Christopher J. De Jonge

Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage

We report studies of preimplantation human embryo development that correlate time-lapse image analysis and gene expression profiling. By examining a large set of zygotes from in vitro fertilization (IVF), we find that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA). These parameters can be reliably monitored by automated image analysis, confirming that successful development follows a set of carefully orchestrated and predictable events. Moreover, we show that imaging phenotypes reflect molecular programs of the embryo and of individual blastomeres. Single-cell gene expression analysis reveals that blastomeres develop cell autonomously, with some cells advancing to EGA and others arresting. These studies indicate that success and failure in human embryo development is largely determined before EGA. Our methods and algorithms may provide an approach for early diagnosis of embryo potential in assisted reproduction.

Semen analysis: looking for an upgrade in class

OBJECTIVE To review the literature database regarding current methods for diagnosing male subfertility and to critique new testing methods that have the potential to be incorporated as part of routine semen analysis. DESIGN Literature database review. SETTING None. PATIENT(S) None. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) None. RESULT(S) Methods for performing high quality semen analysis are standardized and clearly presented in the 2010 World Health Organization (WHO) laboratory manual for the examination and processing of human semen. In spite of the fact that lower reference limits contained therein are now evidence-based, debate persists about their clinical value. One test put forth as being additive to the semen analysis is assessment of DNA integrity. However, due to lack of standardized methods and quality control measures, accessibility to instrumentation, and evidence-based reference values for clinical interpretation this test has not become incorporated into the clinical andrology mainstream. Novel tests that probe molecular function have emerged that also have promise for integration into routine clinical semen analysis. CONCLUSION(S) Semen analysis, when performed according to WHO guidelines, will yield accurate and precise clinical laboratory data on traditional semen parameters. Due to the biological nature of the specimen in question definitive diagnosis of subfertility and its cause(s) remains enigmatic. Novel tests that may be easily standardized for subsequent multi-center, prospective randomized trials need to be integrated so more meaningful clinical diagnoses can be made.

The clinical value of sperm nuclear DNA assessment

Traditional semen analysis is essential for the diagnosis of male infertility. A number of studies over the past decade have reported that a significant contributing factor to male fertility that is not revealed as part of semen analysis is sperm DNA, specifically its composition and organization. Exogenous and endogenous factors can cause damage to sperm DNA. For example, topoisomerase II activity, which is necessary for sperm DNA packaging, can adversely influence the competence of sperm DNA if the activity of the enzyme is abnormal. Germ cell apoptosis can be induced by oxygen radicals produced from environmental (for example cigarette smoke) or testicular (for example localized ischaemia) sources. Several assays have been developed that are useful for assessing sperm DNA composition and organization. To date, each of these assays has revealed that when sperm DNA has been damaged or packaged improperly there is a concomitant and often significant decline in male fertility.Traditional semen analysis is essential for the diagnosis of male infertility. A number of studies over the past decade have reported that a significant contributing factor to male fertility that is not revealed as part of semen analysis is sperm DNA, specifically its composition and organization. Exogenous and endogenous factors can cause damage to sperm DNA. For example, topoisomerase II activity, which is necessary for sperm DNA packaging, can adversely influence the competence of sperm DNA if the activity of the enzyme is abnormal. Germ cell apoptosis can be induced by oxygen radicals produced from environmental (for example cigarette smoke) or testicular (for example localized ischaemia) sources. Several assays have been developed that are useful for assessing sperm DNA composition and organization. To date, each of these assays has revealed that when sperm DNA has been damaged or packaged improperly there is a concomitant and often significant decline in male fertility.

