Baseline DNA damage in spermatozoa from fertile and infertile men was compared using a modified alkali single cell gel electrophoresis (comet) assay. Semen from normozoospermic fertile, normozoospermic infertile and asthenozoospermic infertile (World Health Organization criteria, 1992) samples were studied. No significant difference was observed in levels of baseline damage between the three groups. A median value for baseline damage of approximately 20% (80% head DNA) was obtained in all samples. Irradiation with X-rays (5-30 Gy) produced no additional damage in fertile samples when median values were examined. However, irradiation with 30 Gy X-rays produced significant damage in both infertile groups. Hydrogen peroxide (40 microM) treatment induced significant damage in the asthenozoospermic group, whereas 100 microM H2O2 was required to cause significant damage in the normozoospermic fertile and infertile samples. Within the fertile population a subgroup in which percentage head DNA was greater than 80% was observed in both treated and untreated specimens. This subgroup significantly decreased with treatment in both infertile groups. We conclude that the asthenozoospermic infertile group is more susceptible to damage than the normozoospermic infertile group, which in turn is more susceptible than the fertile group. The fertile group contains a resistant subpopulation of spermatozoa with relatively intact DNA.

/pdf/a-comparison-of-baseline-and-induced-dna-damage-in-human-5emlb7pdlf.pdf

A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay.

The aim of the present prospective study was to determine whether subtle sperm morphological characteristics affect the outcome of intracytoplasmic sperm injection (ICSI), and if so, to identify those that are relevant. For this purpose, we developed a new method, the motile sperm organelle morphology examination (MSOME). The examination is performed in real time using an inverted light microscope equipped with high-power Nomarski optics enhanced by digital imaging to achieve a magnification up to 6300x. MSOME was applied to the leftover sperm fraction selected for microinjection in 100 random couples referred for ICSI treatment at 3 major in vitro fertilization centers. We found that the morphological normalcy of the entire sperm cell, according to MSOME criteria, was positively associated with ICSI fertilization rate (area under the receiver operating characteristics [ROC] curve, 88%) but not with pregnancy outcome. The morphological normalcy of the sperm nucleus, defined by MSOME, was significantly and positively associated with both fertilization rate and pregnancy outcome (areas under the ROC curve, 72% and 74%, respectively). These findings indicate that ICSI-associated pregnancy rate may be affected by subtle morphological malformations of the sperm nucleus, which may remain undetected by the embryologist during the routine selection procedure.

Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome.

The aim of the present prospective study was to de- termine whether subtle sperm morphological characteristics affect the outcome of intracytoplasmic sperm injection (ICSI), and if so, to identify those that are relevant. For this purpose, we developed a new method, the motile sperm organelle morphology examination (MSOME). The examination is performed in real time using an in- verted light microscope equipped with high-power Nomarski optics enhanced by digital imaging to achieve a magnification up to 6300. MSOME was applied to the leftover sperm fraction selected for mi- croinjection in 100 random couples referred for ICSI treatment at 3 major in vitro fertilization centers. We found that the morphological normalcy of the entire sperm cell, according to MSOME criteria, was positively associated with ICSI fertilization rate (area under the re- ceiver operating characteristics (ROC) curve, 88%) but not with preg- nancy outcome. The morphological normalcy of the sperm nucleus, defined by MSOME, was significantly and positively associated with both fertilization rate and pregnancy outcome (areas under the ROC curve, 72% and 74%, respectively). These findings indicate that ICSI-associated pregnancy rate may be affected by subtle morpho- logical malformations of the sperm nucleus, which may remain un- detected by the embryologist during the routine selection procedure.

/pdf/breakthroughs-in-andrology-real-time-fine-morphology-of-3pnl4mgx1l.pdf

Breakthroughs in Andrology Real-Time Fine Morphology of Motile Human Sperm Cells is Associated With IVF-ICSI Outcome

https://www.europeanurology.com/article/S0302-2838(11)00642-7/pdf/varicocele-and-male-factor-infertility-treatment-a-new-meta-analysis-and-review-of-the-role-of-varicocele-repair

Varicocele and male factor infertility treatment: a new meta-analysis and review of the role of varicocele repair.

