Publications by authors named "Sheena E M Lewis"

36 Publications

What does a varicocele do to a man's fertility? There is much more than meets the eye.

Int Braz J Urol 2021 Mar-Apr;47(2):284-286

ANDROFERT, Andrology and Human Reproduction Clinic, Campinas, SP, Brasil.

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http://dx.doi.org/10.1590/S1677-5538.IBJU.2019.0827.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857774PMC
December 2020

Sperm DNA fragmentation testing: Summary evidence and clinical practice recommendations.

Andrologia 2021 Mar 27;53(2):e13874. Epub 2020 Oct 27.

Faculty of Health, Aarhus University, Aarhus, Denmark.

We herein summarise the evidence concerning the impact of sperm DNA fragmentation in various clinical infertility scenarios and the advances on sperm DNA fragmentation tests. The collected evidence was used to formulate 41 recommendations. Of these, 13 recommendations concern technical aspects of sperm DNA fragmentation testing, including pre-analytical information, clinical thresholds and interpretation of results. The remaining 28 recommendations relate to indications for sperm DNA fragmentation testing and clinical management. Clinical scenarios like varicocele, unexplained infertility, idiopathic infertility, recurrent pregnancy loss, intrauterine insemination, in vitro fertilisation/intracytoplasmic sperm injection, fertility counselling for men with infertility risk factors and sperm cryopreservation have been contemplated. The bulk evidence supporting the recommendations has increased in recent years, but it is still of moderate to low quality. This guideline provides clinicians with advice on best practices in sperm DNA fragmentation testing. Also, recommendations are provided on possible management strategies to overcome infertility related to sperm DNA fragmentation, based on the best available evidence. Lastly, we identified gaps in knowledge and opportunities for research and elaborated a list of recommendations to stimulate further investigation.
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http://dx.doi.org/10.1111/and.13874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988559PMC
March 2021

Sperm DNA fragmentation is a novel biomarker for early pregnancy loss.

Reprod Biomed Online 2021 Jan 22;42(1):175-184. Epub 2020 Sep 22.

Centre for Reproductive Immunology and Pregnancy, Bramshott House, 137/139 High Street, Epsom KT19 8EH, UK; Maternal Medicine at Epsom and St Helier University Hospitals NHS Trust, Dorking Road, Epsom KT18 7EG, UK.

Research Question: Spontaneous pregnancy loss affects 10-15% of couples, with 1-2% suffering recurrent pregnancy loss and 50% of miscarriages remaining unexplained. Male genomic integrity is essential for healthy offspring, meaning sperm DNA quality may be important in maintaining a pregnancy. Does sperm DNA fragmentation measured by alkaline Comet assay act as a biomarker for early pregnancy loss?

Design: Sperm DNA fragmentation was measured by alkaline Comet test in 76 fertile donors and 217 men whose partners had recently experienced miscarriage. Couples were divided into five groups for analysis: one miscarriage after spontaneous conception; two or more miscarriages after spontaneous conception; one miscarriage after fertility treatment; two or more miscarriages after fertility treatment and biochemical pregnancy.

Results: Receiver operator characteristic curve analysis was used to determine ability of the average Comet score (ACS), low Comet score (LCS) and high Comet score (HCS) to diagnose miscarriage and develop clinical thresholds comparing men whose partners have miscarried with men with recently proven fertility. Male partners of women who had miscarried had higher sperm DNA damage (ACS 33.32 ± 0.57%) than fertile men (ACS 14.87 ± 0.66%; P < 0.001). Average Comet score, HCS and LCS all have promise as being highly predictive of sporadic and recurrent miscarriage using clinical thresholds from comparisons with fertile men's spermatozoa: receiver operating characteristic curve AUC for ACS ≥26%, 0.965; LCS ≤70%, 0.969; HCS ≥2%, 0.883; P <0.0001.

Conclusions: Sperm DNA damage measured by the alkaline Comet has promise as a robust biomarker for sporadic and recurrent miscarriage after spontaneous or assisted conception, and may provide novel diagnoses and guidance for future fertility pathways.
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http://dx.doi.org/10.1016/j.rbmo.2020.09.016DOI Listing
January 2021

SARS-CoV-2 pandemic and repercussions for male infertility patients: A proposal for the individualized provision of andrological services.

Andrology 2021 01 22;9(1):10-18. Epub 2020 May 22.

Andrology Center, Department of Urology, Cleveland Clinic, Cleveland, OH, USA.

The prolonged lockdown of health facilities providing non-urgent gamete cryopreservation-as currently recommended by many reproductive medicine entities and regulatory authorities due to the SARS-CoV-2 pandemic will be detrimental for subgroups of male infertility patients. We believe the existing recommendations should be promptly modified and propose that the same permissive approach for sperm banking granted for men with cancer is expanded to other groups of vulnerable patients. These groups include infertility patients (eg, azoospermic and cryptozoospermic) undergoing medical or surgical treatment to improve sperm quantity and quality, as well as males of reproductive age affected by inflammatory and systemic auto-immune diseases who are about to start treatment with gonadotoxic drugs or who are under remission. In both scenarios, the "fertility window" may be transitory; postponing diagnostic semen analysis and sperm banking in these men could compromise the prospects of biological parenthood. Moreover, we provide recommendations on how to continue the provision of andrological services in a considered manner and a safe environment. Our opinion is timely and relevant given the fact that fertility services are currently rated as of low priority in most countries.
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http://dx.doi.org/10.1111/andr.12809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7267121PMC
January 2021

Novel use of COMET parameters of sperm DNA damage may increase its utility to diagnose male infertility and predict live births following both IVF and ICSI.

