Sub-chronic cadmium and lead compound exposure induces reproductive toxicity and development of testicular germ cell neoplasia in situ in murine model: Attenuative effects of resveratrol.

Cadmium and lead are widespread, nonbiodegradable heavy metals of perpetual environmental concerns. The present study aimed to evaluate whether sub-chronic exposure to cadmium chloride (CdCl2 ) and lead acetate [Pb(CH3 COO)2 ] induces reproductive toxicity and development of testicular germ cell neoplasia in situ (GCNIS) in swiss albino mice. The effects of resveratrol to reverse the metal-induced toxicity were also analyzed. The mice were randomly divided into four groups for metal treatments and two groups received two different doses of each metal, CdCl2 (0.25 and 0.5 mg/kg) and Pb(CH3 COO)2 (3 and 6 mg/kg). The fourth group received oral doses of 20 mg/kg resveratrol in combination with 0.5 mg/kg CdCl2 or 6 mg/kg Pb(CH3 COO)2 for 16 weeks. Toxic effects of both metals were estimated qualitatively and quantitatively by the alterations in sperm parameters, oxidative stress markers, testicular histology, and protein expressions of the treated mice. Pronounced perturbation of sperm parameters, cellular redox balance were observed with severe distortion of testicular histo-architecture in metal exposed mice. Significant overexpression of Akt cascade and testicular GCNIS marker proteins were recorded in tissues treated with CdCl2 . Notable improvements were observed in all the evaluated parameters of resveratrol cotreated mice groups. Taken together, the findings of this study showed that long-term exposure to Cd and Pb compounds, induced acute reproductive toxicity and initiation of GCNIS development in mice. Conversely, resveratrol consumption abrogated metal-induced perturbation of spermatogenesis, testicular morphology, and the upregulation of Akt cascade proteins along with GCNIS markers, which could have induced the development of testicular cancer.

Lead and cadmium exposure induces male reproductive dysfunction by modulating the expression profiles of apoptotic and survival signal proteins in tea-garden workers.

The aim of this study was to evaluate the impact of Lead (Pb) and Cadmium (Cd) exposure at the molecular level on the reproductive status of tea garden workers in North-East India. Using semen samples, we experimentally determined sperm analysis as well as oxidative stress parameters in all samples and evaluated the expression levels of apoptotic and cell survival proteins [p53, phospho-Akt, nuclear factor-κB (NF-κB, p50 subunit) and B cell lymphoma 2 (Bcl2)]. Our data revealed significant differences in the average heavy metal concentrations and various semen analysis profile between the infertile and normal groups. Increasing Pb and Cd concentrations in semen samples of patients showed positive associations with increasing number of multiple defects in sperm and the level of seminal oxidative stress markers in the high Pb and Cd concentration groups. These groups also exhibited positive correlations between high metal concentrations and the average p53 expression levels, but negative correlations with the mean p-Akt cascade protein levels in sperm cells. In the low Pb and Cd concentrations groups, we also observed reverse mean range and correlation patterns. Therefore, our findings may suggest that graded levels of metal exposure significantly influence the relative fluctuation in the levels of p53 and Akt cascade proteins in the sperm cells of infertile subjects. Furthermore, this may be a regulating factor of sperm cell fate, in turn, determining the fertility outcome of the men working in the tea gardens.

Effect of transfer of a poor quality embryo along with a top quality embryo on the outcome during fresh and frozen in vitro fertilization cycles.

