Showing papers in "Systems Biology in Reproductive Medicine in 2012"

Effect of smoking on semen quality, FSH, testosterone level, and CAG repeat length in androgen receptor gene of infertile men in an Indian city

Abstract: This study was conducted as part of an epidemiological survey of 126 nonsmokers and 178 smokers, showing primary infertility residing around Kolkata region of Eastern India. Their lifestyle history including smoking habits along with semen and blood were collected. The study examined the association of cigarette smoking with the risk of infertility, by determining the semen quality, follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone levels, and androgen receptor (AR)-CAG repeat length in a group of smokers compared with a control group (non smokers). Based on conventional WHO criteria, lower sperm motility (P < 0.001) and increased sperm morphological defects (P < 0.0001) were associated with smoking habits. Binary logistic regression analysis for the effect of smoking status on sperm DNA integrity demonstrated significant positive correlation (p = 0.006). Serum FSH and LH levels were higher for smokers compared with non-smokers while the testosterone level decreased significantly ...

The contribution of proteomics to understanding epididymal maturation of mammalian spermatozoa.

Abstract: The acquisition of the ability of the male gamete to fertilize an ovum is the result of numerous and sequential steps of differ- entiation of spermatozoa that occur as they transit from the testis to the end of the epididymal tubule. The post gonadal sperm modifications are mostly related to motility, egg bind- ing, and penetration processes. As the activity of the epididy- mis and its luminal fluid composition are believed to be directly related to 'sperm maturation', a review on epididymal proteins is presented. Comparative studies have shown that the epididymal activities are species specific. Nevertheless, for all mammalian species studied, similarities exist in the sequential proteomic changes of the luminal composition of the epididy- mal tubule and proteins on the sperm surface. The potential roles of these modifications are discussed.

4-vinylcyclohexene diepoxide: a model chemical for ovotoxicity.

Abstract: The occupational chemical 4-vinylcyclohexene diepoxide (VCD) has been shown to cause selective destruction of ovarian small pre-antral (primordial and primary) follicles in rats and mice by accelerating the natural, apoptotic process of atresia. Chemicals that destroy primordial follicles are of concern to women because exposure can result in premature ovarian failure (early menopause). In`itial studies using in vivo exposure of rats determined that VCD specifically targets primordial and primary (small pre-antral) follicles and that repeated dosing is required. Through a method of isolation of ovarian small follicles, biochemical and molecular studies determined that intracellular pro-apoptotic pathways are activated following VCD dosing in rats. Subsequently an in vitro system using cultured whole neonatal rat ovaries was developed to provide more mechanistic information. That approach was used to demonstrate that the cell survival c-kit//kit ligand signaling pathway is the direct target for VCD-induced...

Proteomic insights into the maturation and capacitation of mammalian spermatozoa

Abstract: Spermatozoa represent the epitome of terminally differentiated, highly specialized cells. They are transcriptionally and translationally silent and yet manage to undergo a complete functional transformation after they leave the testes, entirely fuelled by post-translational modifications occurring during epididymal maturation and capacitation. The latter have been recognized as biological processes for more than half a century. However, the biochemical mechanisms that drive these events have remained elusive, as have the pathological mechanisms that lead to defective sperm function and infertility. In the past decade the combined power of advanced proteomics, biochemistry, and functional genomics has permitted an unprecedented improvement in our understanding of sperm cell biology. We can also predict that a systems-biology approach, in concert with the new tools provided by the 'omics' revolution, will lead to dramatic gains in our understanding in the near future. As a result of such advances, insights will be generated that should ultimately lead to significant improvements in our capacity to diagnose and treat the infertile male.