Hum Reprod 2019 10;34(10):1915-1923

Urology department, Charing Cross Hospital, Imperial College Healthcare NHS Trust, London UK.

Study Question: Do the Comet parameters of the proportions of sperm with low or high DNA damage improve the power of the test in the diagnosis of male infertility and/or prediction of IVF and ICSI live birth rates?

Summary Answer: The mean Comet score and the scores for proportions of sperm with high or low DNA damage were useful in diagnosing male infertility and provided additional discriminatory information for the prediction of both IVF and ICSI live births.

What Is Known Already: Sperm DNA damage impacts adversely on male fertility and IVF outcomes.

Study Design, Size, Duration: A retrospective study was performed involving a total of 457 participants (381 patients and 76 fertile donors). Data was collected from a fertility clinic between 2015 and 2017.

Participants/materials, Setting, Methods: A total of 381 consecutive male partners of couples attending for ART and 76 fertile donors were included in the study. DNA fragmentation was measured by the alkaline Comet assay. Receiver operator characteristic curve analysis (area under the ROC curve (AUC)) was used to determine the value of average Comet score (ACS), low Comet score (LCS) and high Comet score (HCS) to diagnose male factor infertility. In total, 77 IVF and 226 ICSI cycles were included to determine thresholds for each parameter (AUC analysis) and to compare live birth rates (LBRs) following each ART.

Main Results And The Role Of Chance: ACS, HCS and LCS were predictive of male infertility (AUC > 0.9, P < 0.0001). IVF LBRs declined once DNA damage exceeded the threshold levels. HCS showed the sharpest decline. Following ICSI, the highest LBRs were in men whose DNA damage levels approached the fertile range. Trends differed in IVF. LBRs decreased as damage increased whereas in ICSI the LBRs decreased but then remained stable.

Limitations, Reasons For Caution: Since this is the first study to show the impact of sperm DNA damage on ICSI live births, a prospective study should be performed (stratifying patients to IVF or ICSI based on these thresholds) to validate this study.

Wider Implications Of The Findings: Our study presents novel information towards elucidating the genetic basis of male infertility and secondly on relevance of the extent of DNA damage as an impending factor in both IVF and ICSI success.

Study Funding/competing Interest(s): This study was supported by Examenlab Ltd, The Lister Clinic, Cryos International and Imperial College London NHS Trust. No external funding was obtained for this study. SL and KL are employees of Examenlab Ltd, a university spin-out company with a commercial interest in sperm DNA damage. No other author has a conflict of interest to declare.

Trial Registration Number: Non-applicable.
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http://dx.doi.org/10.1093/humrep/dez151DOI Listing
October 2019

Sperm DNA fragmentation testing is the way forward.

Authors:
Sheena E M Lewis

Transl Androl Urol 2017 Sep;6(Suppl 4):S331

Professor Emeritus, Queen's University, Belfast, UK. (Email:

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http://dx.doi.org/10.21037/tau.2017.08.07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643710PMC
September 2017

Should sperm DNA fragmentation testing be included in the male infertility work-up?

Authors:
Sheena E M Lewis

Reprod Biomed Online 2015 Aug 15;31(2):134-7. Epub 2015 May 15.

Centre for Public Health, Queen's University, Belfast BT12 6 BJ, Northern Ireland, UK. Electronic address:

A response to the editorial 'Are we ready to incorporate sperm DNA fragmentation testing into our male infertility work-up? A plea for more robust studies' by Erma Drobnis and Martin Johnson.
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http://dx.doi.org/10.1016/j.rbmo.2015.05.006DOI Listing
August 2015

The paternal genome and the health of the assisted reproductive technology child.

Asian J Androl 2015 Jul-Aug;17(4):616-22

Centre for Public Health, Queen's University Belfast, Grosvenor Road, BT12 6BJ,NI, UK.

As a number of children born by assisted reproductive technology (ART) are increasing each year across the developed world, the health of such offspring is a matter of public concern. Does the integrity of the paternal genome impact on offspring health? In societal terms, as birth rates fall, and the Western population become unsustainable, do the benefits outweigh the costs of creating and providing for this ART conceived subpopulation? There are little data to date to answer these questions. The long-term health of such children has largely been ignored, and success measured only by early (prebirth) outcomes such as embryo quality or pregnancy. However, there are powerful paradigms such as ageing and smoking that give vital clues as to the potential impact of unhealthy spermatozoa on disease risk, mental and physical health, fertility and mortality of these offspring.
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http://dx.doi.org/10.4103/1008-682X.153301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4492053PMC
March 2016

Sperm DNA fragmentation and base oxidation.

