.428 (95% CI 1.054 - 1.936) (p = 0.002) and lower live birth rate (LBR) (21.9 vs. 13.5%) OR 1.685 (95% CI 1.143 - 2.485) (p = 0.008). Conclusions Ovarian cystectomy did not negatively impact the pregnancy rate or the live birth rate compared to controls.

Iron chronically limits aquatic photosynthesis, especially in marine environments, and the correct perception and maintenance of iron homeostasis in photosynthetic bacteria, including cyanobacteria, is therefore of global significance. Multiple adaptive mechanisms, responsive promoters, and posttranscriptional regulators have been identified, which allow cyanobacteria to respond to changing iron concentrations. However, many factors remain unclear, in particular, how iron status is perceived within the cell. Here we describe a cyanobacterial ferredoxin (Fed2), with a unique C-terminal extension, that acts as a player in iron perception. Fed2 homologs are highly conserved in photosynthetic organisms from cyanobacteria to higher plants, and, although they belong to the plant type ferredoxin family of [2Fe-2S] photosynthetic electron carriers, they are not involved in photosynthetic electron transport. As deletion of fed2 appears lethal, we developed a C-terminal truncation system to attenuate protein function. Disturbed Fed2 function resulted in decreased chlorophyll accumulation, and this was exaggerated in iron-depleted medium, where different truncations led to either exaggerated or weaker responses to low iron. Despite this, iron concentrations remained the same, or were elevated in all truncation mutants. Further analysis established that, when Fed2 function was perturbed, the classical iron limitation marker IsiA failed to accumulate at transcript and protein levels. By contrast, abundance of IsiB, which shares an operon with isiA , was unaffected by loss of Fed2 function, pinpointing the site of Fed2 action in iron perception to the level of posttranscriptional regulation., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Aim: To examine the effects of genetic variation, parental age and BMI on parental allele-specific methylation of imprinted genes in fetal cord blood samples., Methodology: We have developed SNP genotyping and deep bisulphite sequencing assays for six imprinted genes to determine parental allele-specific methylation patterns in diploid somatic tissues., Results: Multivariate linear regression analyses revealed a negative correlation of paternal age with paternal MEG3 allele methylation in fetal cord blood. Methylation of the maternal PEG3 allele showed a positive correlation with maternal age. Paternal BMI was positively correlated with paternal MEST allele methylation. In addition to parental origin, allele-specific methylation of most imprinted genes was largely dependent on the underlying SNP haplotype., Conclusion: Our study supports the idea that parental factors can have an impact, although of small effect size, on the epigenome of the next generation, providing an additional layer of complexity to phenotypic diversity.

In tropical agriculture, the vigorously growing Bracken fern causes severe problems by invading pastures and out-competing the common pasture grasses. Due to infestation by that weed, pastures are abandoned after a few years, and as a fatal consequence, the biodiversity-rich tropical forest is progressively cleared for new grazing areas. Here we present a broad physiological comparison of the two plant species that are the main competitors on the pastures in the tropical Ecuadorian Andes, the planted forage grass Setaria sphacelata and the weed Bracken (Pteridium arachnoideum). With increasing elevation, the competitive power of Bracken increases as shown by satellite data of the study region. Using data obtained from field measurements, the annual biomass production of both plant species, as a measure of their competitive strength, was modeled over an elevational gradient from 1800 to 2800 m. The model shows that with increasing elevation, biomass production of the two species shifts in favor of Bracken which, above 1800 m, is capable of outgrowing the grass. In greenhouse experiments, the effects on plant growth of the presumed key variables of the elevational gradient, temperature and UV radiation, were separately analyzed. Low temperature, as well as UV irradiation, inhibited carbon uptake of the C4-grass more than that of the C3-plant Bracken. The less temperature-sensitive photosynthesis of Bracken and its effective protection from UV radiation contribute to the success of the weed on the highland pastures. In field samples of Bracken but not of Setaria, the content of flavonoids as UV-scavengers increased with the elevation. Combining modeling with measurements in greenhouse and field allowed to explain the invasive growth of a common weed in upland pastures. The performance of Setaria decreases with elevation due to suboptimal photosynthesis at lower temperatures and the inability to adapt its cellular UV screen., Competing Interests: The authors have declared that no competing interests exist.

