The proportion of total hip arhtoplasties (THAs) associated with corticosteroid use is uncertain, and the mechanisms of corticosteroid-induced osteonecrosis remain unknown. We sought to evaluate the clinical and radiographic outcomes, complications and satisfaction with THA among patients with corticosteroid-induced osteonecrosis. We retrospectively assessed functional outcome at a minimum 1-year follow-up ...

The sex of mammalian offspring can be predetermined by flow sorting relatively pure living populations of X- and Y-chromosome-bearing sperm. This method is based on precise staining of the DNA of sperm with the nucleic acid-specific fluorophore, Hoechst 33342, to differentiate between the subpopulations of X- and Y-sperm. The fluorescently ...

Testicular tissue grafting and male germ cell transplantation are techniques that offer unprecedented opportunities to study testicular function and development. While testicular tissue grafting allows recapitulation of testis development and spermatogenesis from immature males of different mammalian species in recipient mice, germ cell transplantation results in donor-derived spermatogenesis in recipient ...

High quality DNA extractions developed for mammalian somatic cells are ineffective for sperm, due mainly to the high degree of nuclear compaction in sperm. The highly specialized nuclear proteins in sperm create a chromatin structure that is at least six times denser than histone bound DNA. Unlike somatic cells, sperm ...

Immunohistochemistry is an important technique that uses specific antibodies to determine the cellular localization of proteins/antigens in highly complex organs and tissues. While most immunohistochemistry experiments target protein epitopes, nonprotein antigens including BrdU may also be detected. Briefly, tissues are fixed, processed, sectioned, and then probed by a primary antibody ...

Human spermatozoa for clinical procedures such as IUI or IVF, or for diagnostic or research studies of sperm fertilizing ability, must be separated from the seminal plasma environment not only as soon as possible after ejaculation but also as efficiently as possible, minimizing seminal plasma and bacterial carryover. Furthermore, in ...

Charge is a fundamental property of all forms of matter that exhibit attraction or repulsion in the presence of another charged particle. This electrokinetic property occurs when the particles exhibiting a net negative or positive charge are subjected to an external electric field that exerts an electrostatic force between them. ...

An improved enhanced chemiluminescence antioxidant assay utilizes horseradish peroxidase conjugate and luminol to produce a cell-free oxygen radical generating system. We introduce the use of a peroxidase enzyme stabilizer to prolong the production of oxygen radicals at a steady rate. Addition of antioxidants temporarily interrupts oxygen radical generation, resulting in ...

The paternal genome in many animal taxa is efficiently packaged into the sperm nucleus by switching from a histone (nucleosome)-based chromatin configuration to one using predominantly protamines. Nonetheless, various studies have shown that some nucleosomes, often containing modified histones are retained in mature sperm and bind DNA with distinct sequence ...

Progressive motility is a vital functional characteristic of ejaculated human spermatozoa that governs their ability to penetrate into, and migrate through, both cervical mucus and the oocyte vestments, and ultimately fertilize the oocyte. A detailed protocol, based on the most common computer-aided sperm analysis (CASA) system with phase contrast microscope ...

Flow cytometry allows the assessment of multiple sperm parameters following a diverse number of protocols. Here, we describe three methods for evaluating critical aspects of sperm quality: the double stain SYBR-14 and propidium iodide (PI) for assessing sperm viability; the double stain PNA-FITC (peanut agglutinin-fluorescein isothiocyanate) and PI for assessing ...

There are multiple clinical situations where cryopreservation of sperm is required including sperm banking prior to chemotherapy or radiation treatment for cancer, donor sperm for couples without a male partner, and various etiologies of male factor infertility. Bunge and Sherman presented the first pregnancy resulting from the thawing of cryopreserved ...

Proteomics is the study of the proteins of cells or tissues. Sperm proteomics aims at the identification of the proteins that compose the sperm cell and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput for the identification and study of the ...

Although sperm morphology evaluation is one of the most important aspects of the semen analysis if done correctly and accurately, a trend is developing in which many laboratories or clinicians no longer regard sperm morphology as relevant due to the very low normal reference value of only 4% morphological normal ...

The SCSA(®) is the pioneering assay for the detection of damaged sperm DNA and altered proteins in sperm nuclei via flow cytometry of acridine orange (AO) stained sperm. The SCSA(®) is considered to be the most precise and repeatable test providing very unique, dual parameter data (red vs. green fluorescence) ...

The isolation of mammalian sperm heads from their tails is complicated by the relatively high density of the tails, but facilitated by the fact that protamine condensation of the sperm chromatin and the insolubility of the perinuclear theca make the sperm nucleus stable in sodium dodecyl sulfate. Two methods are ...