Authors:
Sheena E M Lewis

Adv Exp Med Biol 2014 ;791:103-16

Centre for Public Health, Queen's University Belfast, Room 0017, Ground Floor, Belfast, Northern Ireland, UK,

Sperm DNA damage has been shown to be a valuable diagnostic and prognostic biomarker for male infertility and assisted reproductive treatment (ART) outcome. It is linked to every fertility checkpoint from reduced fertilization rates, lower embryo quality and pregnancy rates to higher rates of spontaneous miscarriage and childhood diseases. It is more robust than conventional semen parameters.The aim of this chapter is to provide an overview of current laboratory tests and relationships between sperm DNA damage and clinical outcomes. The conclusion is that sperm DNA damage is an important indicator of semen quality, and its routine use in the fertility clinic would improve ART success rates.
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http://dx.doi.org/10.1007/978-1-4614-7783-9_7DOI Listing
March 2014

The impact of sperm DNA damage in assisted conception and beyond: recent advances in diagnosis and treatment.

Reprod Biomed Online 2013 Oct 11;27(4):325-37. Epub 2013 Jul 11.

Centre for Public Health, Institute of Clinical Sciences, Queen's University Belfast, Grosvenor Road, Belfast BT12 6BJ, Northern Ireland, UK. Electronic address:

Sperm DNA damage is a useful biomarker for male infertility diagnosis and prediction of assisted reproduction outcomes. It is associated with reduced fertilization rates, embryo quality and pregnancy rates, and higher rates of spontaneous miscarriage and childhood diseases. This review provides a synopsis of the most recent studies from each of the authors, all of whom have major track records in the field of sperm DNA damage in the clinical setting. It explores current laboratory tests and the accumulating body of knowledge concerning the relationship between sperm DNA damage and clinical outcomes. The paper proceeds to discuss the strengths, weaknesses and clinical applicability of current sperm DNA tests. Next, the biological significance of DNA damage in the male germ line is considered. Finally, as sperm DNA damage is often the result of oxidative stress in the male reproductive tract, the potential contribution of antioxidant therapy in the clinical management of this condition is discussed. DNA damage in human spermatozoa is an important attribute of semen quality. It should be part of the clinical work up and properly controlled trials addressing the effectiveness of antioxidant therapy should be undertaken as a matter of urgency. Sperm DNA damage is a useful biomarker for male infertility diagnosis and prediction of assisted reproduction outcomes. It is associated with reduced fertilization rates, embryo quality and pregnancy rates, and higher rates of spontaneous miscarriage and childhood diseases. With all of these fertility check points, it shows more promise than conventional semen parameters from a diagnostic perspective. Despite this, few infertility clinics use it routinely. This review provides a synopsis of the most recent studies from each of the authors, all of whom have major track records in the field of sperm DNA damage in the clinical setting. It explores current laboratory tests and the accumulating body of knowledge concerning the relationship between sperm DNA damage and clinical outcomes. The paper proceeds to discuss the strengths and weaknesses and clinical applicability of current sperm DNA fragmentation tests. Next, the biological significance of DNA damage in the male germ line is considered. Finally, as sperm DNA damage is often the result of increased oxidative stress in the male reproductive tract, the potential contribution of antioxidant therapy in the clinical management of this condition is discussed. As those working in this field of clinical research, we conclude that DNA damage in human spermatozoa is an important attribute of semen quality which should be carefully assessed in the clinical work up of infertile couples and that properly controlled trials addressing the effectiveness of antioxidant therapy should be undertaken as a matter of urgency.
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http://dx.doi.org/10.1016/j.rbmo.2013.06.014DOI Listing
October 2013

Differences in the endocannabinoid system of sperm from fertile and infertile men.

PLoS One 2012 17;7(10):e47704. Epub 2012 Oct 17.

School of Medicine, Centre for Public Health, Queen's University Belfast, Institute of Clinical Science, Belfast, United Kingdom.

Male infertility is a major cause of problems for many couples in conceiving a child. Recently, lifestyle pastimes such as alcohol, tobacco and marijuana have been shown to have further negative effects on male reproduction. The endocannabinoid system (ECS), mainly through the action of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) at cannabinoid (CB(1), CB(2)) and vanilloid (TRPV1) receptors, plays a crucial role in controlling functionality of sperm, with a clear impact on male reproductive potential. Here, sperm from fertile and infertile men were used to investigate content (through LC-ESI-MS), mRNA (through quantitative RT-PCR), protein (through Western Blotting and ELISA) expression, and functionality (through activity and binding assays) of the main metabolic enzymes of AEA and 2-AG (NAPE-PLD and FAAH, for AEA; DAGL and MAGL for 2-AG), as well as of their binding receptors CB(1), CB(2) and TRPV1. Our findings show a marked reduction of AEA and 2-AG content in infertile seminal plasma, paralleled by increased degradation: biosynthesis ratios of both substances in sperm from infertile versus fertile men. In addition, TRPV1 binding was detected in fertile sperm but was undetectable in infertile sperm, whereas that of CB(1) and CB(2) receptors was not statistically different in the two groups. In conclusion, this study identified unprecedented alterations of the ECS in infertile sperm, that might impact on capacitation and acrosome reaction, and hence fertilization outcomes. These alterations might also point to new biomarkers to determine male reproductive defects, and identify distinct ECS elements as novel targets for therapeutic exploitation of ECS-oriented drugs to treat male fertility problems.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047704PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3474715PMC
April 2013

Relationships between human sperm protamines, DNA damage and assisted reproduction outcomes.

Reprod Biomed Online 2011 Dec 10;23(6):724-34. Epub 2011 Sep 10.

Centre for Public Health, Reproductive Medicine, Institute of Clinical Science, Queens University of Belfast, Grosvenor Road, Belfast BT12 6BJ, Northern Ireland, UK.