Imprinted genes show parent-specific activity (functional haploidy), which makes them particularly vulnerable to epigenetic dysregulation. Here we studied the methylation profiles of oppositely imprinted genes at single DNA molecule resolution by two independent parental allele-specific deep bisulfite sequencing (DBS) techniques. Using Roche (GSJunior) next generation sequencing technology, we analyzed the maternally imprinted MEST promoter and the paternally imprinted MEG3 intergenic (IG) differentially methylated region (DMR) in fetal cord blood, adult blood, and visceral adipose tissue. Epimutations were defined as paternal or maternal alleles with >50% aberrantly (de)methylated CpG sites, showing the wrong methylation imprint. The epimutation rates (range 2-66%) of the paternal MEST and the maternal MEG3 IG DMR allele, which should be completely unmethylated, were significantly higher than those (0-15%) of the maternal MEST and paternal MEG3 alleles, which are expected to be fully methylated. This hypermethylation of the non-imprinted allele (HNA) was independent of parental origin. Very low epimutation rates in sperm suggest that HNA occurred after fertilization. DBS with Illumina (MiSeq) technology confirmed HNA for the MEST promoter and the MEG3 IG DMR, and to a lesser extent, for the paternally imprinted secondary MEG3 promoter and the maternally imprinted PEG3 promoter. HNA leads to biallelic methylation of imprinted genes in a considerable proportion of normal body cells (somatic mosaicism) and is highly variable between individuals. We propose that during development and differentiation maintenance of differential methylation at most imprinting control regions may become to some extent redundant. The accumulation of stochastic and environmentally-induced methylation errors on the non-imprinted allele may increase epigenetic diversity between cells and individuals.

Study Question: Does ICSI induce specific DNA methylation changes in the resulting offspring?, Summary Answer: Although several thousand analyzed CpG sites (throughout the genome) displayed significant between-group methylation differences, both ICSI and spontaneously conceived children varied within the normal range of methylation variation., What Is Known Already: Children conceived by ART have increased risks for medical problems at birth and to the extent of present knowledge also in later life (i.e. impaired metabolic and cardiovascular functions). One plausible mechanism mediating these ART effects are epigenetic changes originating in the germ cells and/or early embryos and persisting during further development., Study Design, Size, Duration: We compared the cord blood methylomes and candidate gene methylation patterns of newborns conceived through ICSI or spontaneously., Participants/materials, Setting, Methods: Umbilical cord bloods were obtained from healthy newborn singletons conceived spontaneously (53 samples), through ICSI (89) or IVF (34). Bisulfite-converted DNA samples of 48 ICSI and 46 control pregnancies were used for genome-wide analyses with Illumina's 450K methylation arrays. Candidate genes from the methylation screen were analyzed in all three groups by bisulfite pyrosequencing., Main Results and the Role of Chance: Altogether, 4730 (0.11%) of 428 227 analyzed CpG sites exhibited significant between-group methylation differences, but all with small (β < 10%) or very small (β < 1%) effect size. ICSI children showed a significantly decreased DNA methylation age at birth, lagging approximately half a week behind the controls. ART-susceptible CpGs were enriched in CpG islands with low methylation values (0-20%) and in imprinting control regions (ICRs). Eighteen promoter regions (six in microRNA and SNORD RNA genes), four CpG islands (three in genes including one long non-coding RNA), and two ICRs contained multiple significant sites. Three differentially methylated regions were studied in more detail by bisulfite pyrosequencing. ATG4C and SNORD114-9 could be validated in an independent ICSI group, following adjustment for maternal age and other confounding factors. ATG4C was also significant in the IVF group., Large Scale Data: N/A., Limitations, Reasons for Caution: The observed epigenetic effects are small and there are numerous potential confounding factors such as parental age and infertility. Although our study meets current standards for epigenetic screens, sample size is still two orders of magnitude below that of genome-wide association studies., Wider Implications of the Findings: Our study suggests an impact of ICSI on the offspring's epigenome(s), which may contribute to phenotypic variation and disease susceptibility in ART children. Epigenetic regulation of gene expression by different classes of non-coding RNAs may be a key mechanism for developmental programming through ART., Study Funding/competing Interest(s): This work was supported by a research grant (no. 692185) from the European Union (ERA of ART). There are no competing interests., (©The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Children of older fathers carry an increased risk for developing autism and other disorders. To elucidate the underlying mechanisms, we investigated the correlation of sperm DNA methylation with paternal age and its impact on the epigenome of the offspring. Methylation levels of nine candidate genes and LINE-1 repeats were quantified by bisulfite pyrosequencing in sperm DNA of 162 donors and 191 cord blood samples of resulting children (conceived by IVF/ICSI with the same sperm samples). Four genes showed a significant negative correlation between sperm methylation and paternal age. For FOXK1 and KCNA7, the age effect on the sperm epigenome was replicated in an independent cohort of 188 sperm samples. For FOXK1, paternal age also significantly correlated with foetal cord blood (FCB) methylation. Deep bisulfite sequencing and allele-specific pyrosequencing allowed us to distinguish between maternal and paternal alleles in FCB samples with an informative SNP. FCB methylation of the paternal FOXK1 allele was negatively correlated with paternal age, whereas maternal allele was unaffected by maternal age. Since FOXK1 duplication has been associated with autism, we studied blood FOXK1 methylation in 74 children with autism and 41 age-matched controls. The FOXK1 promoter showed a trend for accelerated demethylation in the autism group. Dual luciferase reporter assay revealed that FOXK1 methylation influences gene expression. Collectively, our study demonstrates that age-related DNA methylation changes in sperm can be transmitted to the next generation and may contribute to the increased disease risk in offspring of older fathers.

