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Objectives: The aim of the study was to check the quality of computer-assisted sperm analysis (CASA) system in comparison to the reference manual method as well as standardization of the computer-assisted semen assessment., Material and Methods: The study was conducted between January and June 2015 at the Andrology Laboratory of the Division of Infertility and Reproductive Endocrinology, Poznań University of Medical Sciences, Poland. The study group consisted of 230 men who gave sperm samples for the first time in our center as part of an infertility investigation. The samples underwent manual and computer-assisted assessment of concentration, motility and morphology. A total of 184 samples were examined twice: manually, according to the 2010 WHO recommendations, and with CASA, using the program set-tings provided by the manufacturer. Additionally, 46 samples underwent two manual analyses and two computer-assisted analyses. The p-value of p < 0.05 was considered as statistically significant., Results: Statistically significant differences were found between all of the investigated sperm parameters, except for non-progressive motility, measured with CASA and manually. In the group of patients where all analyses with each method were performed twice on the same sample we found no significant differences between both assessments of the same probe, neither in the samples analyzed manually nor with CASA, although standard deviation was higher in the CASA group., Conclusions: Our results suggest that computer-assisted sperm analysis requires further improvement for a wider application in clinical practice.

Phosphatidylserine membrane translocation (PST) is considered to be a marker of apoptosis; however, numerous studies have reported on its role in processes not related to cell death. The purpose of the study was to investigate: (1) what is the impact of PST on the motility of spermatozoa, and (2) does the swim-up isolation involve the percentage of cells presenting PST? Semen of 28 normozoospermic men (WHO criteria) was analyzed. High motility spermatozoa were isolated by the swim-up technique. The percentage of spermatozoa with PST in neat semen and after swim-up isolation was assessed with Annexin-V labeled with fluorescein, using flow cytometry technique. The spermatozoas' motility was measured with a computer-assisted analysis system. The kinetic subpopulations of spermatozoa were identified with dedicated software and analyzed regarding PST. Vital spermatozoa with PST demonstrated progressive movement. The motion analysis system revealed a very strong positive correlation between the percentage of vital spermatozoa with PST and the percentage of spermatozoa belonging to the slow subpopulation (r = 0.83; p < 0.05), as well as a very strong negative correlation between the percentage of vital spermatozoa with PST and the percentage of spermatozoa belonging to the rapid subpopulation (r = -0.86; p < 0.05). After the swim-up isolation, the percentage of vital spermatozoa presenting PST significantly decreased (2.4 ± 2.1% vs. 5.2 ± 2.4%; p < 0.05). Spermatozoa with PST present progressive movement; however, their motility is decreased. Isolation of spermatozoa with the swim-up technique eliminates the cells with PST.

Protamine 1 (PRM1) and 2 (PRM2) are major nuclear proteins in spermatozoa known to bind to chromatin during early spermatogenesis. The aim of this study was to evaluate the impact of protamine transcripts and proteins on human spermatozoa and their fertilization ability as well as the development of preimplantation embryos. Oocytes selected from 92 patients were fertilized in vitro (IVF) using their partners' sperm after evaluating its concentration, motility and morphology. Reverse transcription and real-time quantitative PCR (RQ-PCR) were applied to determine protamine mRNA level, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were used to quantify the protamine protein concentration. Next, protamine mRNA and protein levels were correlated with sperm concentration, motility and morphology as well as with the fertilization ability of mature spermatozoa and embryos of different quality obtained from the IVF program. A significant correlation has been found between protamine transcripts/proteins and: sperm concentration, progressive sperm motility, sperm fertilization ability as well as embryo quality. Protamine transcripts and proteins contribute to the quality of spermatozoa, successful fertilization and may have a significant influence on the development of preimplantation embryos.

Introduction: Psychological factors are believed to play an important role in infertility and its treatment Psychological problems may be considered to be risk factors and the cause of reduced infertility, as well as contribute to an unsuccessful outcome of infertility treatment., Objectives: The aim of the study was to investigate the correlation between semen parameters and quality of life in male patients with fertility problems., Material and Methods: 70 patients with infertility treated in Andrology Clinic were divided into two groups according to semen parameters: bad and good quality of semen. The quality of life has been with the help of Campbell questionnaires., Results: Significantly lower questionnaire score in bad quality of semen group has been observed, in comparison with normozoospermic men. High correlation between concentration, motility and morphology in comparison with the quality of life has been observed in the studied group of patients., Conclusion: The quality of life is directly associated with semen parameters such as concentration, motility and morphology of sperm.