OBJECTIVE: To evaluate the impact of a poor quality embryo (PQE) during double ET (DET) with a top quality embryo (TQE) on IVF outcome. DESIGN: A review of prospectively collected data. SETTING: Tertiary level fertility clinic. PATIENT(S): All patients undergoing blastocyst transfers as part of fresh IVF (n = 939) and frozen ET (n = 1,009) cycles performed between 2010 and 2016. INTERVENTION(S): Single ET (SET) with TQE (group 1) was set as control and compared with outcomes for SET with PQE (group 2), DET with 2 TQEs (group 3), PQE plus TQE (group 4), and 2 PQE (group 5). MAIN OUTCOME MEASURE(S): Live births and multiple births. RESULT(S): The live birth rates for group 4 were statistically similar to group 1 during fresh IVF (26.5% vs. 33.7%; odds ratio [OR], 0.95; 95% confidence interval [CI] 0.53-1.7) and frozen ET (24.2% vs. 32.7%; OR, 0.75; 95% CI 0.48-1.2), although there was a trend for lower success. Conversely, multiple births were higher in group 4 for fresh IVF (19% vs. 4.7%; OR, 2.9; 95% CI 1.3-6.6) and frozen ET (10.3% vs. 2.6%; OR, 2.4; 95% CI 1.2-4.9). The live birth rates for group 2 (12.2% for fresh IVF and 14.6% for frozen ET) and group 5 (21.2% for fresh IVF and 14% for frozen ET) were lower and for group 3 were higher (40.8% for fresh IVF and 40.3% for frozen ET) when compared with group 1. Multiple births were significantly higher with DET. CONCLUSION(S): This study does not support DET with one PQE along with a TQE, when there is only one TQE and one or more PQEs available for fresh IVF or frozen ET.

OMICS: Current and future perspectives in reproductive medicine and technology.

Many couples present fertility problems at their reproductive age, and although in the last years, the efficiency of assisted reproduction techniques has increased, these are still far from being 100% effective. A key issue in this field is the proper assessment of germ cells, embryos and endometrium quality, in order to determine the actual likelihood to succeed. Currently available analysis is mainly based on morphological features of oocytes, sperm and embryos and although these strategies have improved the results, there is an urgent need of new diagnostic and therapeutic tools. The emergence of the - OMICS technologies (epigenomics, genomics, transcriptomics, proteomics and metabolomics) permitted the improvement on the knowledge in this field, by providing with a huge amount of information regarding the biological processes involved in reproductive success, thereby getting a broader view of complex biological systems with a relatively low cost and effort.

Laboratory handling of epididymal and testicular spermatozoa: What can be done to improve sperm injections outcome.

Spermatozoa from azoospermic males can be retrieved from either the epididymis or the testis, depending on the type of azoospermia, using different surgical methods such as percutaneous epididymal sperm aspiration (PESA), testicular sperm aspiration (TESA), testicular sperm extraction (TESE), and microsurgical testicular sperm extraction (micro- TESE). After collecting the epididymal fluid or testicular tissue, laboratory techniques are used to remove contaminants, cellular debris, noxious microorganisms, and red blood cells. Processed spermatozoa may be used for intracytoplasmic sperm injection or eventually be cryopreserved. However, spermatozoa collected from either the epididymis or the testis are often compromised and more fragile than ejaculated ones. Therefore, sperm processing techniques should be used with great caution to avoid jeopardizing the sperm fertilizing potential in treatment cycles. In this review, we describe the current methods for processing surgically-retrieved specimens, either fresh or frozen- thawed, and provide the tips and pitfalls for facilitating the handling of such specimens. In addition, we present the available laboratory tools to aid in the identification of viable immotile spermatozoa to be used in conjunction with assisted reproductive techniques. Review of the literature was carried out using PubMed and Science Direct search engines.

Oocyte developmental competence and embryo development: impact of lifestyle and environmental risk factors.

Oocyte development is the end result of a sophisticated biological process that is hormonally regulated and produced by highly specialized cellular lines that differentiate in early embryo/fetal development. Embryo development is initially regulated by maternal transcripts until replaced by embryonic genomic expression. Then, an assortment of hormones and local environmental factors in various concentrations along the reproductive tract (e.g. fallopian tube, endometrial lining) provide the protection, nutrients and means of communication for the embryo to implant and develop. Both oocytes and embryos are susceptible to environmental, occupational and lifestyle exposures that can exert direct toxic effects and disrupt hormones. While some exposures may produce reversible changes, others, especially those damaging germinal cells in utero or during prepuberty, may result in permanent sequelae that continue in future generations. This article reviews the main factors that affect female fertility and their possible influence on human reproduction. Some lifestyles, xeno-oestrogens and heavy metals are already known to compromise female reproductive function. Nonetheless, many questions remain and little is known about the effect of many other factors on female fertility.