Background: Histone to protamine exchange and the hyperacetylation of the remaining histones are hallmarks of spermiogenesis. Acetylation of histone H4 at lysine 12 (H4K12ac) was observed prior to full decondensation of sperm chromatin after fertilization suggesting an important role for the regulation of gene expression in early embryogenesis. Similarly, DNA methylation may contribute to gene silencing of several developmentally important genes. Following the identification of H4K12ac-binding promoters in sperm of fertile and subfertile patients, we aimed to investigate whether the depletion of histone-binding is associated with aberrant DNA methylation in sperm of subfertile men. Furthermore, we monitored the transmission of H4K12ac, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) from the paternal chromatin to the embryo applying mouse in vitro fertilization and immunofluorescence., Results: Chromatin immunoprecipitation (ChIP) with anti-H4K12ac antibody was performed with chromatin isolated from spermatozoa of subfertile patients with impaired sperm chromatin condensation assessed by aniline blue staining. Fertile donors were used as control. DNA methylation analysis of selected H4K12ac-interacting promoters in spermatozoa was performed by pyrosequencing. Depletion of binding sites for H4K12ac was observed within the following developmentally important promoters: AFF4, EP300, LRP5, RUVBL1, USP9X, NCOA6, NSD1, and POU2F1. We found 5% to 10% hypomethylation within CpG islands of selected promoters in the sperm of fertile donors, and it was not significantly altered in the subfertile group. Our results demonstrate that the H4K12ac depletion in selected developmentally important promoters of subfertile patients was not accompanied by a change of DNA methylation. Using a murine model, immunofluorescence revealed that H4K12ac co-localize with 5mC in the sperm nucleus. During fertilization, when the pronuclei are formed, the paternal pronucleus exhibits a strong acetylation signal on H4K12, while in the maternal pronucleus, there is a permanent increase of H4K12ac until pronuclei fusion. Simultaneously, there is an increase of the 5hmC signal and a decrease of the 5mC signal., Conclusions: We suggest that aberrant histone acetylation within developmentally important gene promoters in subfertile men, but not DNA methylation, may reflect insufficient sperm chromatin compaction affecting the transfer of epigenetic marks to the oocyte.

