Douglas T. Carrell

DISTINCTIVE CHROMATIN IN HUMAN SPERM PACKAGES GENES FOR EMBRYO DEVELOPMENT

Because nucleosomes are widely replaced by protamine in mature human sperm, the epigenetic contributions of sperm chromatin to embryo development have been considered highly limited. Here we show that the retained nucleosomes are significantly enriched at loci of developmental importance, including imprinted gene clusters, microRNA clusters, HOX gene clusters, and the promoters of stand-alone developmental transcription and signalling factors. Notably, histone modifications localize to particular developmental loci. Dimethylated lysine 4 on histone H3 (H3K4me2) is enriched at certain developmental promoters, whereas large blocks of H3K4me3 localize to a subset of developmental promoters, regions in HOX clusters, certain noncoding RNAs, and generally to paternally expressed imprinted loci, but not paternally repressed loci. Notably, trimethylated H3K27 (H3K27me3) is significantly enriched at developmental promoters that are repressed in early embryos, including many bivalent (H3K4me3/H3K27me3) promoters in embryonic stem cells. Furthermore, developmental promoters are generally DNA hypomethylated in sperm, but acquire methylation during differentiation. Taken together, epigenetic marking in sperm is extensive, and correlated with developmental regulators.

Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss.

Previous studies have indicated that sperm quality may be related to unexplained recurrent pregnancy loss. This study evaluated the degree of sperm DNA fragmentation using the TUNEL assay on sperm from 24 couples with unexplained recurrent pregnancy loss (RPL) compared to sperm from 2 control groups: donors of known fertility and unscreened men from the general population. The percentage of sperm staining positive for DNA fragmentation was increased ( p <.001) in the RPL group (38±4.2) compared to the donor (11.9±1.0) or general population (22±2.0) control groups. In the RPL group, no correlation was observed between semen quality parameters and the TUNEL data. These data indicate that some RPL patients have a significant increase of sperm DNA fragmentation, which may be causative of pregnancy loss in some patients.

Male obesity and alteration in sperm parameters

OBJECTIVE To study the effect of male obesity on sperm parameters and erectile dysfunction. DESIGN Retrospective analysis. SETTING Referral fertility center. PATIENT(S) Couples presenting for infertility treatment. INTERVENTION(S) On presentation, all men reported their weight and height and filled out an intake form that includes questions regarding factors that affect male infertility, including presence of erectile dysfunction. Body mass index (BMI) was divided into three groups: normal (BMI <25 kg/m(2)), overweight (25 kg/m(2) <or= BMI < 30 kg/m(2)), and obese (BMI >or=30 kg/m(2)). Sperm parameters reviewed included sperm concentration and progressively motile sperm count. MAIN OUTCOME MEASURE(S) Oligozoospermia, low progressively motile sperm count, and self-reported erectile dysfunction. RESULT(S) The mean age of the study population was 32.8 +/- 0.3 years. Among the 526 patients, 10.2% (54 of 526) were excluded because of the presence of a male factor known to affect fertility. The incidence of oligozoospermia increased with increasing BMI: normal weight = 5.32%, overweight = 9.52%, and obese = 15.62%. The prevalence of a low progressively motile sperm count was also greater with increasing BMI: normal weight = 4.52%, overweight = 8.93%, and obese = 13.28%. The incidence of erectile dysfunction did not vary across BMI categories when corrected for potential contributing factors. CONCLUSION(S) Male obesity is associated with increased incidence of low sperm concentration and low progressively motile sperm count.

Sperm DNA: organization, protection and vulnerability: from basic science to clinical applications—a position report

This article reports the results of the most recent in a series of EHSRE workshops designed to synthesize the current state of the field in Andrology and provide recommendations for future work (for details see Appendix). Its focus is on methods for detecting sperm DNA damage and potential application of new knowledge about sperm chromatin organization, vulnerability and repair to improve the diagnosis and treatment of clinical infertility associated with that damage. Equally important is the use and reliability of these tests to identify the extent to which environmental contaminants or pharmaceutical agents may contribute to the incidence of sperm DNA damage and male fertility problems. A working group (for workshop details, see Appendix) under the auspices of ESHRE met in May 2009 to assess the current knowledgebase and suggest future basic and clinical research directions. This document presents a synthesis of the working groups understanding of the recent literature and collective discussions on the current state of knowledge of sperm chromatin structure and function during fertilization. It highlights the biological, assay and clinical uncertainties that require further research and ends with a series of 5 key recommendations.