Pregnancy rates are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection

The aim of this study was to develop a new male fertility diagnostic profile based on quantitative ultramorphology parameters and to determine the contribution of this profile to the enhancement of the routine semen analysis index reported previously. Semen samples from 208 males of known fertility and suspected infertility were evaluated for the ultrafine structure of the following sperm cell organelles: acrosome, post-acrosomal lamina, nucleus, neck, axonema, mitochondrial and fibrous sheaths. For each of these organelles, four pathological states (agenesis, incomplete genesis, malformation and degradation) and an intact state were defined. A quantitative ultramorphology index based on the incidence of intact nucleus, acrosome and fibrous sheath malformations enabled high accuracy in the classification (97% sensitivity and 90% specificity) of 74% of the cases. A combined semen quality index based on a proportional combination of the semen analysis and quantitative ultramorphology indices was found to increase the percentage of cases classified correctly to 80%. It was proposed that semen specimens of males whose fertility status cannot be predicted clearly using routine semen analysis should be fixed and sent for quantitative ultramorphology analysis to specialized laboratories so that their fertility potential can be determined more accurately using the semen quality index.

Improved diagnosis of male fertility potential via a combination of quantitative ultramorphology and routine semen analyses.

Summary. Varicocele of spermatic veins is considered to be one of the major causes of male infertility associated with reduction of sperm quality. The pathophysiology of this condition is not yet completely understood. The aim of this study was to shed light on the pathophysiology of varicocele by identifying semen parameters, especially sperm ultramorphology, which improve following high ligation of the spermatic vein. Seventy-five males with diagnosed varicocele were included in this study. Semen parameters were assessed prospectively using light microscopy, semen biochemistry and sperm quantitative ultramorphological analysis, before high ligation and 3–9 months after high ligation. The control group consisted of twenty-five untreated varicocele patients who underwent two semen examinations within 3–9 months. No statistical difference in any of the examined variables was found between the two examinations in the control group. The treated patients exhibited a significant improvement in sperm density, progressive motility, percentage of normally formed spermatozoa, agenesis of sperm acrosome, chromatin condensation and incidence of amorphous heads compared with the pretreatment condition (P≤0.01). In contradiction, no significant improvement was observed following treatment in any of the sperm tail subcellular organelles. It is concluded that varicocele may cause deleterious alterations in early spermatid head differentiation during spermiogenesis and that varicocele patients with a high incidence of sperm acrosome and nucleus malformations are appropriate candidates for varicocele correction.

Sperm ultramorphology as a pathophysiological indicator of spermatogenesis in males suffering from varicocele.

Summary An in vitro nuclear chromatin decondensation test, and quantitative nuclear ultramorphology analysis, were performed on 18 males judged to be infertile, by two failures in in vitro fertilization, and 16 fertile males These two clinical groups only differed significantly in (1) the direction of their chromatin stability change, which took place 30–120 min post-ejaculation while stored in the seminal plasma, and (2) in the incidence of the hypoelongated sperm-head Generally, the fertile male group exhibited positive chromatin stability change after prolonged storage, and low incidence of hypoelongated sperm heads, and vice versa in the unexplained infertile group When the nuclear chromatin decondensation test and quantitative ultramorphology analysis were performed in step-wise fashion, it was possible to correctly classify 94% of the fertile cases with 6% of false-negative, and 89% of the unexplained infertile cases with 11% of false-positive Therefore, it appears that these tests might be of benefit clinically for identifying functional properties of sperm-cells in unexplained infertile males, which cannot be detected by routine semen analysis

Sperm head ultramorphology and chromatin stability of males with unexplained infertility who fail to fertilize normal human ova in vitro.