The genetic causes of male factor infertility: a review.

OBJECTIVE: To illustrate the necessity for an enhanced understanding of the genetic basis of male factor infertility, to present a comprehensive synopsis of these genetic elements, and to review techniques being utilized to produce new insights in fertility research. BACKGROUND: Male factor infertility is a complex disorder that affects a large sector of the population; however, many of its etiologies are unknown. By elucidating the underlying genetic basis of infertile phenotypes, it may be possible to discover the causes of infertility and determine effective treatments for patients. METHOD(S): The PubMed database was consulted for the most relevant papers published in the last 3 years pertaining to male factor infertility using the keywords "genetics" and "male infertility." RESULT(S): Advances have been made in the characterization of the roles of specific genes, but further research is necessary before these results can be used as guidelines for diagnosing and treating male factor infertility. The accurate transmission of epigenetic information also has considerable influence on fertility in males and on the fertility of their offspring. CONCLUSION(S): Analysis of the genetic factors that impact male factor infertility will provide valuable insights into the creation of targeted treatments for patients and the determination of the causes of idiopathic infertility. Novel technologies that analyze the influence of genetics from a global perspective may lead to further developments in the understanding of the etiology of male factor infertility through the identification of specific infertile phenotype signatures.

The in vitro effect of benzo[a]pyrene on human sperm hyperactivation and acrosome reaction.

OBJECTIVE: To evaluate the in vitro effect of benzo[a]pyrene on sperm hyperactivation and acrosome status in normozoospermic semen samples of nonsmokers analyzed by computer-assisted semen analysis (CASA). DESIGN: Experimental in vitro study. SETTING: Andrology laboratory. PATIENT(S): Thirteen proven fertile, normozoospermic, and nonsmoking men. INTERVENTION(S): Spermatozoa were washed free of seminal plasma and were treated with different concentrations of benzo[a]pyrene and compared with controls treated with medium alone. The benzo[a]pyrene concentrations were: 100, 50, 25, and 12.5 microg/mL. MAIN OUTCOME MEASURE(S): Effect of varying concentrations of benzo[a]pyrene on sperm hyperactivation and acrosomal reaction. RESULT(S): A statistically significant increase in sperm hyperactivation was observed in presence of benzo[a]pyrene at concentrations of >or=50 microg/mL. The result of the acrosome halo test showed that concentrations of benzo[a]pyrene >or=25 microg/mL statistically significantly decreased the percentage of halo formation, indicating an inappropriate (false) acrosome reaction. CONCLUSION(S): Benzo[a]pyrene statistically significantly affected sperm functional competence as evidenced by increased hyperactivation as well as premature acrosomal reaction.

Semen quality and age-specific changes: a study between two decades on 3,729 male partners of couples with normal sperm count and attending an andrology laboratory for infertility-related problems in an Indian city.

OBJECTIVE: To compare the semen quality and age-specific changes in men between the 1980s and 2000s. DESIGN: Prospective study. SETTING: Andrology laboratory, University of Calcutta, India. PATIENT(S): A semen sample was obtained from 3729 men presenting for infertility problems in two distinct decades, that is, between 1981-85 and 2000-2006. INTERVENTION(S): Subjects with sperm count >20 x 10(6)/mL without any extreme pathological disorders were selected. Samples having a major liquefaction problem were excluded. MAIN OUTCOME MEASURE(S): A standard World Health Organization procedure for semen analysis was performed that included assessment of volume, sperm concentration, and percentage motility. The motility parameters were further classified into forward progressive motility and nonprogressive motility. RESULT(S): The present large-scale study confirms a significant decline in the sperm motility parameters and seminal volume in the present decade. However, no change in overall sperm concentration was noted. A decline was seen in sperm motility with increasing age in both decades. CONCLUSION(S): There are significant changes in sperm motility and volume between the two decades, and the age-related changes in semen parameters are also different in the two decades.

