BACKGROUND: DNA damage in human spermatozoa is known to be associated with a variety of adverse clinical outcomes affecting both reproductive efficiency and the health and wellbeing of the offspring. However, the origin of this damage, its biochemical nature and strategies for its amelioration, still await resolution. METHODS: Using novel methods to simultaneously assess DNA fragmentation (modified TUNEL assay), DNA-base adduct formation (8-hydroxy-2'-deoxyguanosine [80HdG]) and cell vitality, spermatozoa from a cohort of 50 assisted conception patients were examined and compared with a group of donors. Receiver operating characteristic (ROC) curve analysis was then used to examine the frequency distribution of the data and to determine optimized thresholds for identifying patients exhibiting abnormally high levels of DNA damage. RESULTS: BOHdG formation and DNA fragmentation were highly correlated with each other and frequently associated with cell death. Percoll centrifugation improved sperm quality but, unexpectedly, increased BOHdG formation in live cells, as did sperm fractionation using Puresperm ® gradients. ROC analysis indicated that the frequency distribution of 8OHdG and DNA fragmentation data were significantly different between patients and donors (P < 0.001), permitting the development of thresholds that would allow the accurate diagnosis of DNA damage in the male germ line. CONCLUSION: The aetiology of DNA damage in spermatozoa involves a cascade of changes that progress from the induction of oxidative stress and oxidized DNA base adduct formation to DNA fragmentation and cell death. Preparation of spermatozoa on discontinuous density gradients aggravates the problem by stimulating the formation of 8OHdG in live cells. However, the development of novel methods and optimized thresholds for diagnosing oxidative DNA damage in human spermatozoa should assist in the clinical management of this pathology.

Analysis of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: development of diagnostic criteria

The cryopreservation of spermatozoa was introduced in the 1960s as a route to fertility preservation. Despite the extensive progress that has been made in this field, the biological and biochemical mechanisms involved in cryopreservation have not been thoroughly elucidated to date. Various factors during the freezing process, including sudden temperature changes, ice formation and osmotic stress, have been proposed as reasons for poor sperm quality post-thaw. Little is known regarding the new aspects of sperm cryobiology, such as epigenetic and proteomic modulation of sperm and trans-generational effects of sperm freezing. This article reviews recent reports on molecular and cellular modifications of spermatozoa during cryopreservation in order to collate the existing understanding in this field. The aim is to discuss current freezing techniques and novel strategies that have been developed for sperm protection against cryo-damage, as well as evaluating the probable effects of sperm freezing on offspring health.

Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches.

BACKGROUND Cryopreservation has been extensively used in assisted reproductive technology, agriculture and conservation programmes for endangered species. The literature reports largely positive results regarding the survival of frozen-thawed cells and clinical outcomes. Nonetheless, it is unclear whether or not cryopreservation of sperm, oocytes and embryos causes any disruption in their genetic integrity. Drawing on the available published evidence, this review paper describes in detail the physical and biochemical factors of cryopreservation that could potentially affect genomic integrity. METHODS A critical review of the published literature using PubMed with particular emphasis on studies which include assessment of genetic stability after cryopreservation of oocyte, sperm and embryos. The search was performed in 2014 and covered the period from the beginning of electronic records until July 2014. No language restrictions were applied. RESULTS Cryopreservation is associated with extensive damage to cell membranes, and results in alteration of the functional and metabolic status of the cells and mitochondria. Some evidence suggests an increase in DNA single-strand breaks, and degree of DNA condensation or fragmentation in sperm after cryopreservation. The extent of these changes may vary between different individuals and different techniques. The addition of antioxidants to the cryopreservation media and the use of well-controlled cooling regimes could potentially improve such outcomes. Limited numbers of studies on oocytes provide controversial results regarding the effect on DNA fragmentation, sister chromatid exchange (SCE) and aneuploidy. The only study on human embryos suggested that vitrification affects DNA integrity to a much lesser extent than slow freezing. Animal studies show increases in mitochondrial DNA mutations in embryos after cryopreservation. The limited numbers of long-term follow-up studies in humans provide reassurance that derives mostly from retrospective studies with some methodological weaknesses. CONCLUSIONS This review provides an overview of studies performed to date on the effect of cryopreservation on the oocyte, sperm and embryos. Controversy of the reported data has highlighted the gaps in our knowledge not only for clinical studies, but also for basic research in human embryos. New perspectives for future research are proposed.

