Viviana Kozina

A One-Step Real-Time Multiplex PCR for Screening Y-Chromosomal Microdeletions without Downstream Amplicon Size Analysis

Backgound Y-chromosomal microdeletions (YCMD) are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR) is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers. Principal Findings The widely established “EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004)” were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size. Significance With these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection.

Macrophages and Leydig cells in testicular biopsies of azoospermic men.

A number of studies have indicated that testicular macrophages play an important role in regulating steroidogenesis of Leydig cells and maintain homeostasis within the testis. The current paper deals with macrophages (CD68 positive cells) and Leydig cells in patients with nonobstructive azoospermia (NOA). Methods employed included histological analysis on semi- and ultrathin sections, immunohistochemistry, morphometry, and hormone analysis in the blood serum. Histological analysis pointed out certain structural changes of macrophages and Leydig cells in NOA group of patients when compared to controls. In the testis interstitium, an increased presence of CD68 positive cells has been noted. Leydig cells in NOA patients displayed a kind of a mosaic picture across the same bioptic sample: both normal and damaged Leydig cells with pronounced vacuolisation and various intensity of expression of testosterone have been observed. Stereological analysis indicated a significant increase in volume density of both CD68 positive and vacuolated Leydig cells and a positive correlation between the volume densities of these cell types. The continuous gonadotropin overstimulation of Leydig cells, together with a negative paracrine action of macrophages, could result in the damage of steroidogenesis and deficit of testosterone in situ.

Cryptorchidism (Undescended Testis)

The undescended testis may be present at birth (congenital cryptorchidism) or, more frequently, may ascend during childhood (ascending testis, acquired cryptorchidism). The acquired undescended testis frequently is confused with the retractile testis (a variant of the normal organ). If a retractile or an undescended testis is still present in adulthood, it may be the cause of infertility and even a place for the development of a testicular carcinoma.

Key molecules in the GABA signalling pathway are present in mouse and human cervical tissue

Cervical mucus modulates fertility by cyclical changes of its biophysical and functional properties. Based on an analogy with bronchial goblet cells we set out to investigate the possible role of the gamma-aminobutyric acid (GABA) signalling pathway in the mediation of oestrogen-induced mucus secretion from endocervical secretory cells. The aim of the study was to examine the existence of GABAA receptor (GABAAR), glutamic acid decarboxylase 65/67 (GAD65/67) and vesicular GABA transporter (VGAT) in human and mouse cervical tissue. The mouse cervical tissue expressed GabaAR mRNA transcripts throughout the oestrous cycle. GABAAR-positive immunolabelling was present in the superficial layer of the mouse cervico-vaginal epithelium in pro-oestrus. Human cervical tissue showed the presence of GABAAR, GAD67 and VGAT mRNA transcripts and clear immunofluorescent signals of all three molecules were detected in the endocervical secretory epithelium. The results of this study confirmed that elements of the GABA signalling pathway are present in the secretory epithelium of mouse and human cervical tissue and that GABA signalling pathway could be considered a possible mediator in oestrogen regulation of mucus secretion in the endocervical glands.

Normal Morphology of the Human Testis and Epididymis

The aim of this chapter is to present the histologic architecture of the human testis and epididymis to clinicians who deal with spermatozoa (micro)surgical retrieval procedures, such as testicular sperm aspiration (TESA), testicular sperm extraction (TESE), percutaneous sperm aspiration (PESA), microsurgical epididymal aspiration (MESA), and similar techniques.

Histologic Procedures and Testicular Biopsy Freezing

The purpose of this chapter is to describe the biopsy handling and histologic processing of testicular tissue. This chapter is intended to serve as a practical guide to performing routine procedures in the histopathologic examination and cryopreservation of the testicular biopsy specimen. Some basic staining and step-by-step laboratory procedures are described.

New Approaches to Diagnosing Male Infertility, Part II: The Role of Confocal Microscopy and Three-Dimensional Reconstruction in Visualization of Reinke’s Crystals

The aim of this chapter is to illustrate the use of confocal microscopy and three-dimensional (3D) reconstruction in the visualization of various testicular structures. As an example, Reinke’s crystals are pointed out. Reinke’s crystals resemble hexagonal prisms of various sizes (mostly 2–3 μm). They are composed of parallel 5- to 10-nm filaments and do not have a unit membrane. Sometimes, a single crystal may be found in the cytoplasm and nucleus of Leydig cells. However, very often, two or even more crystals are present that may cause a deformity of the nucleus and infolding of the nuclear membrane. Occasionally, one may note electron-dense rod-shaped structures inside the crystal. Besides the remarkable Reinke crystals, other paracrystalline inclusions may be found within human Leydig cells.