The exchange of histones with protamines in sperm DNA results in sperm chromatin compaction and protection. Variations in sperm protamine expression are associated with male infertility. The aim of this study was to investigate relationships between DNA fragmentation, sperm protamines and assisted reproduction treatment. Semen and spermatozoa prepared by density-gradient centrifugation (DGC) from 73 men undergoing IVF and 24 men undergoing intracytoplasmic sperm injection (ICSI) were included in the study. Nuclear DNA fragmentation was assessed using the alkaline Comet assay and protamines were separated by acid-urea polyacrylamide gels. Sperm DNA fragmentation and protamine content (P1-DNA, P2-DNA, P1+P2-DNA) decreased in spermatozoa after DGC. Abnormally high and low P1/P2 ratios were associated with increased sperm DNA fragmentation. Couples with idiopathic infertility had abnormally high P1/P2 ratios. Fertilization rates and embryo quality decreased as sperm DNA fragmentation or protamines increased. Sperm DNA fragmentation was lower in couples achieving pregnancies after IVF, but not after ICSI. There was no correlation between protamine content (P1-DNA, P2-DNA, P1+P2-DNA) or P1/P2 ratios and IVF or ICSI pregnancies. Increased sperm DNA fragmentation was associated with abnormal protamination and resulted in lower fertilization rates, poorer embryo quality and reduced pregnancy rates.
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http://dx.doi.org/10.1016/j.rbmo.2011.08.010DOI Listing
December 2011

Sperm DNA damage or progressive motility: which one is the better predictor of fertilization in vitro?

Syst Biol Reprod Med 2011 Jun 8;57(3):133-8. Epub 2011 Feb 8.

Centre for Public Health, Reproductive Medicine, Institute of Clinical Science, Queens University of Belfast, Northern Ireland, UK.

Sperm progressive motility has been reported to be one of the key factors influencing in vitro fertilization rates. However, recent studies have shown that sperm DNA fragmentation is a more robust predictor of assisted reproductive outcomes including reduced fertilization rates, embryo quality, and pregnancy rates. This study aimed to compare the usefulness of sperm progressive motility and DNA damage as predictive tools of in vitro fertilization rates. Here, 136 couples provided 1,767 eggs with an overall fertilization rate of 64.2%. The fertilization rate in vitro correlated with both sperm progressive motility (r² = 0.236; P = 0.002) and DNA fragmentation (r² = -0.318; P < 0.001). The relative risk of a poor fertilization rate was 9.5 times higher in sperm of men with high DNA fragmentation (>40%) compared with 2.6 times in sperm with poor motility (<40%). Further, sperm DNA fragmentation gave a higher specificity (93.3%) in predicting the fertilization rate than progressive motility (77.8%). Finally, the odds ratio to determine fertilization rate (>70%) was 4.81 (1.89-12.65) using progressive motility compared with 24.18 (5.21-154.51) using DNA fragmentation. This study shows that fertilization rates are directly dependent upon both sperm progressive motility and DNA fragmentation, but sperm DNA fragmentation is a much stronger test.
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http://dx.doi.org/10.3109/19396368.2011.553984DOI Listing
June 2011

Clinical implications of sperm DNA damage.

Hum Fertil (Camb) 2010 Dec;13(4):201-7

Centre for Public Health, Institute of Clinical Science, Queens University of Belfast, Northern Ireland, UK.

Traditionally, the diagnosis of male infertility has relied upon microscopic assessment and biochemical assays to determine human semen quality. These tests are essential to provide the fundamental information on which clinicians base their initial diagnosis. However, none of these parameters addresses sperm function and their clinical value in predicting fertility is questionable. The advent of intracytoplasmic sperm injection (ICSI) has further reduced the significance and perceived need for sperm quality tests since ICSI requires only one sperm for the procedure to be successful. Even the conventional measures of sperm quality in terms of normal morphology or motility are not necessary for successful ICSI. Funding of andrological research has been neglected and improvement in assisted reproductive technology (ART) success has suffered as a consequence. Testing of sperm DNA damage shows much promise both as a diagnostic test for male infertility and a prognostic test for ART outcomes. It has been shown to be closely associated with numerous fertility outcomes including negative relationships with fertilization, embryo quality, implantation and positive relationships with miscarriage and childhood diseases. Here we report the relationships between in vitro fertilisation, ICSI pregnancy rates and sperm DNA damage, using the Comet assay to measure DNA fragmentation and also a novel test to measure modified bases, as a indication of oxidative DNA injury.
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http://dx.doi.org/10.3109/14647273.2010.528823DOI Listing
December 2010

Sperm DNA damage measured by the alkaline Comet assay as an independent predictor of male infertility and in vitro fertilization success.

Fertil Steril 2011 Feb 22;95(2):652-7. Epub 2010 Sep 22.

Centre for Public Health, Reproductive Medicine, Institute of Clinical Science, Queens University of Belfast, Northern Ireland, United Kingdom.

Objective: To evaluate sperm DNA fragmentation and semen parameters to diagnose male factor infertility and predict pregnancy after IVF.

Design: Prospective study.

Setting: Academic research laboratory.

Patient(s): Seventy-five couples undergoing IVF and 28 fertile donors.

Intervention(s): Sperm DNA fragmentation was measured by the alkaline Comet assay in semen and sperm after density gradient centrifugation (DGC). Binary logistic regression was used to analyze odds ratios (OR) and relative risks (RR) for IVF outcomes.