Male germ cells in all mammals are arranged within the seminiferous epithelium of the testicular tubules in a set of well-defined cell associations called stages. The cellular associations found in these stages and characteristics of the cells used to identify the stages have been well described. Here we present a ...

The chemiluminescence method is the most commonly employed technique as a direct measurement of reactive oxygen species (ROS) generation by spermatozoa. This assay is capable of quantifying both intracellular and extracellular ROS. Moreover, the use of various probes allows for differentiation between superoxide and hydrogen peroxide production by spermatozoa. When ...

In some cases, human spermatozoa to be used for in vitro fertilization are processed from testicular or epididymal biopsies collected in the clinic or operating room. An appropriately equipped Andrology or Embryology Laboratory is required. Sterility must be maintained at all stages from collection and transport to identification and processing ...

In this chapter, the laboratory methods for detection of sperm biomarkers that are aimed at identifying arrested sperm development are summarized. These probes include sperm staining with aniline blue for persistent histones, representing a break in the histone-transition protein-protamine sequence, immunocytochemistry with cytoplasmic sperm proteins, highlighting cytoplasmic retention during spermiogenesis, ...

The isolation of spermatozoal RNA is a challenging procedure due to the intrinsic heterogeneous population of cells present in the ejaculate and the small quantity of RNA present in sperm. The transcriptome of these gametes includes a wide variety of messenger RNAs (mRNAs), small noncoding RNAs (sncRNAs), and highly fragmented ...

Approximately 40% of sterility in couples can be attributed to male subfertility and intracytoplasmic sperm injection (ICSI) has become a powerful tool in assisted reproduction to overcome male infertility. Intracytoplasmic morphologically selected sperm injection (IMSI) is an advanced and sophisticated method of ICSI, where prior to sperm injection the morphology ...

A functional membrane is requisite for the fertilizing ability of spermatozoa, as it plays an integral role in sperm capacitation, acrosome reaction, and binding of the spermatozoon to the egg surface. The hypo-osmotic swelling (HOS) test evaluates the functional integrity of the sperm's plasma membrane and also serves as a ...

Measurement of sperm DNA damage is a useful tool in the evaluation of male infertility, as the sperm nucleus lacks protection against oxidative stress and is vulnerable to oxidation-mediated DNA damage. The Comet assay or single-cell gel electrophoresis is a relatively simple and sensitive method for measuring strand breaks in ...

PCR multiplex assays are the method of choice for quickly revealing genomic microdeletions in the large repetitive genomic sequence blocks on the long arm of the human Y chromosome. They harbor the Azoospermia Factor (AZF) genes, which cause male infertility when functionally disrupted. These protein encoding Y genes are expressed ...

Epigenetic modifications on the DNA sequence (DNA methylation) or on chromatin-associated proteins (i.e., histones) comprise the "cellular epigenome"; together these modifications play an important role in the regulation of gene expression. Unlike the genome, the epigenome is highly variable between cells and is dynamic and plastic in response to cellular ...

Sperm vitality is a reflection of the proportion of live, membrane-intact spermatozoa determined by either dye exclusion or osmoregulatory capacity under hypo-osmotic conditions. In this chapter we address the two most common methods of sperm vitality assessment: eosin-nigrosin staining and the hypo-osmotic swelling test, both utilized in clinical Andrology laboratories.

This chapter describes the approach to define the cause of male infertility in a genetically modified male mouse. It provides a guide to the establishment of the infertility status and whether it is due to the failure of mating or due to abnormalities of the sperm output, motility, and morphology. ...

Sperm aneuploidy screening has been used as a tool in diagnosis and determining treatment options for male factor infertility since the development of human sperm karyotyping by injection into hamster and mouse oocytes in the 1970s. From these studies and subsequent work with interphase chromosome analysis, at risk populations of ...

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 ...

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling or the TUNEL assay is an important technique in the assessment of DNA damage. Semen samples are routinely assessed microscopically to assess their fertilization ability. In addition to routine semen analysis, the use of the TUNEL assay can provide information on the level ...

Transmission electron microscopy (TEM) studies have provided the basis for an in-depth understanding of the cell biology and normal functioning of the testis and male gametes and have opened the way to characterize the functional role played by specific organelles in spermatogenesis and sperm function. The development of the scanning ...

The continuous production of spermatazoa throughout the reproductive lifetime of a male depends on the maintenance of a pool of progenitor cells called spermatogonial stem cells (SSCs). SSCs represent a very small fraction of the cellular population in the testes and lack definitive molecular markers for their identification. The discovery ...

Protamines, sperm-specific nuclear proteins, are essential for sperm chromatin condensation and DNA stabilization. They are small, highly basic, and rich in disulfide bonds. Under reducing conditions, protamines, along with other basic proteins, are soluble in acid solutions. Because of their small and similar molecular weights, SDS-PAGE cannot resolve protamine 1 ...