Comparison of epigenetic mediator expression and function in mouse and human embryonic blastomeres

A map of human embryo development that combines imaging, molecular, genetic and epigenetic data for comparisons to other species and across pathologies would be greatly beneficial for basic science and clinical applications. Here, we compared mRNA and protein expression of key mediators of DNA methylation and histone modifications between mouse and human embryos, embryos from fertile/infertile couples, and following growth factor supplementation. We observed that individual mouse and human embryos are characterized by similarities and distinct differences in DNA methylation and histone modification patterns especially at the single-cell level. In particular, while mouse embryos first exhibited sub-compartmentalization of different histone modifications between blastomeres at the morula stage and cell sub-populations in blastocysts, differential histone modification expression was detected between blastomeres earlier in human embryos at the four- to eight-cell stage. Likewise, differences in epigenetic mediator expression were also observed between embryos from fertile and infertile couples, which were largely equalized in response to growth factor supplementation, suggesting that select growth factors might prevent alterations in epigenetic profiles during prolonged embryo culture. Finally, we determined that reduced expression via morpholino technologies of a single histone-modifying enzyme, Rps6ka4/Msk2, resulted in cleavage-stage arrest as assessed by time-lapse imaging and was associated with aneuploidy generation. Taken together, data document differences in epigenetic patterns between species with implications for fertility and suggest functional roles for individual epigenetic factors during pre-implantation development.

Clinical relevance of sperm DNA assessment: an update

For sperm DNA assessments to be widespread effective clinical tools, key objectives need to be urgently achieved. These include robust standardized methods, systems for training staff and ongoing quality control; and rigorous examination of the clinical outcomes. We discuss the significance of these objectives and conclude that, if achieved, the clinical utility of DNA damage assessment will be fully realized.

The diagnosis of male infertility: an analysis of the evidence to support the development of global WHO guidance—challenges and future research opportunities

Abstract BACKGROUND Herein, we describe the consensus guideline methodology, summarize the evidence-based recommendations we provided to the World Health Organization (WHO) for their consideration in the development of global guidance and present a narrative review of the diagnosis of male infertility as related to the eight prioritized (problem or population (P), intervention (I), comparison (C) and outcome(s) (O) (PICO)) questions. Additionally, we discuss the challenges and research gaps identified during the synthesis of this evidence. OBJECTIVE AND RATIONALE The aim of this paper is to present an evidence-based approach for the diagnosis of male infertility as related to the eight prioritized PICO questions. SEARCH METHODS Collating the evidence to support providing recommendations involved a collaborative process as developed by WHO, namely: identification of priority questions and critical outcomes; retrieval of up-to-date evidence and existing guidelines; assessment and synthesis of the evidence; and the formulation of draft recommendations to be used for reaching consensus with a wide range of global stakeholders. For each draft recommendation the quality of the supporting evidence was then graded and assessed for consideration during a WHO consensus. OUTCOMES Evidence was synthesized and recommendations were drafted to address the diagnosis of male infertility specifically encompassing the following: What is the prevalence of male infertility and what proportion of infertility is attributable to the male? Is it necessary for all infertile men to undergo a thorough evaluation? What is the clinical (ART/non ART) value of traditional semen parameters? What key male lifestyle factors impact on fertility (focusing on obesity, heat and tobacco smoking)? Do supplementary oral antioxidants or herbal therapies significantly influence fertility outcomes for infertile men? What are the evidence-based criteria for genetic screening of infertile men? How does a history of neoplasia and related treatments in the male impact on (his and his partners) reproductive health and fertility options? And lastly, what is the impact of varicocele on male fertility and does correction of varicocele improve semen parameters and/or fertility? WIDER IMPLICATIONS This evidence synthesis analysis has been conducted in a manner to be considered for global applicability for the diagnosis of male infertility.

Methods for the Assessment of Sperm Capacitation and Acrosome Reaction Excluding the Sperm Penetration Assay

Assessing the ability of human spermatozoa to acquire fertilizing potential (capacitation) by stimulating exocytosis of the contents of the acrosome (acrosome reaction) is thought to have diagnostic potential (De Jonge, Reprod Med Rev 3:159-178, 1994). Calcium-mobilizing agents, such as calcium ionophores (A23187) and progesterone, stimulate the acrosome reaction in vitro (Brucker and Lipford, Hum Reprod Update 1:51-62, 1995). Acrosomal status is easily detected using Pisum sativum Agglutinin labeled with fluorescein isothiocyanate (Cross and Meizel, Biol Reprod 41:635-641, 1989). Herein we describe a procedure for assessing capacitation and the acrosome reaction of human spermatozoa in vitro.