Main Outcome Measure(s): Semen parameters and sperm DNA fragmentation in semen and DGC sperm compared with fertilization rates, embryo quality, and pregnancy.

Result(s): Men with sperm DNA fragmentation at more than a diagnostic threshold of 25% had a high risk of infertility (OR: 117.33, 95% confidence interval [CI]: 12.72-2,731.84, RR: 8.75). Fertilization rates and embryo quality decreased as sperm DNA fragmentation increased in semen and DGC sperm. The risk of failure to achieve a pregnancy increased when sperm DNA fragmentation exceeded a prognostic threshold value of 52% for semen (OR: 76.00, CI: 8.69-1,714.44, RR: 4.75) and 42% for DGC sperm (OR: 24.18, CI: 2.89-522.34, RR: 2.16).

Conclusion(s): Sperm DNA testing by the alkaline Comet assay is useful for both diagnosis of male factor infertility and prediction of IVF outcome.
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http://dx.doi.org/10.1016/j.fertnstert.2010.08.019DOI Listing
February 2011

Cannabinoid receptor 1 influences chromatin remodeling in mouse spermatids by affecting content of transition protein 2 mRNA and histone displacement.

Endocrinology 2010 Oct 1;151(10):5017-29. Epub 2010 Sep 1.

Dipartimento di Medicina Sperimentale, Sez. Bottazzi, Seconda Università di Napoli, 80138 Naples, Italy.

Marijuana smokers and animals treated with Δ9-tetrahydrocannabinol, the principal component of marijuana, show alterations of sperm morphology suggesting a role for cannabinoids in sperm differentiation and/or maturation. Because the cannabinoid receptor 1 (CNR1) activation appears to play a pivotal role in spermiogenesis, the developmental stage where DNA is remodeled, we hypothesized that CNR1 receptors might also influence chromatin quality in sperm. We used Cnr1 null mutant (Cnr1-/-) mice to study the possible role of endocannabinoids on sperm chromatin during spermiogenesis. We demonstrated that CNR1 activation regulated chromatin remodeling of spermatids by either increasing Tnp2 levels or enhancing histone displacement. Comparative analysis of wild-type, Cnr1+/-, and Cnr1-/- animals suggested the possible occurrence of haploinsufficiency for Tnp2 turnover control by CNR1, whereas histone displacement was disrupted to a lesser extent. Furthermore, flow cytometry analysis demonstrated that the genetic loss of Cnr1 decreased sperm chromatin quality and was associated with sperm DNA fragmentation. This damage increased during epididymal transit, from caput to cauda. Collectively, our results show that the expression/activity of CNR1 controls the physiological alterations of DNA packaging during spermiogenesis and epididymal transit. Given the deleterious effects of sperm DNA damage on male fertility, we suggest that the reproductive function of marijuana users may also be impaired by deregulation of the endogenous endocannabinoid system.
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http://dx.doi.org/10.1210/en.2010-0133DOI Listing
October 2010

Clinical significance of sperm DNA damage in assisted reproduction outcome.

Hum Reprod 2010 Jul 6;25(7):1594-608. Epub 2010 May 6.

Centre for Public Health, Reproductive Medicine, Institute of Clinical Science, Queens University of Belfast, Grosvenor Road, Belfast BT12 6BJ, UK.

Background: Sperm DNA damage shows great promise as a biomarker of infertility. The study aim is to determine the usefulness of DNA fragmentation (DF), including modified bases (MB), to predict assisted reproduction treatment (ART) outcomes.

Methods: DF in 360 couples (230 IVF and 130 ICSI) was measured by the alkaline Comet assay in semen and in sperm following density gradient centrifugation (DGC) and compared with fertilization rate (FR), embryo cumulative scores (ECS(1)) for the total number of embryos/treatment, embryos transferred (ECS(2)), clinical pregnancy (CP) and spontaneous pregnancy loss. MB were also measured using formamidopyrimidine DNA glycosylase to convert them into strand breaks.

Results: In IVF, FR and ECS decreased as DF increased in both semen and DGC sperm, and couples who failed to achieve a CP had higher DF than successful couples (+12.2% semen, P = 0.004; +9.9% DGC sperm, P = 0.010). When MB were added to existing strand breaks, total DF was markedly higher (+17.1% semen, P = 0.009 and +13.8% DGC sperm, P = 0.045). DF was not associated with FR, ECS or CP in either semen or DGC sperm following ISCI. In contrast, by including MB, there was significantly more DNA damage (+16.8% semen, P = 0.008 and +15.5% DGC sperm, P = 0.024) in the group who did not achieve CP.

Conclusions: DF can predict ART outcome for IVF. Converting MB into further DNA strand breaks increased the test sensitivity, giving negative correlations between DF and CP for ICSI as well as IVF.
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http://dx.doi.org/10.1093/humrep/deq103DOI Listing
July 2010

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

Hum Reprod 2010 Apr 6;25(4):824-38. Epub 2010 Feb 6.

Reproductive and Developmental Biology, Maternal and Child Health Science Laboratories, Centre for Oncology and Molecular Medicine, Ninewells Hospital, University of Dundee, Dundee DD1 9SY, UK.

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 group's 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.
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http://dx.doi.org/10.1093/humrep/dep465DOI Listing
April 2010

Sperm DNA tests as useful adjuncts to semen analysis.