The aim of this study was to prospectively investigate the spermatozoa ultrastructure in relation to the results of in vitro fertilization-embryo transer (IVF-ET). Forty-nine consecutive couples admitted for IVF-ET were prospectively evaluated for electron microscopic spermatozoa morphology and the outcome of IVF-ET. Thirty-four couples revealed successful fertilization, defined as presence of two pronuclei 14-16 hours after spermatozoa administration, while the remaining 15 formed the failure group. Spermatozoa fixed with 2.5% glutaraldehyd and embedded in Spurr's resin were analyzed with JAM 100 S transmission electron microscope (TEM) for the following ultrastructure abnormalities: head deformity, cytoplasmic residues, chromatin condensation failures, acrosomal alterations, neck defects, mid-piece defects, principal piece and end-piece defects and immature forms. Successful IVF-ET couples revealed a significantly higher percentage of normal spermatozoa utrastructure (32.0 +/- 13.1% versus 17.1 +/- 13.4%, p < 0.001). Failed IVF-ET couples represented a significantly higher percentage of chromatin condensation failures (9.8 +/- 5.1% versus 5.7 +/- 5.3%, p < 0.05) and tail defects (16.7 +/- 11.5% versus 7.2 +/- 7.2%, p < 0.001). A positive correlation between normal ultrastructure spermatozoa percentage and fertilized oocytes percentage was found (r = 0.35, p < 0.05). Our data suggest that spermatozoa TEM findings correlate with IVF-ET results. Ultrastructural estimation of spermatozoa can improve the diagnosis of male fertility and may explain some reasons of failure in assisted reproduction methods. We consider systematic TEM spermatozoa examination in cases with failed IVF-ET prior to intracytoplasmic sperm injection (ICSI).

Spermatid-specific linker histone H1-like protein (HILS1), transition proteins 1 and 2 (TNP1 and TNP2), and protamines 1 and 2 (PRM1 and PRM2) contribute to considerable dense packing of spermatid chromatin during spermiogenesis. We evaluated the HILS1, TNP1, and TNP2 transcript levels in spermatozoa isolated from normozoospermic and asthenozoospermic men. Human ejaculates from normozoospermic (n = 70) and asthenozoospermic (n = 100) donors were purified by centrifugation through a discontinuous Percoll density gradient. RNA was isolated from spermatozoa according to the Chomczynski and Sacchi method, treated with DNase I and reverse-transcribed into cDNA. Quantitative analysis of HILS1, TNP1, and TNP2 transcripts was performed by real-time quantitative (RQ-PCR) SYBR green I analysis. We found significantly lower levels of HILS1, TNP1, and TNP2 transcripts in spermatozoa from asthenozoospermic men compared to normozoospermic men. Our observations suggest that a reduction in HILS1, TNP1, and TNP2 transcripts may be associated with asthenozoospermia.

We determined the CCR5 chemokine receptor and cytochrome P450 aromatase (P450arom) transcript copies number in swim-up sperm isolated from fertile and infertile men. The ejaculates were purified by centrifugation through discontinuous Percoll density gradient and swim-up techniques. RNA was isolated from sperm, treated with DNase I and reverse-transcribed into cDNA. Quantitative analysis of CCR5 and P450arom cDNA were performed by real-time quantitative (RQ-PCR) SYBR Green I analysis. There was a higher content of CCR5 and P450arom transcripts copy number in swim-up sperm of fertile than from infertile donors. The decrease in CCR5 and P450arom transcripts in swim-up sperm may be associated with male infertility.

A proportion of fertilized oocytes during classical in vitro fertilization (IVF) procedure was analysed depending on the following factors: number of mature oocytes, seminological criteria such as sperm morphology in raw semen and after its selection in a density gradient (six structural defects of a male gamete were taken into consideration), sperm concentration, motility parameters according to World Health Organization criteria and the functional tests: hypo-osmotic swelling assay and acrosomal reaction induced by calcium ionophore. Evaluation of DNA content in sperm by image cytometry and determination of malonyldialdehydes in seminal plasma were also performed. Seventy-nine semen samples from patients undergoing IVF were assessed. Apart from significant correlations obtained for selected semen parameters and proportion of fertilized eggs, logistic regression analysis showed that the best predictive factors for oocyte fertilization were normal morphology of sperm before and after gradient selection, grade B and C of sperm movement in raw semen, and DNA content after density gradient centrifugation, which all accounted for 76.7% of fertilization predictive value.