/pdf/the-effect-of-cryopreservation-on-the-genome-of-gametes-and-pisg9trds7.pdf

The effect of cryopreservation on the genome of gametes and embryos: principles of cryobiology and critical appraisal of the evidence

Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay and the single cell gel electrophoresis (Comet) assay. We performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting chance of ongoing pregnancy with IVF or ICSI. Out of 658 unique studies, 30 had extractable data and were thus included in the meta-analysis. Overall, the sperm DNA fragmentation tests had a reasonable to good sensitivity. A wide variety of other factors may also affect the IVF/ICSI outcome, reflected by limited to very low specificity. The constructed hierarchical summary receiver operating characteristic (HSROC) curve indicated a fair discriminatory capacity of the TUNEL assay (area under the curve (AUC) of 0.71; 95% CI 0.66 to 0.74) and Comet assay (AUC of 0.73; 95% CI 0.19 to 0.97). The SCSA and the SCD test had poor predictive capacity. Importantly, for the TUNEL assay, SCD test and Comet assay, meta-regression showed no differences in predictive value between IVF and ICSI. For the SCSA meta-regression indicated the predictive values for IVF and ICSI were different. The present review suggests that current sperm DNA fragmentation tests have limited capacity to predict the chance of pregnancy in the context of MAR. Furthermore, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment, there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and the methodological weakness and design of the included studies, we do urge for further research on the predictive value of sperm DNA fragmentation for the chance of pregnancy after MAR, also in comparison with other predictors of pregnancy after MAR.

/pdf/measuring-sperm-dna-fragmentation-and-clinical-outcomes-of-1df7ncfpvm.pdf

Measuring sperm DNA fragmentation and clinical outcomes of medically assisted reproduction: A systematic review and meta analysis

There has been an increase in the use of sperm DNA and chromatin integrity tests in the evaluation of the infertile man with the hypothesis that these tests may better diagnose infertility and predict reproductive outcomes. This review discusses the etiology of sperm DNA damage, briefly describing the tests of sperm DNA damage, and evaluates the relationship between sperm DNA damage and reproductive outcomes. A systematic review of the literature allows us to conclude that sperm DNA damage is associated with lower natural, intra-uterine insemination (IUI), and in vitro fertilization (IVF) pregnancy rates. Studies to date have not shown a clear association between sperm DNA and chromatin defects and pregnancy outcomes after intra-cytoplasmic sperm injection (ICSI). However, we cannot exclude the possibility that very high levels of DNA damage will impact on ICSI outcomes. In couples undergoing IVF or ICSI, there is evidence to show that sperm DNA damage is associated with an increased risk of pregnancy loss. A limitation of this systematic review and meta-analysis is that it does not address the heterogeneity of the individual study characteristics. Although the clinical utility of tests of sperm DNA damage remains to be firmly established, the data suggest that there is clinical value in testing couples prior to assisted reproductive technologies (ARTs IUI, IVF, and ICSI) and in those couples with recurrent miscarriages. Additional, well-designed prospective studies are needed before testing becomes a routine part of patient care.

Are sperm chromatin and DNA defects relevant in the clinic

Cryopreservation of human spermatozoa—introduced in the 1960's—has been recognized as an efficient procedure for management of male fertility before therapy for malignant diseases, vasectomy or surgical infertility treatments, to store donor and partner spermatozoa before assisted reproduction treatments and to ensure the recovery of a small number of spermatozoa in severe male factor infertility. Despite the usefulness of it, cryopreservation may lead to deleterious changes of sperm structure and function: while the effects of cryopreservation on cells are well documented, to date there is no agreement in the literature on whether or not cryopreservation affects sperm chromatin integrity or on the use of a unique and functional protocol for the freezing-thawing procedure. Therefore, sperm cryopreservation is an important component of fertility management and much of its successful application seems to affect the reproductive outcome of assisted reproduction technologies (ART): appropriate use of cryoprotectants before and sperm selection technologies after cryopreservation seem to have the greatest impact on preventing DNA fragmentation, thus improving sperm cryosurvival rates.