‘Man Up’: the importance and strategy for placing male reproductive health centre stage in the political and research agenda

Abstract Approximately 1 in 20 young men today have sperm counts low enough to impair fertility, whereas this may not have been the case historically. The cause(s) of such a decline in male reproductive health is unknown, despite it being a global health issue. Concomitantly, little progress has been made in answering fundamental questions in andrology or in developing new diagnostic tools or alternative management strategies to ICSI in infertile men. We advocate formulation of a detailed roadmap for male reproductive health to facilitate development of a research agenda that highlights the present unmet needs and key unanswered questions, and seeks to deliver effective funding and investment to address them. This vision we term ‘a Male Reproductive Health Ecosystem’.

Paternal age and sperm methylation status

The elaboration of knowledge regarding the human genome has simultaneously given rise to a plethora of additional areas of research interest and for which an appreciation of their influence(s) on human genesis has grown. One area, termed epigenetics, focuses on nongenomic influences on gene function and that reside outside the DNA sequence. Put simply, methylation near specific loci (CpG islands) adjacent or even distal (CpG island shores) from gene promotors regulates gene expression. In healthy individuals, CpG islands are, in general, methylation free. CpG areas in the genome not associated with CpG islands are methylated. Methylation is involved in gene silencing. The article by Jenkins et al. (1) used donor semen from healthy normal males to investigate paternal aging and associated alterations (methylation status) in epigenetic regulatory loci, for which changes in regulatory function may have a potential impact on the well-being of offspring in either adolescence or adulthood. It is becoming reasonably well-established that paternal age at offspring birth is associated with an increased risk of not only spontaneous abortion and birth defects but also neurogenic disorders such as autism (2). The causative reason(s) is/are not well-established, but certainly documented candidate suspects include increased aneuploidies, chromatin anomalies, and DNA damage, to name a few (3). Even more obscure is the question of whether there is a paternal agerelated association with epigenetic abnormalities and the subsequent well-being of offspring. Answers to this question have become all the more pressing given the availability of assisted reproductive technologies to help couples of more advanced age procreate. A recent review (3) surveyed the literature database for evidence to link epigenetic alterations and male fertility. It is interesting that abnormalities in methylation, via loss or gain on imprinted genes, histone retention, and transcript deficiencies involved in spermatogenesis appear to all be linked. The consequence(s) of this ‘‘perfect storm’’ may have an impact not only on male fertility but also embryo development. For the former, it was reported that poor-quality spermatozoa demonstrate broad epigenetic abnormalities, as reflected by hypermethylation that extends beyond expected imprinted loci (i.e., global methylation) (4). In regards to paternal influences on embryo development, a recent article (5) provided data using donor sperm that suggests modulation of different regulatory pathways depending onwhether sperm are in a hypoor hypermethylated state. The former condition (hypomethylation) is associated with regulators of cell renewal, and the latter hyper state is suggested to be a mechanism for ‘‘locking in’’ aspects of differentiation associated with the developmental regulators involved in human embryogenesis. With the emerging literature database reflecting that poorquality spermatozoa contain elevated levels of global methylation, the question arises of what the methylation profile is for spermatozoa from normal healthy sperm donors. Jenkins et al. (1) have mined a donor semen depository and analyzed global methylation alterations of regulatory intermediates: 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine

The future of reproductive cellular engineering in male infertility.

Understanding of the pathophysiology of defective spermatogenesis, spermatozoa, and germ cell function must be advanced so that appropriate rational treatment, such as gene therapy, can be developed. Despite the dramatic advances in ART, one ultimate goal must be to develop treatment so that the couple can conceive naturally. Because such treatment will not be possible in all cases, a complementary step will be the ability to induce the production of haploid, functionally competent germ cells that can be used for ART. The achievement of these goals will be based on advances in many other disciplines. Whatever the future holds, it promises to be exciting.