Spermatogenesis in mammals: proteomic insights

Abstract: Spermatogenesis is a highly sophisticated process involved in the transmission of genetic heritage. It includes halving ploidy, repackaging of the chromatin for transport, and the equipment of developing spermatids and eventually spermatozoa with the advanced apparatus (e.g., tightly packed mitochondrial sheat in the mid piece, elongating of the tail, reduction of cytoplasmic volume) to elicit motility once they reach the epididymis. Mammalian spermatogenesis is divided into three phases. In the first the primitive germ cells or spermatogonia undergo a series of mitotic divisions. In the second the spermatocytes undergo two consecutive divisions in meiosis to produce haploid spermatids. In the third the spermatids differentiate into spermatozoa in a process called spermiogenesis. Paracrine, autocrine, juxtacrine, and endocrine pathways all contribute to the regulation of the process. The array of structural elements and chemical factors modulating somatic and germ cell activity is such that the network linking the various cellular activities during spermatogenesis is unimaginably complex. Over the past two decades, advances in genomics have greatly improved our knowledge of spermatogenesis, by identifying numerous genes essential for the development of functional male gametes. Large-scale analyses of testicular function have deepened our insight into normal and pathological spermatogenesis. Progress in genome sequencing and microarray technology have been exploited for genome-wide expression studies, leading to the identification of hundreds of genes differentially expressed within the testis. However, although proteomics has now come of age, the proteomics-based investigation of spermatogenesis remains in its infancy. Here, we review the state-of-the-art of large-scale proteomic analyses of spermatogenesis, from germ cell development during sex determination to spermatogenesis in the adult. Indeed, a few laboratories have undertaken differential protein profiling expression studies and/or systematic analyses of testicular proteomes in entire organs or isolated cells from various species. We consider the pros and cons of proteomics for studying the testicular germ cell gene expression program. Finally, we address the use of protein datasets, through integrative genomics (i.e., combining genomics, transcriptomics, and proteomics), bioinformatics, and modelling.

Significant improvement of sperm DNA quality after microsurgical repair of varicocele.

Abstract: Varicocele is currently the most common irregularity identified in males that is associated with impaired spermatogenesis. It primarily presents in the form of decreased sperm count and motility, abnormal morphology, and significantly increased sperm DNA fragmentation. Several studies have shown that surgical repair improves semen parameters and increases the odds of spontaneous pregnancy. However the exact effect of surgical repair treatment remains controversial. Therefore, the aim of our study was to evaluate the effectiveness of microsurgical repair by comparing common semen parameters and sperm DNA fragmentation index (DFI). We evaluated infertile men (n = 19) who underwent microsurgical subinguinal varicocelectomy for treatment of clinical varicocele before and 3 months after surgery. Normozoospermic men (n = 19) were considered as the normal control group. Semen parameters improved significantly after surgery when compared with that before surgery, but still significant differences with the normal control group were observed. In comparison, sperm DNA integrity improved significantly after surgery (percentage DFI decreased from 28.4 ± 15.6% before surgery to 22.4 ± 12.9%, at 3 months post surgery) to similar levels as the normal control group. These results suggest that microsurgical repair may be considered as a treatment option in infertile men with palpable varicocele.

Developmental competence in oocytes and cumulus cells: candidate genes and networks.

Abstract: Common aspects of infertility can be seen across several species. In humans, dairy cows, and mares there is only a 25-35% chance of producing a live offspring after a single insemination, whether natural or artificial. Oocyte quality and subsequent embryo development can be affected by factors such as nutrition, hormonal regulation, and environmental influence. The objective of this study was to identify genes expressed in oocytes and/or cumulus cells, across a diverse range of species, which may be linked to the ability an oocyte has to develop following fertilization. Performing a meta-analysis on previously published microarray data on various models of oocyte and embryo quality allowed for the identification of 56 candidate genes associated with oocyte quality across several species, 4 of which were identified in the cumulus cells that surround the oocyte. Twenty-one potential biomarkers were associated with increased competence and 35 potential biomarkers were associated with decreased competence. The upregulation of Metap2, and the decrease of multiple genes linked to mRNA and protein synthesis in models of competence, highlights the importance of de novo protein synthesis and its regulation for successful oocyte maturation and subsequent development. The negative regulation of Wnt signaling has emerged in human, monkey, bovine, and mouse models of oocyte competence. Atrx expression was linked to decreased competence in both oocytes and cumulus cells. Biological networks and transcription factor regulation associated with increased and decreased competence were also identified. These genes could potentially act as biomarkers of oocyte quality or as pharmacological targets for manipulation in order to improve oocyte developmental potential.