Syst Biol Reprod Med 2008 May-Jun;54(3):111-25

School of Medicine, Obstetrics and Gynaecology, Queen's University Belfast, Institute of Clinical Science, Belfast, Northern Ireland, UK.

Male infertility has traditionally been diagnosed by microscopic assessment of concentration, motility and morphology of sperm in the ejaculate. Most laboratories use sperm isolated by various methods such as density gradient centrifugation to enrich for subpopulations of sperm believed to have greater fertilization potential. These tests are essential to provide the fundamental information on which clinicians base their initial diagnosis. However, in the clinical setting, tests with superior prognostic value are needed. Tests showing much promise are those determining sperm DNA integrity, particularly the Comet, TUNEL, and Sperm Chromatin Structure assays. Sperm nuclear DNA fragmentation has been positively correlated with lower fertilization rates in IVF, impaired implantation rates, an increased incidence of abortion and disease in offspring, including childhood cancer. The mitochondrial genome of sperm has also been shown to be a sensitive marker of sperm health. Although the usefulness of these tests is recognized, insufficient resources have been available to develop standardized tests and protocols that could lead to universally accepted clinical thresholds. Associated with the lack of useful prognostic tests is the lack of improvement in assisted conception success rates despite thirty years of worldwide use. International collaborations should be initiated to develop agreed protocols and establish clinical thresholds.
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http://dx.doi.org/10.1080/19396360801957739DOI Listing
September 2008

Using the alkaline comet assay in prognostic tests for male infertility and assisted reproductive technology outcomes.

Mutagenesis 2008 May 5;23(3):163-70. Epub 2008 Mar 5.

Reproductive Medicine, Queen's University of Belfast, Institute of Clinical Science, Grosvenor Road, Belfast, BT12 6BJ, Northern Ireland, UK.

Infertility affects one in six couples in Europe during their reproductive years with dysfunctional sperm being one of the most common causes. Conventional semen analysis has proven variable and lacking in prognostic value so, over the past decade, more useful molecular fertility biomarkers have been explored. Among the tests showing most promise are those measuring sperm DNA quality. Sperm DNA damage has been closely associated with numerous indicators of reproductive health, including, fertilization, embryo quality, implantation, spontaneous abortion and childhood diseases. It therefore has great potential as a prognostic test for assisted reproductive treatment (ART), when couples are presenting with male infertility. Unlike somatic cells, sperm have a unique tightly compacted chromatin structure. Our group has modified the alkaline comet assay for use with sperm. Sperm DNA also differs from somatic cells in its high susceptibility to oxidative damage; this is largely due to the presence of abundant polyunsaturated fatty acids acting as substrates for reactive oxygen species (ROS) and its lack of repair mechanisms. Consequently, the effects of ROS and antioxidant protection on sperm DNA fragmentation have been widely investigated. In this review, the relationship between actual sperm DNA damage as determined by the alkaline comet assay and potential DNA damage as measured by DNA adduct testing will also be examined and the potential of routine clinical practices such as cryopreservation and prolonged incubation to induce further DNA damage was investigated. Finally, the usefulness of sperm DNA tests as prognostic markers and in particular, the opportunities and challenges provided by DNA testing in male fertility determination will be discussed.
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http://dx.doi.org/10.1093/mutage/gem052DOI Listing
May 2008

Sildenafil citrate (Viagra) impairs fertilization and early embryo development in mice.

Fertil Steril 2009 Mar 5;91(3):893-9. Epub 2008 Mar 5.

Obstetrics and Gynaecology, Institute of Clinical Science, School of Medicine, Belfast, United Kingdom.

Objective: To determine the effects of sildenafil citrate, a cyclic monophosphate-specific type 5 phosphodiesterase inhibitor known to affect sperm function, on fertilization and early embryo cleavage.

Design: This acute mammal study included male and female mice assigned randomly, the females sacrificed after mating and their oocytes/embryos evaluated at four time periods after treatment.

Setting: Academic research environment.

Animal(s): Male and female CBAB(6) mice.

Intervention(s): Female mice were injected intraperitoneally with 5 IU gonadotropin (hCG) to stimulate follicular growth and induce ovulation. They were each caged with a male that had been gavaged with sildenafil citrate (0.06 mg/0.05 mL) and allowed to mate. After 12, 36, 60, and 84 h, females were killed, their oviducts were dissected out, and retrieved embryos were assessed for blastomere number and quality.

Main Outcome Measure(s): Fertilization rates and numbers of embryos were evaluated after treatment.

Result(s): Fertilization rates (day 1) were markedly reduced (-33%) in matings where the male had taken sildenafil citrate. Over days 2-4, the numbers of embryos developing in the treated group were significantly fewer than in the control group. There was also a trend for impaired cleavage rates within those embryos, although this did not reach significance.

Conclusion(s): The impairments to fertility caused by sildenafil citrate have important implications for infertility centers and for couples who are using this drug precoitally while attempting to conceive.
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http://dx.doi.org/10.1016/j.fertnstert.2007.12.014DOI Listing
March 2009

Is sperm evaluation useful in predicting human fertility?

Authors:
Sheena E M Lewis

Reproduction 2007 Jul;134(1):31-40

School of Medicine, Obstetrics and Gynaecology, Queen's University Belfast, Institute of Clinical Science, Grosvenor Road, Belfast BT12 6BA, UK.