Impact of male obesity on infertility: a critical review of the current literature

OBJECTIVE To evaluate the current understanding of the effects and potential mechanisms of obesity on male fertility. DESIGN Literature review of articles pertaining to obesity and male infertility. RESULT(S) Recent population-based studies suggest an elevated risk for subfertility among couples in which the male partner is obese and an increased likelihood of abnormal semen parameters among heavier men. Male factor infertility is associated with a higher incidence of obesity in the male partner. Obese men exhibit reduced androgen and SHBG levels accompanied by elevated estrogen levels. Reduced inhibin B levels correlate with degree of obesity and are not accompanied by compensatory increases in FSH. This complexly altered reproductive hormonal profile suggests that endocrine dysregulation in obese men may explain the increased risk of altered semen parameters and infertility. Additional features of male obesity that may contribute to an increased risk for infertility are altered retention and metabolism of environmental toxins, altered lifestyle factors, and increased risks for sexual dysfunction. Neither reversibility of obesity-associated male infertility with weight loss nor effective therapeutic interventions have been studied yet. CONCLUSION(S) The increasing prevalence of obesity calls for greater clinician awareness of its effects on fertility, better understanding of underlying mechanisms, and eventually avenues for mitigation or treatment.

Alterations in sperm DNA methylation patterns at imprinted loci in two classes of infertility

OBJECTIVE To evaluate the associations between proper protamine incorporation and DNA methylation at imprinted loci. DESIGN Experimental research study. SETTING Research laboratory. PATIENT(S) Three populations were tested-abnormal protamine patients, oligozoospermic patients, and fertile donors. INTERVENTION(S) The CpG methylation patterns were examined at seven imprinted loci sequenced: LIT1, MEST, SNRPN, PLAGL1, PEG3, H19, and IGF2. MAIN OUTCOME MEASURE(S) The DNA methylation patterns were analyzed using bisulfite sequencing. The percentage of methylation was compared between fertile and infertile patients displaying abnormal protamination. RESULT(S) At six of the seven imprinted genes, the overall DNA methylation patterns at their respective differentially methylated regions were significantly altered in both infertile patient populations. When comparing the severity of methylation alterations among infertile patients, the oligozoospermic patients were significantly affected at mesoderm-specific transcript (MEST), whereas abnormal protamine patients were affected at KCNQ1, overlapping transcript 1 (LIT1), and at small nuclear ribonucleoprotein polypeptide N (SNRPN). CONCLUSION(S) Patients with male factor infertility had significantly increased methylation alteration at six of seven imprinted loci tested, with differences in significance observed between oligozoospermic and abnormal protamine patients. This could suggest that risk of transmission of epigenetic alterations may be different with diagnoses. However, this study does not provide a causal link for epigenetic inheritance of imprinting diseases, but does show significant association between male factor infertility and alterations in sperm DNA methylation at imprinted loci.

Genome-wide analysis identifies changes in histone retention and epigenetic modifications at developmental and imprinted gene loci in the sperm of infertile men