A comparison of ejaculated and testicular spermatozoa aneuploidy rates in patients with high sperm DNA damage

Abstract: Testicular spermatozoa are utilized to achieve pregnancy in couples with severe male factor infertility. Several studies suggest that aneuploidy rates in spermatozoa are elevated at the testicular level in infertile patients compared to ejaculates of normal controls. However, essential data regarding aneuploidy rates between ejaculated and testicular spermatozoa in the same individuals is lacking. The purpose of our study was to compare aneuploidy rates at the testicular and post-testicular level from the same patients with persistently high sperm DNA damage. Ejaculates and testicular biopsies were obtained from eight patients with persistently high DNA damage (>30%). Both ejaculated and testicular samples were analyzed for sperm DNA damage and sperm aneuploidy for chromosomes 13, 18, 21, X, and Y. In addition, semen samples from ten normozoospermic men presenting for fertility evaluation served as a control group. A strong correlation between the alteration of spermatogenesis and chromatin deterioration ...

DHR123: an alternative probe for assessment of ROS in human spermatozoa

Abstract: The aim of this study was to assess the potential of dihydrorhodamine 123 (DHR123) to measure oxidative stress produced by human spermatozoa. The results were compared with 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) that is routinely used for assessment of H2O2 produced by spermatozoa. Fluorescence intensity and percentage R123 and DCF positive sperm were measured by flow cytometry. The optimal condition for assessment of reactive oxygen species (ROS) produced by sperm with DHR123 was 0.05 µM for 1 million sperm per ml for 20 minutes. The results of ROS measurement by DHR123 showed a significant correlation (r = +0.818, P < 0.001) with DCFH-DA staining. Immunofluorescence of sperm stained with DHR123 revealed ROS production in the sperm mid-piece. In addition a significant correlation was observed between oxidant production assessed by DHR123 and semen parameters. Therefore, DHR123 may be considered a suitable probe for estimating oxidants produced by human spermatozoa, and can present heteroge...

Oxidative stress markers in follicular fluid of women undergoing in vitro fertilization and embryo transfer

Abstract: The aim of the study was to evaluate the levels of lipid and protein peroxidation markers, in the follicular fluids (FF) of 82 patients undergoing in vitro fertilization (IVF). This included, thiobarbituric acid-reactive substances (TBARS), protein carbonyl, and thiol groups. The oxidative stress markers were compared between the pregnancy positive and pregnancy negative patient groups. The two patient groups were compared in terms of their age, body mass index (BMI), cause of infertility, and the plasma hormone levels (AMH, E2, peak E2). Protein carbonyl and thiol groups were estimated using an ELISA assay and with Ellman's reagent (5, 5'-dithiobis-2-nitrobenzoic acid, DTNB), respectively. The mean FF TBARS level of 29 pregnant women was 0.954 ± 0.420 µmol/l, whereas it was twice as high (1.961 ± 0.796 µmol/l) in a group of 53 non-pregnant patients (p < 0.0001). In non-pregnant patients, we observed 2-fold elevated levels of protein carbonyl groups when compared to pregnant women (2.969 ± 0.723 vs. 1.523...

Effects of hypoxia on the expression of inflammatory markers IL-6 and TNF-a in human normal peritoneal and adhesion fibroblasts.

