By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous papers and manuals discuss in detail conditions influencing the quality of PCR in general, relatively little has been published about the important experimental factors and the common difficulties frequently encountered with multiplex PCR. We have examined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multiplex PCR assay are the relative concentrations of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome commonly encountered problems.

https://www.future-science.com/doi/pdf/10.2144/97233rr01

Multiplex PCR: critical parameters and step-by-step protocol.

https://uroweb.org/wp-content/uploads/Jungwirth-A-et-al-Eur-Urol-2012-622342-EAU-Guidelines-on-Male-Infertility-the-2012-update.pdf

European Association of Urology Guidelines on Male Infertility: The 2012 Update

A systematic search of the nonrecombining region of the human Y chromosome (NRY) identified 12 novel genes or families, 10 with full-length complementary DNA sequences. All 12 genes, and six of eight NRY genes or families previously isolated by less systematic means, fell into two classes. Genes in the first group were expressed in many organs; these housekeeping genes have X homologs that escape X inactivation. The second group, consisting of Y-chromosomal gene families expressed specifically in testes, may account for infertility among men with Y deletions. The coherence of the NRY's gene content contrasts with the apparently haphazard content of most eukaryotic chromosomes.

https://science.sciencemag.org/content/sci/278/5338/675.full.pdf

Functional coherence of the human Y chromosome.

Regulation of Spermatogenesis

Recent advances in understanding of male infertility have implicated two major causative factors, oxidative stress and Y chromosome deletions. A major cause of oxidative stress appears to be the high rate of reactive oxygen species generation associated with the retention of excess residual cytoplasm in the sperm midpiece. Other possible causes include the redox cycling of xenobiotics, and antioxidant depletion or apoptosis. Oxidative stress induces peroxidative damage in the sperm plasma membrane and DNA damage in both the mitochondrial and nuclear genomes. Nuclear DNA damage in the germ line of the father may be associated with pathology in the offspring, including childhood cancer and infertility. Gene deletions on the non-recombining region of the Y chromosome account for the infertility observed in about 15% of patients with azoospermia and 5-10% of subjects with severe oligozoospermia. The Y chromosome is particularly susceptible to gene deletions because of the inability of the haploid genome to deploy recombination repair in retrieving lost genetic information. Aberrant recombination, defective chromatin packaging, abortive apoptosis and oxidative stress may all be involved in the aetiology of DNA damage in the germ line. The factors responsible for Y chromosome deletions in spermatozoa remain unresolved but may be one facet of a central reproductive problem: controlling the amount of oxidative stress experienced by germ cells during their differentiation and maturation in the male reproductive tract.

/pdf/oxidative-stress-dna-damage-and-the-y-chromosome-3dr9oq8vcx.pdf

Oxidative stress, DNA damage and the Y chromosome.

In a large collaborative screening project, 370 men with idiopathic azoospermia or severe oligozoospermia wereanalysed for deletions of 76 DNA loci in Yq11. In 12 individuals, we observed de novo microdeletions involvingseveral DNA loci, while an additional patient had an inherited deletion. They were mapped to three differentsubregions in Yq11. One subregion coincides to the AZF region defined recently in distal Yq11. The second andthird subregion were mapped proximal to it, in proximal and middle Yq11, respectively. The different deletionsobserved were not overlapping but the extension of the deleted Y DNA in each subregion was similar in eachpatient analysed. In testis tissue sections, disruption of spermatogenesis was shown to be at the same phasewhen the microdeletion occurred in the same Yq11 subregion but at a different phase when the microdeletionoccurred in a different Yq11 subregion. Therefore, we propose the presence of not one but three spermatogenesisloci in Yq11 and that each locus is active during a different phase of male germ cell development. As the mostsevere phenotype after deletion of each locus is azoospermia, we designated them as: AZFa, AZFb and AZFc.Their probable phase of function in human spermatogenesis and candidate genes involved will be discussed. INTRODUCTIONGenes for male germ cell development are present on the Ychromosome in different species groups (1–3). In men, theposition of a spermatogenesis locus was mapped in theeuchromatic part of the long Y arm (Yq11). It was called‘azoospermia factor’ (AZF), as the first six men observed withterminal deletions in Yq were azoospermic (4). Mature spermcells were not detectable in their seminal fluid. In all cases, the Ydeletions included the large heterochromatin block of the long Yarm (Yq12) and an undefined amount of the adjacent euchromatin(Yq11). Subsequently, the presence of AZF in Yq11 wasconfirmed by numerous studies at both cytogenetic (5) andmolecular level (6–8). However, the genetic complexity of AZFcould not be revealed by these analyses.This first became possible by the detection of sterile patientswith small interstitial deletions (i.e. microdeletions) in Yq11. Ina study with 13 sterile men suffering from idiopathic azoospermiatwo different microdeletions in Yq11 were observed (9). Theywere mapped to two non overlapping positions in Yq11 interval6 (10). However, further studies of Yq11 microdeletionsassociated to the phenotype of male sterility, only confirmed theposition of an AZF locus in distal Yq11 (11,12). The mostextensive study was performed by Reijo et al. (13) on 89 sterile

Human Y Chromosome Azoospermia Factors (AZF) Mapped to Different Subregions in Yq11