BACKGROUND The sperm chromatin of fertile men retains a small number of nucleosomes that are enriched at developmental gene promoters and imprinted gene loci. This unique chromatin packaging at certain gene promoters provides these genomic loci the ability to convey instructive epigenetic information to the zygote, potentially expanding the role and significance of the sperm epigenome in embryogenesis. We hypothesize that changes in chromatin packaging may be associated with poor reproductive outcome. METHODS Seven patients with reproductive dysfunction were recruited: three had unexplained poor embryogenesis during IVF and four were diagnosed with male infertility and previously shown to have altered protamination. Genome-wide analysis of the location of histones and histone modifications was analyzed by isolation and purification of DNA bound to histones and protamines. The histone-bound fraction of DNA was analyzed using high-throughput sequencing, both initially and following chromatin immunoprecipitation. The protamine-bound fraction was hybridized to agilent arrays. DNA methylation was examined using bisulfite sequencing. RESULTS Unlike fertile men, five of seven infertile men had non-programmatic (randomly distributed) histone retention genome-wide. Interestingly, in contrast to the total histone pool, the localization of H3 Lysine 4 methylation (H3K4me) or H3 Lysine 27 methylation (H3K27me) was highly similar in the gametes of infertile men compared with fertile men. However, there was a reduction in the amount of H3K4me or H3K27me retained at developmental transcription factors and certain imprinted genes. Finally, the methylation status of candidate developmental promoters and imprinted loci were altered in a subset of the infertile men. CONCLUSIONS This initial genome-wide analysis of epigenetic markings in the sperm of infertile men demonstrates differences in composition and epigenetic markings compared with fertile men, especially at certain imprinted and developmental loci. Although no single locus displays a complete change in chromatin packaging or DNA modification, the data suggest that moderate changes throughout the genome exist and may have a cumulative detrimental effect on fecundity.

Monozygotic twinning associated with assisted reproductive technologies: a review

Twin birth rates have increased markedly in developed countries since the 1970s for two primary reasons: increasing maternal age and the advent and increasing use of fertility treatments. In addition, monozygotic (MZ) twin pregnancies have been reported to occur at a significantly higher rate following assisted reproductive technologies (ART) procedures compared with the natural incidence. Twin pregnancies are of concern due to a dramatically increased risk of associated complications. Monozygotic twin pregnancies carry a 10-20% risk of twin-twin transfusion syndrome, and monoamniotic monochorionic twins are additionally at risk for cord entanglement. While the mechanisms and contributory factors for dizygotic twinning are well established, very little is known about the mechanisms involved in MZ twinning or the factors that contribute to its occurrence. In this review, we will discuss a number of potential mechanisms involved in MZ twinning and explore factors that may be contributing to the increased incidence of ART-associated MZ twins. An improved understanding of the factors that contribute to increased MZ twinning associated with ART will help to elucidate the poorly understood mechanisms involved in the process and will further aid in reducing the overall incidence of multiple pregnancies with their associated risks following ART procedures.

The human sperm epigenome and its potential role in embryonic development

Along with many of the genome-wide transitions in chromatin composition throughout spermatogenesis, epigenetic modifications on histone tails and DNA are continuously modified to ensure stage specific gene expression in the maturing spermatid. Recent findings have suggested that the repertoire of epigenetic modifications in the mature sperm may have a potential role in the developing embryo and alterations in the epigenetic profile have been associated with infertility. These changes include DNA demethylation and the retention of modified histones at important developmental, signaling and micro-RNA genes, which resemble the epigenetic state of an embryonic stem cell. This review assesses the significance of epigenetic changes during spermatogenesis, and provides insight on recent associations made between altered epigenetic profiles in the mature sperm and its relationship to infertility.

Elevated sperm chromosome aneuploidy and apoptosis in patients with unexplained recurrent pregnancy loss.

OBJECTIVE To evaluate sperm chromosome aneuploidy and semen quality in 24 partners of women with unexplained recurrent pregnancy loss and to analyze the data in relation to sperm apoptosis data. METHODS Semen quality parameters and sperm chromosome aneuploidy for chromosomes X, Y, 13, 18, and 21 were evaluated in the recurrent pregnancy loss patients, fertile controls, and a control group of men from the general population. RESULTS The mean aneuploidy rate in the recurrent pregnancy loss group was 2.77 ± 0.22, significantly higher (P < .005) than in either the general population (1.48 ± 0.12) or in fertile (1.19 ± 0.11) control groups. In the recurrent pregnancy loss patients, the percentage of aneuploid sperm was correlated to the percentage of apoptotic sperm (r = .62, P < .001). Normal morphology was diminished in the patient group, compared with the general population group (P < .01) and the donor group (P < .001). CONCLUSION These data indicate that some recurrent pregnancy loss patients have a significant increase of sperm chromosome aneuploidy, apoptosis, and abnormal sperm morphology. This study demonstrates a new possible cause of recurrent pregnancy loss.