Abstract: Inflammation is known to be involved in the postoperative adhesion development. Interleukin (IL)-6 and tumor necrosis factor (TNF)-α are cytokines that stimulate the acute-phase reaction, which leads to a systemic reaction including inflammation, fever, and activation of the complement and clotting cascades. The goal of this study was to examine the expression of these inflammatory markers, under normal and hypoxic conditions, in normal and adhesion fibroblasts. Primary cultures of fibroblasts were established from normal peritoneum and adhesion tissues from the same patient(s) and cultured under 20% O(2) or hypoxic 2% O(2) conditions for 24 hours. Cells were harvested and total RNA was isolated. Complimentary DNA was generated by reverse transcription and subjected to real-time RT-PCR using specific primers for IL-6 and TNF-α. Both normal peritoneal and adhesion fibroblasts expressed IL-6 and TNF-α. Adhesion fibroblasts exhibited significantly higher levels of IL-6 and TNF-α mRNA as compared to normal peritoneal fibroblasts (p < 0.05). Both IL-6 and TNF-α mRNA levels were upregulated in response to hypoxia in both normal peritoneal and adhesion fibroblasts. The increase in IL-6 and TNF-α mRNA levels of normal fibroblasts reached the levels observed in adhesion fibroblasts. Our results suggest that hypoxia promotes the development of the adhesion phenotype by the induction of inflammatory markers, which may contribute to the development of postoperative adhesions. The inhibition of inflammation may be a potential therapeutic approach in the prevention and/or reduction of postoperative adhesion development.

Proteomic discovery of diverse immunity molecules in mammalian spermatozoa

Abstract: Ongoing proteomic analyses are providing a wealth of new data on the composition of the sperm proteome across a range of mammals and other taxa. Although molecular evolution and functional genomic analyses of the proteome have only begun recently, we now broadly understand the molecular composition of sperm. Systems level analysis has revealed a variety of molecular insights into sperm evolution and function, including a remarkable diversity of immunity-related proteins within the proteome. Using existing mammalian sperm proteomes as a starting point, we provide an overview of this important class of sperm proteins and what is known about their function in sperm maturation, sperm quality, sperm competition, and fertilization. The recent observation that many sperm immunity proteins are rapidly evolving, presumably under the influence of positive selection, suggests that they may be responding not only to selection associated with host immunity defense but also with pleiotropic functions in sperm. In addition to the documented role of sperm in the mediation of female immune response, we propose that the fundamental mechanisms involved in cell-cell recognition and binding in both immune processes and fertilization may underlie the multi-functionality of proteins in immunity and reproductive systems.

Kisspeptins and the control of gonadotrophin secretion.

Abstract: Kisspeptins, the peptide products of the KiSS-1 gene, bind to the G protein coupled receptor 54 (GPR54). Since 2003, research has revealed the important role of kisspeptins in initiating puberty, timing puberty and regulating fertility in adulthood. Specific mutations in GPR54 gene cause either delayed/absent puberty or precocious puberty. The KiSS-1/GPR54 system stimulates the gonadotrophin releasing hormone (GnRH) neurons and is involved in the feedback regulation of the HPG axis by gonadal steroids. Different hypothalamic nuclei are involved in negative (arcuate nucleus; ARC) and positive (anteroventral periventricular nucleus; AVPV) feedback in mice. Continuous administration of kisspeptins down-regulates the HPG axis. During pregnancy, kisspeptins are secreted from the placenta in large amounts and are responsible for the physiological invasion of primary human trophoblast. Kisspeptins have been administered to normal male and female individuals as well as to women with hypothalamic secondary amenorr...

Alternative models in developmental toxicology.

Abstract: In light of various pressures, toxicologists have been searching for alternative methods for safety testing of chemicals. According to a recent policy in the European Union (Regulation, Evaluation Authorisation and Restriction of Chemicals, REACH), it has been estimated that over the next twelve to fifteen years, approximately 30,000 chemicals may need to be tested for safety, and under current guidelines such testing would require the use of approximately 7.2 million laboratory animals [ Hofer et al. 2004 ]. It has also been estimated that over 80% of all animals used for safety testing under REACH legislation would be used for examining reproductive and developmental toxicity [Hofer et al., 2004]. In addition to REACH initiatives, it has been estimated that out of 5,000 to 10,000 new drug entities that a pharmaceutical company may start with, only one is finally approved by the Food and Drug Administration at a cost of over one billion dollars [ Garg et al. 2011 ]. A large portion of this cost is due to animal testing. Therefore, both the pharmaceutical and chemical industries are interested in using alternative models and in vitro tests for safety testing. This review will examine the current state of three alternative models - whole embryo culture (WEC), the mouse embryonic stem cell test (mEST), and zebrafish. Each of these alternatives will be reviewed, and advantages and disadvantages of each model will be discussed. These models were chosen because they are the models most commonly used and would appear to have the greatest potential for future applications in developmental toxicity screening and testing.