Expression of metallothionein-1 (MT-1) mRNA in the rat testes and liver after cadmium injection.

Metallothioneins (MTs) belong to the family of stress proteins that are present in the majority of living organisms. The MTs play an important task in detoxifying heavy metals. The mammalian scrotal testis is known to be susceptible to cadmium (Cd) exposure. The present work focuses on the MT-1 isoform and aims to ascertain and confirm previous findings to answer whether rodent testes indeed contain MT-1 mRNA, whether its level is increased with Cd injection in liver and testes, and lastly what is the relative difference in the expression of MT-1 mRNA in liver and testes both with and without Cd injection. Adult male Wistar rats weighing 270-290 g received a subcutaneous injection of 4.0 mumol Cd/kg and were sacrificed by cervical dislocation 6 h later. RNA was isolated from testes as well as the liver. There were 2 replicates per treatment for RNA analyses. MT-1 mRNA levels were determined by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis and then assessed by densitometry scanning. The results of RT-PCR clearly demonstrated that the rodent testes express MT-1 mRNA. The densitometry data shows that the expression of MT-1 mRNA increases with Cd treatment in testes. The relative level of MT1-mRNA is greater in the control-liver than in the control-testes. However, upon Cd injection, the level of testes MT-1 mRNA increases 2.16 fold. These results suggest that the testes respond to Cd for at least 6 h post injection through a transcriptional mechanism.

Markers of oxidative stress and sperm chromatin integrity.

Oxidative stress (OS) is an imbalance between the amount of reactive oxygen species (ROS) produced and the ability of the antioxidants to scavenge these. OS has been established as a major etiological cause of male infertility. High levels of ROS are harmful and cause damage to sperm nuclear DNA. Evaluation of OS-related damage to spermatozoa is therefore highly relevant in assisted reproductive techniques (ART) such as intracytoplasmic sperm injection (ICSI). ICSI is an effective therapy for severe male factor infertility that bypasses the majority of reproductive tract deficiencies. Despite the controversial findings in the existing literature, there is now enough evidence to show that sperm DNA damage is detrimental to reproductive outcomes. In addition to impairment of fertility, such damage might increase the transmission of genetic diseases to the offspring. Standardization of protocols to assess ROS, antioxidant status, and DNA damage is very important for implementation of these tests in clinical practice. Estimation of seminal ROS levels and extent of sperm DNA damage, especially in an infertile male, may help develop new therapeutic strategies and improve the success of ART.

Current trends, biological foundations and future prospects of oocyte and embryo cryopreservation.

For a long time, the cryopreservation of gametes and embryos remained a major hurdle for the clinicians and scientists in terms of success. However, recent technical advancement in the field of cryobiology has opened up various options for freezing gametes and embryos at different developmental stages. The tendency of the IVF world to switch over to natural-cycle IVF and to elective single-embryo transfer has put cryotechnology in the forefront of research. Still, the intricacies of the cold-induced changes in human gametes and embryos that could affect the intracellular and developmental processes need to be known. The transcriptomics, proteomics and metabolomic platforms hold promise for elucidating these complex processes during cryopreservation processes.

Male gamete survival at stake: causes and solutions.