The concept that an elevation of testicular temperature results in impairment of spermatogenesis is widely accepted. Here, current knowledge concerning genital heat stress and its consequences in men is reviewed. Duration of sitting during work positively correlates with daytime scrotal temperatures and daytime scrotal temperature negatively correlates with semen quality. However, the assumed negative correlation between duration of sitting and semen quality could not be shown in the available studies. Fertility parameters of professional drivers with long periods of sitting in vehicles were impaired; however, for predominantly affected drivers of vans, trucks or industrial heavy machinery potential confounders have to be considered. Wearing tight fitting compared with loose-fitting underwear is associated with significantly higher scrotal temperatures. However, available observations suggesting a link between tight-fitting underwear or trousers and impaired semen quality are not convincing. Studies addressing professional exposure to high temperatures delivered conflicting results concerning fertility parameters. The postulated negative impact of sauna visits on semen quality is not sufficiently underlined by the available studies. Oligozoospermic men with a varicocele have significantly higher scrotal temperatures than normozoospermic men, and according to several studies varicocelectomy normalises scrotal temperatures. A further link has been reported between fever and deteriorated semen quality. Contraception via genital heat stress has been demonstrated using hot sitting baths or insulating suspensors. However, down-regulation of spermatogenesis is inconsistent and unsafe. On the other hand, scrotal and consecutively testicular cooling is able to improve semen quality.

Influence of genital heat stress on semen quality in humans.

To evaluate the fertilizing capacity of spermatozoa from elderly men, ejaculates from 29 older fathers (mean age 50.3 years) were compared with those from 35 younger fathers (mean age 32.2 years). In addition to conventional semen parameters, sperm functions were studied that have been reported to be positively correlated with the fertilization rate: progressive motility, acrosin activity, inducible acrosome reaction, and chromosome condensation. Sperm concentration and follicle stimulating hormone concentration differed significantly in both groups. With regard to sperm functions there were no differences between older men and younger men, except for decreased sperm motility in the older group which, however, reached nearly normal values according to World Health Organization criteria. Decreased fertility of older couples is obviously more dependent on the age of the female partner. The significance of genetic risks remains to be clarified, especially when methods of assisted reproduction are applied.

/pdf/ageing-and-sperm-function-ubz825y3my.pdf

Ageing and sperm function

BACKGROUND Testicular temperature correlates highly with scrotal temperature. The aim of this study was to evaluate the influence of the type of undertrousers on scrotal temperature during standardized periods of sitting and walking. METHODS Fifty volunteers without a history of infertility and normal andrological examination were included for scrotal temperature evaluation. Temperatures were measured every minute with a portable data recorder connected with two thermistor temperature sensors, which were attached on either side of the scrotum. Ambient temperature in the study room was adjusted to 20 degrees C throughout the whole experiment. All volunteers started the experiment at the same time of day. Clothing of the volunteers consisted of standardized cotton wool trousers and shirts fitting to body size. Each volunteer performed six periods of 45 min, either walking on a treadmill (3.0 km/h) or sitting, and wearing in a standardized and randomized manner either tight, loose fitting or no undertrousers respectively. RESULTS The following interactions were demonstrated by means of multivariate analysis of variance for repeated measurements: scrotal temperatures were significantly higher for tight versus loose fitting versus absent undertrousers. Furthermore, significantly lower scrotal temperatures were identified for walking versus sitting as well as for the right versus the left scrotal side. CONCLUSIONS The present study suggests that wearing tight fitting undertrousers is associated with higher scrotal and consequently testicular temperatures than wearing loose fitting undertrousers or none.

/pdf/influence-of-the-type-of-undertrousers-and-physical-activity-i8obzuscdn.pdf

Influence of the type of undertrousers and physical activity on scrotal temperature

The effects of age on semen quality were analysed in patients referred to an andrology outpatient clinic in a retrospective study covering a period of 3 years. Semen analyses of older men (n = 66; > or = 50 years; median: 53) were compared with those of young patients (n = 134; 21-25 years; median 24). The duration of sexual abstinence was longer among older patients (median: 5.5 vs. 5.0 days; + 10%, P < 0.05). Even after adjustment for duration of sexual abstinence, progressive motility (- 27%, P < 0.01), percentage of morphologically normal spermatozoa (- 44%, P < 0.01) and semen volume (- 29%, P < 0.0005) were significantly lower in older than in younger men. Impaired sperm morphology was mainly attributed to abnormal staining of flagella (+ 50%, P < 0.05) and an increased portion of coiled or bent tails (+ 44%, P = 0.001) indicating epididymal dysfunction. Total sperm count and sperm concentration were unaffected by age. Age-dependent changes in accessory gland function, as indicated by fructose concentration (- 27%, P < 0.05), was correlated with lower semen volume. Moreover, serum testosterone levels were significantly reduced in the group of older men (median: 3.0 vs. 3.6 ng ml-1; -17%, P < 0.005).

A. Jung

Papers

Human Y Chromosome Azoospermia Factors (AZF) Mapped to Different Subregions in Yq11

Influence of genital heat stress on semen quality in humans.

Ageing and sperm function

Influence of the type of undertrousers and physical activity on scrotal temperature

Comparison of semen quality in older and younger men attending an andrology clinic.