Natural cycle is superior to hormone replacement therapy cycle for vitrificated-preserved frozen-thawed embryo transfer

Abstract: We undertook this retrospective variables-control analysis to compare the reproductive outcomes of frozen-thawed embryo transfer using endometrial preparation with either natural cycle or hormone replacement therapy cycle. Patients were divided into three subgroups. Subgroup A (n = 32) consisted of patients having three 8-cell post-thawed embryos transferred. Subgroup B (n = 404) consisted of patients having three good quality post-thawed embryos transferred. Subgroup C (n = 578) consisted of patients having two or three all intact and mitosis resumption post-thawed embryos transferred. Implantation rate, biochemical pregnancy rate, and clinical pregnancy rate were measured. In subgroup A, significantly higher implantation rate, clinical pregnancy rate ongoing pregnancy rate, and lower biochemical pregnancy rate were observed in the natural cycle compared with hormone replacement therapy (HRT) cycle. Subgroup B, had a significantly higher rate of implantation, ongoing pregnancy, and a significantly lower rate of biochemical pregnancy in natural cycle compared with HRT cycle. The natural cycle had a higher trend of clinical pregnancy rate without reaching statistical significance. No statistical difference in reproductive outcomes between natural cycle and HRT cycle was observed in subgroup C. The results suggest the superiority of the natural cycle as compared with the HRT cycle under certain circumstances in a selected population of patients.

Spermatozoa as biomarkers for the assessment of human male infertility and genotoxicity

Abstract: Establishing specific biomarkers for the assessment of human male fertility status is an important goal to ensure the fitness of the male contribution so as to support the birth of a healthy child. Spermatozoa are considered an optimal surrogate tissue for the evaluation of spermatogenic function. Unlike the cells of the testis, spermatozoa do not require invasive procedures to procure a sample. A broad range of sperm biomarkers and tests have been described as useful for the assessment of the sperm function. However, these approaches appear limited considering the current state of the art of molecular diagnostics that could be developed for this purpose. In this review, we outline the suite of sperm biomarkers that are currently in use to assess human male fertility status. Their use as indicators of genotoxic exposure will be discussed.

Immunolocalization of aquaporin 7 in human sperm and its relationship with semen parameters.

Abstract: Aquaporins (AQPs) are a family of 13 small hydrophobic trans-membrane proteins expressed in numerous tissues and cells. Some AQPs work as strict water channels, others are permeable to a range of substances, including glycerol. In the male reproductive system their localization in testis, efferent ducts, epididymis, and spermatozoa has been described. We studied the distribution of AQP7 in ejaculated human sperm and the relationship between AQP7 labeling and sperm characteristics. Semen samples from 33 men were examined by light and transmission electron microscopy (TEM). TEM data were quantified using a mathematical formula that calculates a fertility index (FI) and the percentages of sperm apoptosis, immaturity, and necrosis. Immunocytochemistry with a polyclonal antibody anti-AQP7 was performed on the sperm samples. Normal sperm were labeled in the pericentriolar area, midpiece, equatorial segment, and weakly in the tail (grade 1). Abnormal sperm showed a diffuse low intensity of fluorescence evident i...

Key functional genes of spermatogenesis identified by microarray analysis.

Abstract: At present many couples face difficulties when trying to conceive that may have a genetic basis. The male factor is the cause of infertility as often as the female. Therefore it is important to identify key genes involved in spermatogenesis which may be linked to male infertility. This review discusses the identification of a range of genes associated with male fertility using microarrays. Based on differences in gene expression profiles between fertile and infertile male subgroups or between fetal and adult male gonads, many genes important for spermatogenesis have been discovered. Genes that are critical at particular stages of spermatogenesis were defined and can be considered as potential male fertility biomarkers. The studies described showed that microarrays may be potentially used as a diagnostic platform to increase the efficacy of diagnosis and perhaps treatment of infertile males.

Emerging evidence for the role of genomic instability in male factor infertility.