The molecular basis of male infertility is poorly understood, the majority of cases remaining unsolved. The association of aberrant sperm DNA methylation patterns and compromised semen parameters suggests that disturbances in male germline epigenetic reprogramming contribute to this problem. So far there are only few data on the epigenetic heterogeneity of sperm within a given sample and how to select the best sperm for successful infertility treatment. Limiting dilution bisulfite sequencing of small pools of sperm from fertile donors did not reveal significant differences in the occurrence of abnormal methylation imprints between sperm with and without morphological abnormalities. Intracytoplasmic morphologically selected sperm injection was not associated with an improved epigenetic quality, compared to standard intracytoplasmatic sperm injection. Deep bisulfite sequencing (DBS) of 2 imprinted and 2 pluripotency genes in sperm from men attending a fertility center showed that in both samples with normozoospermia and oligoasthenoteratozoospermia (OAT) the vast majority of sperm alleles was normally (de)methylated and the percentage of epimutations (allele methylation errors) was generally low (<1%). However, DBS allowed one to identify and quantify these rare epimutations with high accuracy. Sperm samples not leading to a pregnancy, in particular in the OAT group, had significantly more epimutations in the paternally methylated GTL2 gene than samples leading to a live birth. All 13 normozoospermic and 13 OAT samples leading to a child had <1% GTL2 epimutations, whereas one (7%) of 14 normozoospermic and 7 (50%) of 14 OAT samples without pregnancy displayed 1-14% GTL2 epimutations.

Purpose: Although intrauterine insemination is one of the oldest techniques in reproductive medicine, its significance is still controversially discussed. Many factors have been reported as influencing pregnancy rates after IUI. The aim of this retrospective analysis is to evaluate the success rate of repeated inseminations depending on the type of ovarian stimulation., Methods: Patients who underwent intrauterine insemination in Wiesbaden Kinderwunschzentrum between 1998 and 2010, not older than 45 years of age, with male subfertility were included in this study. On the whole, 5,346 inseminations on 2,180 patients were analyzed retrospectively., Results: Females' mean age was 34.1, ranging from 19-45 years. In 433 cycles an insemination was performed during a natural cycle. 4,020 cycles were stimulated with recombinant FSH, 596 cycles with clomiphene, 194 with urinary FSH, 103 with HMG. The pregnancy rates range from 7.4% in the clomiphene group to 14.4% in the urinary FSH group. Clomiphene stimulation seems to offer the significantly lowest pregnancy rate (p = 0.03). The other types of stimulation do not differ significantly from each other concerning the pregnancy rate. Patients under 39 years of age do not profit from any ovarian stimulation. In 40 and more years of old patients, pregnancy rates are higher, if any stimulation was performed., Conclusion: To sum up, clomiphene stimulation showed to offer significantly lower pregnancy rates in comparison to the natural cycle, FSH stimulation and HMG stimulation in IUI treatment. While women younger than 40 seem not to profit from any ovarian stimulation, women over 40 do profit.

Antiphospholipid syndrome (APS) is defined as an autoimmune disorder characterized by recurrent thrombosis or obstetrical morbidity. These features are linked to the presence in blood of autoantibodies against negatively charged phospholipids or phospholipid-binding proteins. Obstetric morbidity includes recurrent abortion (early and late) and severe pre-eclampsia (P-EC)/hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome, and/or severe placental insufficiency. Criteria that define the major clinical and laboratory events were published in revised forms in the Sydney recommendations in 2006. We analyzed the blood of patients with severe P-EC according to the subgroups based on the 2006 revised criteria definition and compared these results with women after uncomplicated pregnancy and delivery. We found 20% elevated antiphospholipid antibodies (APAs) in women with severe P-EC (group I, 7.5%; group IIa, 5.0%; group IIb, 5.0%; group IIc, 2.5%). The increased APAs were observed only in women with severe P-EC (odds ratio: 2.45; 95% confidence interval, 1.01 to 4.3) and not in patients with severe P-EC at >34 weeks of gestation. According to our retrospective observation, we recommend the determination of anticardiolipin antibodies, lupus anticoagulant, and β-2 glycoprotein-1 antibodies in patients with severe P-EC at <34 weeks of gestation., (© Thieme Medical Publishers.)

