Denny Sakkas

Nature of DNA Damage in Ejaculated Human Spermatozoa and the Possible Involvement of Apoptosis

Abstract Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa. The nature of this nuclear anomaly and its relationship to patient etiology is however poorly understood. The aim of this study was to investigate the relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas, Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters. We also determined the nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase I. We show that TUNEL positivity and apoptotic markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas and p53 expression. In addition, the DNA damage in ejaculated human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted nature of ejaculated spermatozoa. This DNA protection is poorer in men with abnormal semen parameters. We propose that the presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems in the nuclear remodeling process. Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked to defects in cytoplasmic remodeling during the later stages of spermatogenesis.

Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis

OBJECTIVE To review the mechanisms responsible for DNA fragmentation in human sperm, including those occurring during spermatogenesis and transport through the reproductive tract. The mechanisms examined include: apoptosis in the seminiferous tubule epithelium, defects in chromatin remodeling during the process of spermiogenesis, oxygen radical-induced DNA damage during sperm migration from the seminiferous tubules to the epididymis, the activation of sperm caspases and endonucleases, damage induced by chemotherapy and radiotherapy, and the effect of environmental toxicants. The different tests currently used for sperm DNA fragmentation analysis and the factors that determine the predictive value of sperm DNA fragmentation testing and their implications in the diagnosis and treatment of infertility are also discussed. Finally, we also scrutinize how the presence in the embryonic genome of DNA strand breaks or modifications of DNA nucleotides inherited from the paternal genome could impact the embryo and offspring. In particular we discuss how abnormal sperm could be dealt with by the oocyte and how sperm DNA abnormalities, which have not been satisfactorily repaired by the oocyte after fertilization, may interfere with normal embryo and fetal development. CONCLUSION(S) Sperm DNA can be modified through various mechanisms. The integrity of the paternal genome is therefore of paramount importance in the initiation and maintenance of a viable pregnancy both in a natural conception and in assisted reproduction. The need to diagnose sperm at a nuclear level is an area that needs further understanding so that we can improve treatment of the infertile couple.

Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during spermatogenesis.

The mechanisms responsible for producing abnormal spermatozoa in the ejaculate are relatively unknown. Numerous studies have now shown the presence of nuclear DNA strand breaks in human ejaculated spermatozoa and the abnormal persistence of apoptotic marker proteins. The reason why human spermatozoa, in particular from men with abnormal semen parameters, possess these abnormalities is still not clear. Two processes that have been linked to the presence of nuclear DNA strand breaks in spermatozoa are anomalies in apoptosis during spermatogenesis or problems in the replacement of histones with protamines during spermiogenesis. Understanding the mechanisms responsible for producing abnormal spermatozoa in the human will improve knowledge about certain causes of male infertility.

Noninvasive metabolomic profiling of human embryo culture media using Raman spectroscopy predicts embryonic reproductive potential: a prospective blinded pilot study.

OBJECTIVE To determine if metabolomic profiling of embryonic development was associated with implantation rates in IVF. DESIGN Prospective blinded. SETTING University-affiliated assisted reproductive technology program. PATIENT(S) Unselected assisted reproductive technology population. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Raman-based biospectroscopic metabolomic profiling of spent culture media and delivery rates. RESULTS Forty-one spent media samples from 19 patients with known reproductive potential (0 or 100% delivery rates of each embryo that implanted) were evaluated. Raman-based metabolomic profiling was used to calculate a viability index for each sample. On day 3, the spent media of embryos with proven reproductive potential (n = 33) demonstrated higher viability indices (0.875 +/- 0.12) than those that failed to implant (0.56 +/- 0.09). Similar findings were present in spent media from embryos transferred on day 5 (n = 8) (-0.40 +/- -0.21 vs. -0.81 +/- -0.08). Receiver operating characteristic curve analyses were used to select thresholds with the greatest ability to discriminate outcomes. Overall diagnostic accuracy for predicting delivery or a failed implantation was 80.5%. CONCLUSIONS There is a clear relationship between the reproductive potential of human embryos and their modification of their culture media as detected by Raman biospectroscopy-based metabolomic profiling. This technology offers great potential for development as a tool to allow rapid noninvasive assessment of embryonic reproductive potential before transfer.

Fertility testing and ICSI sperm selection by hyaluronic acid binding: clinical and genetic aspects

The testis-expressed chaperone protein, HspA2 (previously creatine kinase M isoform) was established as a measure of human sperm cellular maturity, function and fertility. The presence of HspA2 in the synaptonemal complex is likely to link low HspA2 expression and increased frequency of chromosomal aneuploidies in arrested-maturity spermatozoa. A relationship also exists between HspA2 expression in elongating spermatids and the associated spermatogenetic events, including plasma membrane remodelling and the formation of zona pellucida and hyaluronic acid (HA) binding sites. The HA receptor of mature spermatozoa, when coupled with HA-coated slides and/or Petri dishes, allows visual observation of sperm-HA binding, providing a basis for sperm maturity testing, a major improvement in semen evaluation, and selection of mature spermatozoa for intracytoplasmic sperm injection (ICSI). Thus, in HA-selected spermatozoa the frequency of chromosomal disomy and diploidy is reduced 4- to 6-fold compared with semen sperm fractions. This reduction is similar to the increase in numerical chromosomal aberrations in ICSI children. Combined studies of sperm shape and chromosome probes demonstrated that sperm morphology does not aid selection of haploid spermatozoa. The HA-mediated sperm selection is a novel and efficient technique that may alleviate potential problems related to ICSI fertilization with visually selected spermatozoa.