Abstract: Male infertility is a common and complex pathology affecting0about 7% of men of reproductive age. Given its complexity, the underlying etiology for male infertility is often unknown. A growing amount of evidence suggests genomic instability may be an important factor in some cases of male factor infertility. While some specific manifestations of genomic instability, such as increased sperm aneuploidy rates and increased somatic translocations and inversions in infertile men, are well established, other facets of genomic instability associated with male infertility have not been thoroughly investigated. A limited body of recent work has identified a potential association between microsatellite instability and spermatogenic failure. In addition, mutations in mismatch repair and tumor suppressor genes, which could potentially lead to genomic instability, have been identified in some infertile men and animal models. In addition, results of two epidemiologic studies suggest spermatogenic defects might be just ...

Computer-assisted sperm analysis parameters in young fertile sperm donors and relationship with age

Abstract: Sperm parameter values have been shown to decline with age, according to conventional sperm analysis. However, the effect of age on sperm kinematic parameters has been rarely studied, especially in young fertile men. Here, we studied Computer-Assisted Sperm Analysis (CASA) parameters in a large cohort of men with proven fertility, in order to determine if there is a decline with age in this young fertile population. This retrospective analysis of CASA parameters was conducted on all donors included in the sperm donor programme in the Assisted Reproductive Techniques (ART) Centre of the University Hospital of Nantes between 2006 and 2009. Sperm concentration, motility, and kinetic parameters were recorded by a HTM-Ceros system and compared in 3 groups of sperm donors according to their age: <35 years, 36–40 years, and 41–44 years. A total of 362 ejaculates from 138 donors were analyzed. Values for ALH, VCL, LIN, and STR significantly decreased with age. Sperm concentration, motile sperm proportion, and oth...

Comparison of pregnancy outcomes in natural cycle IVF/M treatment with or without mature oocytes retrieved at time of egg collection.

Abstract: The objective of this study is to compare the pregnancy and live birth rates of a natural cycle in vitro fertilization (IVF) combined with in vitro maturation (IVM) treatment (natural cycle IVF/M) by the presence or absence of mature oocytes retrieved. Infertile women were divided into two groups: (A) patients with mature oocytes found at retrieval and (B) patients with only immature oocytes at retrieval. Patients of group A were further divided into three subgroups: (A1) mature oocytes retrieved from both the leading and the small follicles, (A2) mature oocytes retrieved from the leading follicles only, and (A3) mature oocytes retrieved from the small follicles only. Pregnancy and implantation rates were compared. The results indicate that the clinical pregnancy rates were 40.1% (126/314) and 34.5% (19/55) for groups A and B, respectively. There were no differences in pregnancy rates among the subgroups: A1 = 44.0% (66/150), A2 = 34.9% (30/86), and A3 = 38.5% (30/78). In addition there were no difference...

Toxic stress prioritizes and imbalances stem cell differentiation: implications for new biomarkers and in vitro toxicology tests

Abstract: This hypothesis and review introduces rules of stem cell stress responses that provide biomarkers and alternative testing that replaces or reduces gestational tests using whole animals. These rules for the stress responses of cultured stem cells validate the organismal strategy of the stress response and show that it emulates what must happen if the conceptus implants during a response to stress in vivo. Specifically there is a profound threshold during a stress dose response where stem cell accumulation is significantly reduced. Below this threshold stress enzymes manage the stress response by converting anabolic to catabolic processes and by suppressing apoptosis, without affecting differentiation. However above this threshold the stem cell survival response converts to an organismal survival response where stress enzymes switch to new substrates and mediate loss of potency factors, gain of early essential differentiated lineages, and suppression of later essential lineages. Stressed stem cells ‘compens...

Relationship of seminal plasma antioxidants and serum male hormones with sperm chromatin status in male factor infertility.