/pdf/human-sperm-cryopreservation-update-on-techniques-effect-on-53qv8y9v3l.pdf

Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART

Irregular cleavage divisions are expected to produce chromosomally deviant embryos. We investigated whether embryos from irregular cleavages could develop into euploid blastocysts, and, if so, whether any evidence existed of a self-correction mechanism of the embryo. We also investigated the role of different dynamic aspects of morula compaction in this process. A total of 791 embryos from 141 patients undergoing pre-implantation genetic screening were retrospectively analysed using a time-lapse imaging system, and multiple cell divisions were evaluated. A total of 276 embryos developed into blastocysts suitable for biopsy and chromosome screening through array-comparative genomic hybridization. As well as testing trophectoderm biopsy specimens for aneuploidy, excluded cells of 18 blastocysts, which developed from partially compacted morulas, were also analysed. Unique data on the developmental fate of embryos with cleavage abnormalities are presented, and a potential mechanism of 'aneuploidy rescue' is postulated through which mosaic embryos may form partially compacted morulas to exclude aneuploid cells. In addition, this process seems to be less efficient in older women. The data obtained also provide further evidence that excluded cells should not be used to infer the cytogenetic status of the embryo.

Embryos with morphokinetic abnormalities may develop into euploid blastocysts

Sperm protamine deficiency and DNA damage were analysed employing chromomycin A 3 (CMA 3 ) staining and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay, respectively, in 132 patients (82 IVF, 50 intracytoplasmic sperm injection [ICSI]). The antioxidant ability of seminal plasma was analysed in 10 men, using the total oxidant scavenging capacity assay. A significant negative correlation was found between abnormal protamination and sperm parameters, including sperm DNA fragmentation ( P P = 0.004 and P P = 0.031). Finally, there was a negative correlation between chromatin under-protamination and the antioxidant ability of seminal plasma ( P 3 analysis only in IVF, whereas DNA fragmentation analysis was prognostic only for ICSI outcome. Data are also provided to support the idea of a relationship between defective antioxidant system activity and impairment of chromatin packaging.

Anomalies in sperm chromatin packaging: implications for assisted reproduction techniques

Sperm protamine deficiency and DNA damage were analysed employing chromomicyn A 3 (CMA 3 ) staining and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay, respectively, in 132 patients (82 IVF, 50 intracytoplasmic sperm injection (ICSI)). The antioxidant ability of seminal plasma was analysed in 10 men, using the total oxidant scavenging capacity assay. A significant negative correlation was found between abnormal protamination and sperm parameters, including sperm DNA fragmentation (P < 0.01). A close relationship was found between sperm protamination and fertilization and pregnancy only in IVF (P = 0.004 and P < 0.04, respectively); in ICSI there was a correlation between DNA fragmentation and pregnancy (P = 0.031). Finally, there was a negative correlation between chromatin under-protamination and the antioxidant ability of seminal plasma (P < 0.01). Results of this study underline that, despite sperm abnormal protamination and DNA fragmentation being positively correlated, they affect the reproductive outcome in different ways: in particular there was good prognostic value for CMA 3 analysis only in IVF, whereas DNA fragmentation analysis was prognostic only for ICSI outcome. Data are also provided to support the idea of a relationship between defective antioxidant system activity and impairment of chromatin packaging.

Anomalies in sperm chromatin packaging: implications for assisted reproduction

To date, several publications have focused their attention on a new method for observing spermatozoa called 'motile sperm organelle morphology examination' (MSOME), which enables the evaluation of the fine nuclear morphology of motile spermatozoa in real time at high magnification (>x6000). As a consequence, a new microinjection procedure called intracytoplasmic morphologically selected sperm injection (IMSI) has been developed. The aim of the present work is therefore to evaluate the efficacy of the IMSI technique in the light of the current literature, focusing attention on the potential clinical application of the selection of strictly morphologically normal spermatozoa in patients undergoing conventional intracytoplasmic sperm injection treatments. In addition, a brief analysis of preliminary data regarding the relationship between IMSI and assisted reproduction treatment outcome is presented.

https://www.rbmojournal.com/article/S1472-6483(10)60283-0/pdf

Marco Nadalini

Papers

Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART

Embryos with morphokinetic abnormalities may develop into euploid blastocysts

Anomalies in sperm chromatin packaging: implications for assisted reproduction techniques

Anomalies in sperm chromatin packaging: implications for assisted reproduction

Impact of intracytoplasmic morphologically selected sperm injection on assisted reproduction outcome: a review