The present study was carried out to investigate the relationship between sperm subpopulation kinetics on in vitro fertilization rate. The ability of human sperm to achieve fertilization oocytes was investigated in relation to particular motility parameters obtained on a computer aided sperm analysis system base. Analysis covers velocity straight linear (VSL), cross beat frequency (CBF), lateral head displacement (LHD) and homogeneity of progressive motility velocity (HPMV) of fresh semen and semen after density gradient selection. Investigation was based on sperm samples from 82 infertile couples undergoing IVF. Two subpopulations were extracted from each sample using the clustering method with respect to VSL parameter: a slow and rapid one. Comparison of obtained results before and after selection shows no significant change of subpopulations percentage. However, this method of selection strongly influences motility parameters of both subpopulations. There was found a positive correlation for VSL, LHD and HPMV and a negative correlation for CBF parameters found in slow fraction of fresh semen and percentage of fertilized oocytes. On the other hand, rapid subpopulation parameters for fresh semen and parameters found for both subpopulations in semen after selection did not correlate with one. This means that information of slow sperm subpopulation kinetics carries important prognostic value of IVF success. Since the current prognosis factors ignore motility parameters of slow sperms, our results show the importance of such an analysis.

Objective: Basic seminological criteria were analyzed to assess a percentage of fertilized oocytes during classical IVF procedure., Design: A prospective study., Materials and Methods: One hundred fifty semen samples from patients undergoing IVF were studied. Sperm morphology in raw semen and after its selection on a density gradient (6 structural defects of a gamete were taken into consideration), sperm concentration and motility parameters according to WHO criteria were analyzed., Results: Patients achieving fertilization had a significantly higher median proportion of normal forms, concentration of sperm, motility grade A, B, C and significantly lower median values for head defects, acrosomal defects, and motility grade D, than those who did not have fertilization. A marked improvement of sperm morphology after density gradient selection was also noted. Apart from significant correlations obtained for selected semen parameters with proportion of fertilized eggs, logistic regression analysis showed that the predictive value for the IVF oocyte fertilization, using normal morphology of sperm before and after selection, grade A, B and C of sperm movement in a raw semen accounted for 62.4% of fertilization., Conclusion: It seems that to obtain a better efficacy of IVF prognosis, functional tests have to be added to routine semen parameters.

It has recently been reported that the Regulated upon Activation of Normal T-cells Expressed and Secreted (RANTES) chemokine may exhibit a chemotactic effect on sperm. The RANTES chemokine acts on target cells by binding to the CCR5 receptor, which is present on the surface of various cells. Spermatozoa contain a complex repertoire of messenger RNAs (mRNAs) that may provide an insight into past events of spermatogenesis. The type and amount of CCR5 chemokine receptor transcript were investigated in spermatozoa that were isolated by the swim-up method from semen samples of men with normozoospermia. Using reverse transcription and real-time quantitative polymerase chain reaction (RQ-PCR) analysis, we found that the CCR5 mRNA isoform in human spermatozoa consists of exons 3 and 4, and is shorter than the transcript in leukocytes. This CCR5 transcript may represent a more stable mRNA isoform; one that is used to biosynthesize the CCR5 receptor in spermatogenesis or the early stages of embryonic development.

In the last years increasing incidence of malignant diseases in young men is observed. Sperm cryopreservation before cancer therapy gives the opportunity for these men to have a child in the future, but it is not know whether pretreatment sperm quality is sufficient to preserve fertility potential after thawing. The aim of this study was to compare the semen parameters (volume, total count, concentration, motility, morphology) in young cancer patients before treatment with healthy men, and examining the differences in sperm quality among patients group. Semen specimens were obtained from 81 patients with different neoplasms (testicular cancer n = 65, others n = 16 (Hodgkin's disease n = 11, lymphoma n = 3, Ewing's sarcoma n = 1, osteosarcoma n = l1). Control group consisted of 43 healthy males at similar age that came to our division as potential sperm donors. The total sperm count, concentration per ml, motility (grade A, B and C) and morphology were significantly lower in the cancer patients compared with normal men (p < 0.05), but still adequate for the future assisted reproductive technologies. Patients with testicular neoplasms had only significantly lower total sperm count and concentration in comparison with other cancer patients (p < 0.05). Our results indicate that routine sperm banking should be offered for men before radio/chemotherapy to preserve future fertility.

In this prospective study different semen parameters of fertile smokers were compared with fertile non-smokers. 58 men with proven fertility (17 smokers and 41 non-smokers) at the same age were evaluated. Smokers had significantly less spermatozoa with motility grade A (p<0.01), B (p<0.05), and C (p<0.05), and more abnormal forms in the ejaculate (p< 0.001). The percentage of sperm with head defects (p<0.05) and cytoplasmic droplets (p<0.001) were also significantly higher in smokers. No differences were found in the assessment of semen volume, total sperm count, hypoosmotic swelling test, induced acrosomal reaction and acidic aniline blue staining test between two groups (p>0.05). However the concentration of leukocytes in semen was significantly higher in smokers (p<0.001), which can suggests oxidative imbalance in the ejaculates of these men. Our data demonstrate that cigarette smoking is associated with reduced semen quality which could worsen fertilizing potential in fertile men.