Abstract: We explored the relationship between sperm chromatin integrity, hormone levels, seminal plasma total antioxidant capacity (TAC), and routine sperm parameters in men with male factor (MF, n = 81) and non-male factor (NMF, n = 52) infertility. Semen and blood were collected and examined from men undergoing evaluation for infertility in the Avicenna Infertility Clinic. We have examined each patient for serum hormones (LH, FSH, E2, DHEA), sperm chromatin damage, level of protamination and seminal plasma TAC. Levels of FSH, LH, sperm chromatin damage, and abnormal protamination were significantly higher in MF vs. NMF groups (p< 0.001). Sperm chromatin damage was correlated with percentage of CMA3positive sperm (r = 0.64, p< 0.001) and with sperm concentration (r = �0.36, p< 0.001), motility (r = �0.21, p< 0.05), and morphologically normal spermatozoa (r =�0.29, p< 0.001). Linear regression showed sperm chromatin damage was related to percentage of CMA3- positive sperm (p< 0.001) in ungrouped patients. It was related to both percentage of CMA3- positive sperm and serum DHEA in the MF group (p< 0.001 and p< 0.05, respectively). Sperm chromatin maturity assessed by CMA3 test was inversely related to sperm chromatin damage assessed by the toludine blue assay. Male factor infertility associated with sperm chromatin damage may be related to sperm protamination and to serum DHEA.

Combined proteomic and in silico approaches to decipher post-meiotic male genome reprogramming in mice.

Abstract: During the post-meiotic maturation of the male gamete, the haploid genome undergoes major structural transitions, leading to its extreme compaction in a unique specialized structure. Although this process is highly conserved and crucial for the production of male gametes suitable for procreation, its molecular basis has remained unexplored for many years. In particular, the factors driving these events remain to be discovered. Our group has combined specific proteomic approaches with in silico analyses of available expression and sequence data in order to identify chromatin-associated factors as candidates involved in post meiotic reprogramming of the male genome of mice. Here we survey these approaches and the major results obtained. The systematic identification and the functional characterization of several of these factors has led to the proposal of the first working models, guiding us toward the understanding of this unique and very fundamental process, which has long remained one of the black boxes ...

DNA fragmentation status in patients with necrozoospermia.

Abstract: The aim of this study was to determine if a relationship exists between the levels of sperm DNA fragmentation and necrospermia in infertile men. Semen samples obtained from 70 men consulting for infertility evaluation were analyzed according to World Health Organization (WHO) guidelines. Patients were subdivided into three groups according to the percentage of necrotic spermatozoa: normozoospermia ( 80%; n = 20). DNA fragmentation was detected by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) assay. The sperm DNA fragmentation index (DFI) was 9.28 ± 2.98% in patients with a normal level of necrotic spermatozoa, 20.25 ± 3.21% in patients with moderate necrozoospermia, and 35.31 ± 5.25% in patients with severe necrozoospermia. There was a statistically significant increase of DNA fragmentation in the necrozoospermic group (P < 0.01). A strong correlation was f...

Economic benefits of using adaptive predictive models of reproductive toxicity in the context of a tiered testing program.

Abstract: A predictive model of reproductive toxicity, as observed in rat multigeneration reproductive (MGR) studies, was previously developed using high throughput screening (HTS) data from 36 in vitro assays mapped to 8 genes or gene-sets from Phase I of USEPA ToxCast research program, the proof-of-concept phase in which 309 toxicologically well characterized chemicals were testing in over 500 HTS assays. The model predicted the effects on male and female reproductive function with a balanced accuracy of 80%%. In a theoretical examination of the potential impact of the model, two case studies were derived representing different tiered testing scenarios to: 1) screen-out chemicals with low predicted probability of effect; and 2) screen-in chemicals with a high probability of causing adverse reproductive effects. We define ‘testing cost efficiency’ as the total cost divided by the number of positive chemicals expected in the definitive guideline toxicity study. This would approach $$2.11 M under the current practic...

Effect of coincubation time of sperm-oocytes on fertilization, embryonic development, and subsequent pregnancy outcome.