The hemizona assay (HZA) has been developed as a diagnostic test for the tight binding of human spermatozoa to the human zona pellucida to predict fertilization potential. In this homologous bioassay, the two matching hemizona halves are functionally equal surfaces allowing controlled comparison of binding from a fertile control versus ...

Progressive motility is a vital functional characteristic of ejaculated human spermatozoa that governs their ability to penetrate into, and migrate through, both cervical mucus and the oocyte vestments, and ultimately fertilize the oocyte. A detailed protocol, based on traditional manual/visual methods, is provided for performing an accurate four-category differential count ...

Research on in vitro spermatogenesis has a long history and remained to be an unaccomplished task until very recently. In 2010, we succeeded in producing murine sperm from primitive spermatogonia using an organ culture method. The fertility of the sperm or haploid spermatids was demonstrated by microinsemination. This organ culture ...

Annexin-V magnetic-activated cell sorting (MACS) is a new tool to optimize sperm selection in assisted reproduction. The technique is based on the binding of superparamagnetic Annexin-microbeads to externalized phosphatidylserine at the outer leaflet of the plasma membrane of sperm with activated apoptosis signaling or membrane damage. The combination of Annexin-V ...

Antisperm antibodies (ASA) are one well-known cause of refractory infertility in both males and females. In females, a sperm immobilization test, which detects sperm-immobilizing antibodies indirectly in the patient's serum, requires complement for the reaction and thus seems to be a more specific immunological reaction. In males, an immunobead test ...

Sperm morphology is an important measure of testicular health, spermiation, and fertility potential. The World Health Organization (WHO) Semen Manuals advocate different sperm morphology schemes, but, like the schemes themselves, do not describe classification sequence or rules that can be unambiguously applied in a standard method. Our novel dichotomous key ...

Proper assessment of the number of spermatozoa is essential not only as an initial step in every clinical infertility investigation [Björndahl et al (2010) A practical guide to basic laboratory andrology, 1st edn. Cambridge University Press, Cambridge] but also when attempting to establish the total sperm production in the testis ...

Sperm selection is an important part of the ICSI procedure. Routinely, sperm selection for ICSI is based solely on sperm morphology and motility. These latter parameters may not be sufficient to select sperm with intact chromatin. Therefore, sperm selected based on sperm functional characteristics may result in the most appropriate ...

Enhancement of sperm motility can effectively improve assisted reproductive technique outcomes. Here we describe two (pentoxifylline and platelet-activating factor) popular sperm motility enhancers and their respective methods.

Rapidly advancing tools for genetic analysis on a genome-wide scale have been instrumental in identifying the genetic bases for many complex diseases. About half of male infertility cases are of unknown etiology in spite of tremendous efforts to characterize the genetic basis for the disorder. Advancing our understanding of the ...

The transgenic technologies represent potent biotechnological tools that allow the generation of genetically modified animals useful for basic research and for biomedical, veterinary, and agricultural applications. Among transgenic techniques, we describe here the sperm-mediated gene transfer methods that is gene transfer based on the spontaneous ability of sperm cells to ...

The sperm penetration assay, or zona-free hamster oocyte penetration assay, is utilized to measure the ability of sperm to undergo capacitation, fuse with the egg membrane, and decondense the sperm head within the cytoplasm of the oocyte, resulting in the formation of the male pronucleus. The test is scored by ...

Xylan-1,4-β-xylosidase (β-xylosidase) hydrolyses xylo-oligomers at their non-reducing ends into individual xylose units. Recently, XylC, a β-xylosidase from Thermoanaerobacterium saccharolyticum JW/SL-YS485, was found to be structurally different from corresponding glycosyl hydrolases in the CAZy database (http://www.cazy.org/), and was subsequently classified as the first member of a novel family of glycoside hydrolases ...

Endovascular repair of blunt traumatic thoracic aortic injuries (BTAI) is common at most trauma centres, with excellent results. However, little is known regarding which injuries do not require intervention. We reviewed the natural history of untreated patients with minimal aortic injury (MAI) at our centre. We conducted a retrospective database ...

Aseptic loosening and periprosthetic osteolysis occur as a result of the biological response to particulate wear debris and are one of the leading causes of arthroplasty failure. Periprosthetic osteolysis originates from chronic inflammatory responses triggered by implant-derived particulate debris, which cause recruitment of cells, including macrophages, fibroblasts, lymphocytes and osteoclasts. ...

Ongoing gastrointestinal bleeding (GIB) following endoscopic therapy and deciding between mesenteric angiography and surgery often challenge surgeons. We sought to identify predictors of positive angiographic study (active contrast medium extravasation) and characterize outcomes of embolization for acute GIB. We retrospectively analyzed angiographies for GIB at 2 teaching hospitals from January ...