Over the years, the development of assisted reproductive technology to bypass male factor infertility has improved drastically. Considered one of the most perplexing disorders in the reproductive field, male factor infertility is prevalent and may be on the rise. Unfortunately, its aetiology remains elusive. One of the main reasons lies in the complex machinery and structure of the hydrodynamic sperm cell. Its polyunsaturated fatty acid cell membrane, the protamines in its genetic material and the absence of antioxidants in its cytoplasm ensure that the spermatozoon is highly susceptible to environmental effects. The spermatozoon's genesis, storage, and transport through the male reproductive tract are also susceptible, genetically and pathologically, to environmental effects. This review aims to include all the possible causes of disruption to this unique cell and their probable solutions, in the hope of clearing up the ambiguity that surrounds male factor infertility.

Cryopreservation/transplantation of ovarian tissue and in vitro maturation of follicles and oocytes: challenges for fertility preservation.

Cryopreservation of ovarian tissue and in vitro follicle maturation are two emerging techniques for fertility preservation, especially in cancer patients. These treatment regimes are opening up more options and allow for more suitable choices to preserve fertility according to the patient's specific circumstances. If these technologies are to become widely accepted, they need to be safe, easy to perform and must obtain favorable results. The generation of healthy eggs with the normal genetic complement and the ability to develop into viable and healthy embryos requires tight regulation of oocyte development and maturation. Novel freezing techniques such as vitrification, along with whole ovary cryopreservation and three-dimensional follicle cultures, have shown favorable outcomes. The scope of this article is to take a comprehensively look at the challenges still faced in order for these novel technologies to be routinely employed with the aim of successful fertility preservation.

Current and future perspectives on intracytoplasmic sperm injection: a critical commentary.

Intracytoplasmic sperm injection (ICSI) is an increasingly popular means of treating infertility in couples who wish to conceive. However, there are many potential complications that can be faced by the clinician while performing ICSI. These complications and other related issues are discussed, with an emphasis on understanding how these issues are being resolved, or how they can be resolved in the future. Matters of sperm selection and injection are discussed, as well as the effect of ICSI on fertilization, embryonic growth and development, and the health of ICSI-conceived children. These aspects are viewed from various perspectives, including genetic, mechanistic, developmental and clinical. Since new studies on ICSI are published regularly, it is important that the established protocol is revised often, and that the role of ICSI in infertility therapy is continually re-evaluated.

Emerging technologies for the molecular study of infertility, and potential clinical applications.

The techniques currently used to treat infertility cases are quite limited in their capabilities, due to an incomplete understanding of the molecular activities of germ cells. Fortunately, several technologies are presently being researched that should aid the understanding of the various molecular causes of germ cell pathologies. This review discusses microarray technology, proteomics, metabolic profiling, the PolScope, atomic force microscopy and microfluidics. These technologies have all seen success in preliminary studies, and promise directly or indirectly to improve the low success rates of IVF and other related therapies. However, their widespread use in laboratories and clinics may not be seen until preliminary studies confirming their safety and effectiveness are published, and until standardized protocols for their utilization are established.

Current trends in evaluation of sperm function: in vitro selection and manipulation of male gametes for assisted conception.

With increasing medical utilization of assisted reproductive technology (ART), scientists and clinicians have been able to study extensively multiple cell functions operating synchronously and flawlessly during the events preceding, before and after fertilization. Critical evaluation of the functional status of spermatozoa for in vitro techniques such as sperm-mucus interaction, acrosome reaction status, sperm-zona pellucida binding and penetration tests, hyaluronic acid binding assay, and computer assisted semen analysis etc. can direct a male partner of an infertile couple to more aggressive forms of treatments. In vitro selection of functionally competent sperm cells is a pre-requisite for successful outcome in in vitro fertilization or in intracytoplasmic sperm injection (ICSI). Direct injections of acrosome-intact spermatozoa into oocyte during ICSI bypassing the normal events of sperm oocyte interaction and fusion events have raised concerns with regard to fertilization abnormalities and genetic issues. The present communication briefly reviews the sperm function tests with emphasis on its correlation with fertility outcome, and the currently employed sperm selection and manipulation procedures which may have implications in assisted conception programs.