Traditionally, the diagnosis of male infertility has relied upon microscopic assessment and biochemical assays to determine human semen quality. The conventional parameters given most importance have been the concentration, motility, and morphology of sperm in the ejaculate. Most laboratories also include 'sperm suitability' tests where the subpopulations of sperm more likely to finish the marathon journey to the oocyte are separated by density centrifugation. These tests are essential to provide the fundamental information on which clinicians base their initial diagnosis. However, none of these parameters addresses sperm function and their clinical value in predicting fertility is questionable. The advent of intracytoplasmic sperm injection (ICSI) has further reduced the significance and perceived need for sperm quality tests since ICSI requires only one sperm, not subject to classic, or indeed any, tests for the procedure to be successful. Over the past decade, a number of laboratory tests have been developed to determine properties of sperm function. These include quantitative sperm motion parameters, capacitation, basal and induced acrosome reactions, sperm-zona pellucida interactions and nuclear and mitochondrial sperm DNA but few have been adopted into routine clinical use. International collaborations should be initiated to develop clinically relevant molecular and functional tests with agreed protocols and clinical thresholds as a matter of urgency.
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http://dx.doi.org/10.1530/REP-07-0152DOI Listing
July 2007

Sildenafil citrate improves sperm motility but causes a premature acrosome reaction in vitro.

Fertil Steril 2007 May 28;87(5):1064-70. Epub 2007 Feb 28.

School of Medicine, Obstetrics and Gynaecology, Queen's University Belfast, Institute of Clinical Science, Belfast, United Kingdom.

Objective: To determine whether sildenafil citrate, a cyclic monophosphate-specific type 5 phosphodiesterase inhibitor, influences sperm motility or the acrosome reaction.

Design: Laboratory analysis of sperm motility after exposure to sildenafil citrate using computer-assisted semen analysis and acrosome reaction by fluorescein isothiocyanate-labeled peanut agglutinin staining.

Setting: An assisted reproductive technology (ART) unit.

Patient(s): Fifty-seven male patients.

Intervention(s): Sperm were divided into 90% (those with the best fertilizing potential used in assisted conception) and 45% (the poorer population) fractions by density centrifugation and incubated with sildenafil citrate (0.67 muM) at 37 degrees C for up to 180 minutes.

Main Outcome Measure(s): Both the number and velocity of progressively motile sperm were significantly increased by sildenafil citrate between 15 and 135 minutes. Furthermore, samples revealed that these effects were consistent in the 90% and 45% populations of sperm. In both populations, sildenafil also caused a significant increase in the proportion of acrosome-reacted sperm-22.1% compared with 11.8% in the control group of the good quality fraction and 16.6% compared with 9.4% in the control group of the poorer quality fraction.

Conclusion(s): The use of sildenafil citrate may adversely affect male fertility.
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http://dx.doi.org/10.1016/j.fertnstert.2006.11.017DOI Listing
May 2007

Reduced sperm yield from testicular biopsies of vasectomized men is due to increased apoptosis.

Fertil Steril 2007 Apr 22;87(4):834-41. Epub 2007 Jan 22.

Obstetrics and Gynaecology, School of Medicine, Queen's University Belfast, Institute of Clinical Science, Belfast, United Kingdom.

Objective: To compare sperm yields, apoptotic indices, and sperm DNA fragmentation from vasectomized men and fertile men undergoing vasectomy.

Design: Testicular biopsies from vasectomized (n = 26) and fertile men (n = 46), were milked to calculate sperm/gram and also formalin-fixed to determine the numbers of developing sperm and incidence and intensities of testicular FasL, Fas, Bax, and Bcl-2. Testicular sperm DNA fragmentation was assessed using the alkaline Comet assay.

Setting: An ART unit.

Patient(s): Twenty-six men attending for intracytoplasmic sperm injection (ICSI) and 46 men attending for vasectomies.

Main Outcome Measure(s): Spermatocyte, spermatid and sperm yields, Fas, FasL, and Bax staining.

Result(s): Sperm yields from men vasectomized >5 years previously were markedly reduced compared to fertile men. Increased intensities of FasL and Bax staining were observed in the seminiferous tubules of vasectomy men. FasL positivity (percentage) also increased in Sertoli cells, and both FasL and Fas positivity (percentage) increased in primary spermatocytes and round spermatids of vasectomized men. Sperm DNA fragmentation, an end point marker of apoptosis, increased significantly in vasectomized men compared to fertile men.

Conclusion(s): Reduced sperm yields after vasectomy are associated with increased apoptosis through the Fas-FasL and Bax pathways. Sperm after vasectomy displayed increased DNA fragmentation.
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http://dx.doi.org/10.1016/j.fertnstert.2006.11.018DOI Listing
April 2007

Sperm and embryo cryopreservation practice in licensed clinics in the UK endorsed by The British Fertility Society.

Hum Fertil (Camb) 2006 Mar;9(1):15-26

Obstetrics and Gynaecology, Queen's University Belfast, UK.

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http://dx.doi.org/10.1080/14647270500230031DOI Listing
March 2006

Effects of delta-9-tetrahydrocannabinol, the primary psychoactive cannabinoid in marijuana, on human sperm function in vitro.

Fertil Steril 2006 Mar;85(3):653-60

Obstetrics & Gynaecology, School of Medicine, Queen's University, Belfast, United Kingdom.