Problem: The purpose of this retrospective, observational study was to investigate whether additional treatment with intravenous immunoglobulin (IVIG) increased the rate of successful pregnancies after repeated implantation failure (RIF). The retrospective data were compared with data of patients without IVIG-therapy from the meta-analysis of Clark et al., Method of Study: A total of 188 women with 226 treatment cycles between 2007 and 2009 were evaluated for IVIG therapy. The percentage of NK cells was measured two times before a new embryo transfer (only women with NK cell percentages >12% were included) and after embryo transfer at a positive pregnancy test., Results: In comparison with the meta-analysis of Clark et al., we observed a pregnancy rate of 50.5%, an implantation rate of 21% and a miscarriage rate of 16.8%. In 42%/IVIG- patient or 34.9%/embryo transfer, we observed a live born baby. The live born rate per embryo was 16.6%. In accordance with the study of Kwak et al., we indicate a decrease in the NK cells in patients with improved pregnancy outcome., Conclusion: In a subgroup of RIF-patients with high level of CD56(+) CD16(+) NK-cells the additional application of IVIG leads to a favourable pregnancy outcome.

Objective: To compare the reproductive outcome of women undergoing intracytoplasmic sperm injection (ICSI) with or without polar body diagnosis of oocytes., Design: Nonrandomized retrospective study., Setting: University-based human genetic institute in collaboration with a private fertility center., Patient(s): Six hundred seven women undergoing ICSI with polar body diagnosis and 591 women undergoing ICSI without polar body diagnosis at the same time in the same fertility center., Intervention(s): Polar body testing of ICSI oocytes by five-color fluorescence in situ hybridization., Main Outcome Measure(s): Pregnancy rate (positive fetal heartbeats) and live-birth rate (of at least one child)., Result(s): The pregnancy and live-birth rates were significantly lower in women undergoing ICSI with polar body diagnosis than in women without polar body diagnosis. The negative effects of polar body diagnosis were evident in all analyzed subgroups, that is, women of different age groups, with one ICSI cycle, with transfer of a high-quality embryo, and with male factor infertility as indication for ICSI., Conclusion(s): Within the legal restrictions of the German embryo protection law aneuploidy testing of oocytes may not improve reproductive outcome., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To compare the chromosome error rate among oocytes from stimulated ovaries after retrieval of 1-5 oocytes, 6-10 oocytes, and >10 oocytes., Design: Retrospective cohort study., Setting: A university-based human genetic institute in collaboration with a private fertility center., Patient(s): Nine hundred thirty-three women undergoing intracytoplasmic sperm injection (ICSI) with a poor prognosis., Intervention(s): Oocyte collection with ovarian stimulation. Polar body testing of ICSI oocytes for common chromosome errors., Main Outcome Measure(s): Chromosome error rate in oocytes, as determined by five-color fluorescence in situ hybridization., Result(s): In women less than 35 years and women between 35 and 40 years undergoing the first ICSI cycle, oocytes from the high-yield group had an increased likelihood for detectable chromosome errors (50.9% and 54.6%, respectively), compared to the intermediate-yield group (34.9% and 43.8%) and the low-yield group (23.3% and 41.2%). The overall high rate (>or=50%) of chromosomally abnormal oocytes in women more than 40 years appeared to be mainly due to the maternal age effect and increased only slightly with oocyte yield., Conclusion(s): Oocyte yield may be considered as an indicator of ovarian response to hormone stimulation. In women up to 40 years a high yield of oocytes after superovulation is associated with an increased chromosome error rate.