Summary. It was suggested that although not related to the standard semen parameters the level of the acrosin enzyme system is related to the fertility potential in men. Recently a simple clinical assay for total acrosin level was recommended for routine semen analysis. The improved clinical assay was analysed on the freshly liquified semen of 198 Astheno-teratozoospermic men and compared with the routine semen parameters including biochemical data and the ultrastructure of the acrosome. Only the sum of the per cent of live, motile, and normal-shaped spermatozoa had positive significant and reasonably high correlation with the acrosin level (r = 0.382, P < 0.0001). Each characteristic exhibits significant but low (< 0.35) correlation. Similarly negative significant and reasonably high correlation was obtained between the acrosin level and the sum of the principle acrosomal malformations observed by TEM (r = 0.396, P < 0.0001) while lower negative correlation was found only with agenesis or loss of the acrosome. Acrosin levels below 8.1 μIU 10−6 cells were obtained in 4 specimens with above 80% round-form associated with more than 95% of agenesis of the acrosome. The possible significance of the low correlation obtained between the acrosin levels and seminal plasma zinc levels, malformations in the acrosomal equatorial region, and the presence of white blood cells is also discussed. We concluded that the acrosin activity reflects an aspect of male fertility which is not diagnosed by the routine semen analysis or by the ultrastructure of the acrosome, and is therefore a useful diagnostic sperm parameter.

Human sperm acrosin activity with relation to semen parameters and acrosomal ultrastructure.

The advantages of quantitative ultramorphological (QUM) sperm analysis in the diagnosis and treatment of male infertility are presented. The QUM methodology is based on three elements: complementary scanning electron microscopy and transmission electron microscopy observations of 7 sperm cell subcellular organelles (acrosome, postacrosomal lamina, nucleus, neck, axoneme, mitochondrial sheath, and outer dense fibers); systematic classification of the specific ultramorphological malformations into 4 pathological and the normal categories, indicating the morphological state of each subcellular organelle; and comparison between well-defined reference groups with opposite fertility status or treatment conditions. QUM has established 2 indices for the in vivo and in vitro male fertility potential: (1) Natural Fertility Index (NFI), with accurate prediction (97% sensitivity and 90% specificity) of 80% of the male patients; and (2) IVF score, with prediction of 76% of the nonfertilizing and 90% of fertilizing IVF groups. QUM has enabled assessment of ultramorphological indications for varicocele and radiation exposure. Varicocele causes defects in sperm head organelles related to early spermatid development, whereas ionizing radiation causes amorphous head shape. QUM established criteria for specific non-in-vitro therapeutic interventions, including varicocelectomy, follicle-stimulating hormone (FSH) administration, and acupuncture. The varicocele index enabled correct classification of 79 and 89% of patients with and without varicocele. Males with idiopathic impairment of sperm acrosome and nucleus are potential responders to FSH treatment, whereas patients exhibiting low sperm activity are candidates for acupuncture treatment. Patients with a low Natural Fertility Index are recommended for an assisted reproduction technique (ART), based on the ultramorphology of the tail axoneme. Patients who achieved pregnancy following intrauterine insemination or in vitro fertilization and those whose wives conceived only following intracytoplasmic sperm injection were classified with accuracy of 78 and 74%, respectively. QUM sperm analysis is clinically informative, nontraumatic, and cost-effective, and is recommended when the male infertility factor cannot be clearly diagnosed by routine tests prior to first ART trial.

M. Reichart

Papers

Improved diagnosis of male fertility potential via a combination of quantitative ultramorphology and routine semen analyses.

Sperm ultramorphology as a pathophysiological indicator of spermatogenesis in males suffering from varicocele.

Sperm head ultramorphology and chromatin stability of males with unexplained infertility who fail to fertilize normal human ova in vitro.

Human sperm acrosin activity with relation to semen parameters and acrosomal ultrastructure.

Quantitative ultramorphological analysis of human sperm: fifteen years of experience in the diagnosis and management of male factor infertility.