Abstract: Several studies have reported improved IVF by shortening the time of sperm-oocyte coincubation from 16–18 hours to 1–4 hours. The objective of this study was to examine the advantages and disadvantages of a shortened sperm-oocyte coincubation time in order to assess the effects of this insemination method for clinical IVF practice. Two insemination methods, the shortened method (4 hours) and the standard method (16–18 hours) of coincubation of sperm-oocytes for two groups of patients based on the quality of sperm were compared. Group I, was composed of couples without male factor; Group II, involved couples with mild male factor. Fertilization, good quality embryos, clinical pregnancy, and implantation rates were compared by two different insemination methods. In Group I, fertilization, clinical pregnancy, and implantation rates were not different between the two insemination methods. However, the polyspermy rate was significantly higher (P < 0.05) in the shortened (7.3%) than in the standard (4.1%) insem...

REACH and reproductive and developmental toxicology: still questions

Abstract: Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) is a new chemicals regulation law in the European Union (EU). The law is supplemented by tens of thousands of pages of guidance documents, and the implementation of REACH is still a work in progress. Requirements for chemical testing are based on the annual volume of a chemical or 'substance' that is produced or imported into the EU. These requirements include reproductive and developmental toxicity testing in experimental animals for an annual volume of 10 metric tonnes or more. However, under REACH, the testing in vertebrate animals may not be performed without permission, and the law encourages the use of alternative methods of filling data gaps on the toxicological properties of chemicals. These alternatives might include in vitro and structure-activity relationship studies, but the REACH technical guidance indicates that these kinds of studies are not adequate to replace reproductive and developmental toxicity testing in whole animals. The most practical opportunity for the avoidance of whole animal testing may be 'read-across,' a process in which gaps are filled using data from related compounds. A method called 'weight of evidence' under REACH may also be used to avoid whole animal reproductive and developmental toxicity testing based on existing data in regulation and non-regulation studies and based on factors such as chemical structure and anticipated exposure. It is also possible that thresholds of toxicological concerns will be accepted under REACH as a method to avoid vertebrate animal testing.

The influence of the redox state of follicular fluid albumin on the viability of aspirated human oocytes

Abstract: A number of reports have suggested that the oxidative state of human albumin in serum and in some body fluids is associated with cell damage. However there are no reports on the redox state of human follicular fluid (FF) and its influence on oocyte viability. The aim of this study was to examine the relationship between the redox state of FF and serum on oocyte viability. The cytoplasmic condition of oocytes was evaluated microscopically at collection in 117 women. Deteriorating oocytes were recognized by degenerative changes in their cytoplasm. The redox state of FFs that yielded degenerated oocytes was evaluated and compared with fluids containing normal oocytes. The redox state of the corresponding FF and serum, at the time of oocyte retrieval, was analyzed by high performance liquid chromatography. The redox state of FF that contained degenerated oocytes was found to have a significantly elevated oxidized state compared with the FFs that yielded normal oocytes. Also the albumin in the FF of patients was found to be predominantly in the reduced state compared with that in their serum at the time of oocyte retrieval. In addition, increasing age and endometriosis were found to shift the redox of serum to the oxidative state. We propose that the reduced state of albumin in FF may play an important role in protecting oocytes from oxidative damage.

A critical evaluation of developmental and reproductive toxicology in nonhuman primates.

Abstract: The nonhuman primates (NHPs) are used in many areas of biomedical research where their similarities to humans make them exclusively valuable animal models. The use of NHPs in pre-clinical testing is expected to increase due to the increase in the development of biological compounds for therapeutic uses. The regulatory agencies around the world including Food and Drug Administration (FDA) generally requires developmental and reproductive toxicity (DART) testing of all new drugs to be used by women of childbearing age or men of reproductive potential. NHPs are most frequently used for DART testing when commonly used rodents and/or rabbits are not pharmacologically relevant species. Animal studies are unique in that assessment of reproduction and development as DART studies are not performed in controlled clinical trials; therefore, pre-clinical safety assessment forms the basis for risk assessment for marketed drug products. This paper provides a critical evaluation of developmental and reproductive toxicity studies in NHPs. The manuscript will focus on species selection, limitation of International Conference for Harmonization stages (A-F) using NHPs as a test system, study designs, logistical/technical challenges, and strength, and limitations. It will also pinpoint confounding factors inherent to the test system that may complicate the interpretation of the NHP DART data.