Objective: To investigate effects of delta-9-tetrahydrocannabinol (THC) on human sperm function in vitro.

Design: Laboratory analysis of sperm motility after exposure to THC using computer-assisted semen analysis and acrosome reaction by fluoroscein isothiocyanate-labeled peanut agglutinin staining.

Setting: An assisted reproductive technology unit.

Patient(s): Seventy-eight male patients.

Intervention(s): Sperm were divided into 90% (the best fertilizing potential used in assisted conception) and 45% (the poorer subpopulation) fractions by density centrifugation and incubated with THC at concentrations equivalent to therapeutic (0.032 microM) and recreational (0.32 and 4.8 microM) plasma levels at 37 degrees C for 3 h.

Main Outcome Measure(s): Sperm motility and spontaneous and induced acrosome reactions.

Result(s): Percentage progressive motility was decreased dose dependently in the 90% fraction (by 2%-21%; P<.05; P<.001). The 45% fraction showed a greater decrease in percentage progressive motility (by 28% at 0.032 microM; 56% at 4.8 microM; P=.004 and P=.01 res). Straight line velocity and the average path velocity also were reduced (by 10%, in the 90% LAYER) in both fractions. Spontaneous acrosome reactions were reduced in the 90% (17% at 0.032 microM, 35% at 4.8 microM P=.004 and P<.001 resp) and more markedly in the 45% fractions (17%-35%; P<.001). When the acrosome reaction was artificially induced (90% fraction) by A23187, THC (4.8 microM) resulted in a 57% inhibition (P<.001).

Conclusion(s): The use of THC as a recreational drug may adversely affect male fertility.
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http://dx.doi.org/10.1016/j.fertnstert.2005.08.027DOI Listing
March 2006

Dietary oestrogens and male fertility potential.

Hum Fertil (Camb) 2005 Sep;8(3):197-207

Obstetrics & Gynaecology, School of Medicine, Institute of Clinical Science, Queen's University Belfast, UK.

Reports of increased incidences of male reproductive abnormalities and falling sperm counts have prompted interest into the nature of these threats to global fertility. Xenoestrogens have been flagged as major culprits but to date, little is known about the effects of dietary phytoestrogens on male reproductive health. These non-steroidal oestrogens of plant origin are potent endocrine disruptors that modulate normal physiological functions. Phytoestrogens have become a major component in the typical Western fast food diet over the last few decades. Soy formula milk is another common source of phytoestrogens, now used increasingly as an alternative to breast or cow's milk for infants with allergies. This use is of particular concern since the most vulnerable periods for oestrogenic insult are thought to be the pre- and neonatal periods when irreversible damage can be inflicted on the developing germinal epithelium. Studies into the safety of phytoestrogens are urgently needed either to allay fears or increase awareness of the effects of our modern diet on future fertility.
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http://dx.doi.org/10.1080/14647270500030266DOI Listing
September 2005

Effects of short and long incubations on DNA fragmentation of testicular sperm.

Fertil Steril 2004 Nov;82(5):1443-5

DNA fragmentation in testicular sperm from men with obstructive azoospermia is increased by 4-hour and 24-hour incubations and after cryopreservation with the effect is intensified by post-thaw incubation. Testicular sperm for use in intracytoplasmic sperm injection (ICSI) should be injected without delay.
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http://dx.doi.org/10.1016/j.fertnstert.2004.04.053DOI Listing
November 2004

Seeds of concern.

Nature 2004 Nov;432(7013):48-52

Discipline of Biological Sciences, School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales 2308, Australia.

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http://dx.doi.org/10.1038/432048aDOI Listing
November 2004

Incidence of Fas positivity and deoxyribonucleic acid double-stranded breaks in human ejaculated sperm.

Fertil Steril 2004 Mar;81 Suppl 1:767-74

Department of Obstetrics and Gynaecology, School of Medicine, Queens University Belfast, Institute of Clinical Science, Grosvenor Road, Belfast BT12 6BJ, N. Ireland, UK.

Objective: To determine the incidence of Fas positivity and DNA double-strand breaks (DSB) as indicators of early- and late-stage apoptosis in ejaculated sperm.

Design: Fas positivity was assessed by flow cytometry and DSB by the neutral Comet assay.

Setting: Andrology Laboratory, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom.

Patient(s) And Intervention(s): Forty-five infertile men undergoing infertility investigations and 10 fertile men undergoing vasectomies.

Main Outcome Measure(s): Percentage Fas-positive cells, percentage DNA fragmentation, olive tail moment.

Result(s): The apoptotic marker Fas was detected in ejaculated sperm, with a higher incidence of Fas positivity in teratozoospermic and asthenozoospermic than in normozospermic semen. No Fas positivity was observed in fertile men's sperm. Deoxyribonucleic acid fragmentation (DSB) was greater in infertile than in fertile men's sperm and also greater in sperm in semen than in sperm prepared for assisted conception. There was an inverse relationship between DSB and both sperm concentration and motility. There was no relationship between Fas positivity and DNA damage.

Conclusion(s): Fas was expressed in sperm of infertile men. In contrast, DNA fragmentation was observed in all sperm of fertile and infertile men and correlated with inadequate concentration and motility, which suggests that sperm DSB are ubiquitous and are not solely associated with apoptosis.
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http://dx.doi.org/10.1016/j.fertnstert.2003.10.013DOI Listing
March 2004