Metabolomics and its application for non-invasive embryo assessment in IVF

Morphology and cleavage rate remain the mainstay of embryo assessment. However, a number of additional technologies for this application are under investigation. These include the measurement of glucose, lactate, pyruvate or amino acid levels in the embryo culture media, assessment of oxygen consumption by the embryo, genomic and proteomic profiling, and most recently, analytical examination of the embryonic metabolome. As the number of assisted reproduction cycles increases worldwide, improvements in the ability to quickly and non-invasively identify the best embryos for transfer remain a critical goal for reproductive medicine. Recent studies suggest that metabolomic profiling of embryo culture media using optical and non-optical spectroscopies may provide a useful adjunct to the current embryo assessment strategies and provide insight into the phenotype of embryos with increasing reproductive potential.

Noninvasive metabolomic profiling of embryo culture media using proton nuclear magnetic resonance correlates with reproductive potential of embryos in women undergoing in vitro fertilization

OBJECTIVE To identify biomarkers associated with reproductive outcome using proton nuclear magnetic resonance ((1)H NMR) metabolomic profiling of embryo culture media. DESIGN Retrospective study. SETTING An academic assisted reproductive technology (ART) program; a university research center. PATIENT(S) Women undergoing ART treatment. INTERVENTION(S) Spent media samples from embryos that resulted in pregnancy and delivery (n = 17) and samples (n = 17) from embryos that failed to implant were individually collected on day 3, and evaluated using (1)H NMR spectroscopy. The spectra obtained were quantified by integrating six biomarker signals in the aliphatic region after baseline subtraction. Using a multivariate analysis, a model that calculates a viability index for each spectrum was developed. Sensitivity and specificity of predicting pregnancy (described as implantation and delivery) were calculated. MAIN OUTCOME MEASURE(S) The (1)H NMR metabolomic profile of embryo culture media and embryo viability. RESULT(S) Glutamate concentrations determined by (1)H NMR were significantly higher in spent culture media of embryos that resulted in pregnancy and delivery compared to those that failed to implant. Similarly, viability indices calculated by (1)H NMR using the weighted coefficients of glutamate and alanine/lactate ratio quantities were higher for embryos that implanted and resulted in a delivery. Proton NMR spectroscopy predicted viability of individual embryos with a sensitivity of 88.2% and a specificity of 88.2%. CONCLUSION(S) Metabolomic profile of spent embryo culture media using (1)H NMR correlates with the reproductive potential of embryos.

Assessment of early cleaving in vitro fertilized human embryos at the 2-cell stage before transfer improves embryo selection.

OBJECTIVE To determine the most viable embryos for transfer. DESIGN Study 1: Preselection of early-cleaving 2-cell embryos for transfer. Study 2: Alternating weeks during which preselection was performed and not performed. SETTING ART program, Birmingham Womens Hospital, Birmingham, United Kingdom. PATIENT(S) Patients undergoing IVF or ICSI cycles with transfer on day 2. INTERVENTION(S) Culture of all fertilized embryos. MAIN OUTCOME MEASURE(S) Number of fertilized embryos cleaving to the 2-cell stage on day 1, embryo quality, implantation rates, and pregnancy rates. RESULT(S) Patients with early-cleaving 2-cell embryos had significantly higher pregnancy and implantation rates (45 of 100 [45.0%] and 58 of 219 [25.5%], respectively) than did patients without early-cleaving 2-cell embryos (31 of 130 [23.8%] and 43 of 290 [14.8%], respectively). In weeks during which preselection was used, the overall pregnancy and implantation rates of the clinic improved. CONCLUSION(S) The presence of early-cleaving 2-cell embryos improves a patients chance of achieving pregnancy. Use of more stringent embryo selection criteria can improve overall pregnancy rates.

Noninvasive metabolomic profiling as an adjunct to morphology for noninvasive embryo assessment in women undergoing single embryo transfer

OBJECTIVE To determine whether metabolomic profiling of spent embryo culture media correlates with reproductive potential of human embryos. DESIGN Retrospective study. SETTING Academic and a private assisted reproductive technology (ART) programs. PATIENT(S) Women undergoing single embryo transfer after IVF. INTERVENTION(S) Spent embryo culture media were collected after single embryo transfer on day 3 (n = 304) or day 2 (n = 181) and analyzed by near infrared spectroscopy. Near infrared spectral regions were correlated to reproductive potential using a genetic algorithm optimization. Models of these spectral regions were used to calculate viability indices, and were validated by blinded analysis of a subset (n = 60) of samples. Implantation rates were also compared between embryos of higher (>or=0.3) and lower (<0.3) viability indices, and within each morphology grade. MAIN OUTCOME MEASURE(S) Viability index and embryo viability. RESULT(S) Mean viability indices of embryos that resulted in positive fetal cardiac activity were significantly higher compared with embryos that did not for both day 2 and day 3 embryos. Blinded validation of the day 2 model proved to be significant. Increasing viability index values correlated with an increase in pregnancy. Viability indices were found to be independent of morphology for both day 2 and day 3 embryos. Implantation rates were significantly higher among embryos with viability indices >or=0.3. CONCLUSION(S) Metabolomic profiling of human embryo culture media using near infrared spectroscopy is independent of morphology and correlates with reproductive potential of embryos.

Failure of elimination of paternal mitochondrial DNA in abnormal embryos

Paternal mitochondrial DNA is normally eliminated from mammalian embryos. We have shown the presence of paternal mtDNA at the blastocyst stage in some abnormal human embryos.