Phosphatidylserine Membrane Translocation in Human Spermatozoa: Topography in Membrane Domains and Relation to Cell Vitality

The complex structure of the human spermatozoa membrane comprises five topographic domains. Transmembrane asymmetry of the distribution of phospholipids including phosphatidylserine (PS) is considered a marker of cell activity. The objective of the study was to determine which cytomembrane domains of human spermatozoa are involved in PS membrane translocation and to identify the possible relationship of PS translocation with spermatozoa morphology and vitality. In normozoospermic semen of 35 donors, annexin-V labeling with fluorescein determined PS translocation. Propidium iodide staining distinguished between vital and dead spermatozoa. Three types of PS membrane translocation have been distinguished: (1) in the midpiece, (2) in the acrosomal part and (3) simultaneously in the midpiece and acrosomal part. In morphologically normal vital spermatozoa, PS translocation occurred in the midpiece but never in the equatorial region. In dead spermatozoa, simultaneous PS translocation in the midpiece and acrosomal part was most often observed. The difference between proportions of, respectively, vital and dead spermatozoa presenting PS translocation located in different domains was significant (P < 0.0001). In vital cells, there was no difference in PS translocation prevalence between morphologically normal and abnormal spermatozoa (P > 0.05). The strict relation of PS translocation to specific membrane domains indicates functional specificity. It seems doubtful to include this phenomenon in physiological mechanisms of elimination of abnormal spermatozoa

Pronuclear scoring as a predictor of embryo quality in in vitro fertilization program.

Many strategies have been proposed for the selection of viable embryos for transfer in human assisted reproduction. These have included morphological scoring criteria for 20, 28, 44 and 68 h after insemination. The embryo selection is based on morphology, degree of fragmentation and development to the 8-cell. All have shown some correlation with implantation. However, the overall success of these methods is still limited, with over 50% of all transferred embryos failing to implant. Pronuclear zygote morphology has gained much attention recently due to its positive value in predicting implantation and pregnancy. This prospective study involved 178 conventional IVF patients only. The key aspects of pronuclear scoring and namely the presence of a cytoplasmic halo were related to day 3 of development and morphology in a retrospective study. The Z-score and the presence/absence of a halo had significant effect on the rate of development on day 3 embryo. Low Z-score result in slow development and poor morphology. The absence of a halo also resulted in slow and poor development, low morphology, increased fragmentation

Association between fertilin beta, protamines 1 and 2 and spermatid-specific linker histone H1-like protein mRNA levels, fertilization ability of human spermatozoa, and quality of preimplantation embryos.

Fertilization involves a series of cellular interactions culminating in the fusion of gamete membranes, creating a zygote and then an embryo. During the process of human fertilization in vivo or in conventional in vitro fertilization (IVF), sperm must be capable of undergoing the acrosome reaction, binding to the zona pellucida (ZP), and penetrating the ZP to fuse with the oolema. The key role in this process is played by fertilin beta. Protamines and histones are the proteins that bind to sperm chromatin and contribute in chromatin remodeling during early spermiogenesis. It has been suggested that these proteins may also participate in successful fertilization and embryo development. Using reverse transcription and real-time quantitative PCR reaction (QR-PCR) methods and zygote and embryo scoring, we compared fertilin beta, protamine 1 (PRM1), protamine 2 (PRM2), spermatid-specific linker histone 1 (HILS1) mRNAs levels, in vitro fertilization ability of mature spermatozoa, and quality of embryos obtained from in vitro fertilization (IVF). We found significantly lower contents of fertilin beta transcript in spermatozoa from patients in which IVF fertilization failed (

Manual vs. computer-assisted sperm analysis: can CASA replace manual assessment of human semen in clinical practice?

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 set­tings provided by the manufacturer. Additionally, 46 samples underwent two manual analyses and two computer-assisted analyses. The p-value of p &lt; 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

Evaluation of protamines 1 and 2 transcript contents in spermatozoa from asthenozoospermic men.

During mammalian spermatogenesis, the chromatin structure undergoes substantial condensation. The key role in this process is played by protamines 1 and 2 (PRM1, PRM2). We attempted to compare the levels of PRM1 and PRM2 transcripts in mature spermatozoa of normospermic and asthenozoospermic men. Human ejaculates from normozoospermic (n=70) and asthenozoospermic (n=100) donors were purified by centrifugation through discontinuous Percoll density gradient. RNA was isolated from spermatozoa according to the ChomczyĂąski and Sacchi method, treated with DNase I, and reverse-transcribed into cDNA. Using reverse transcription and real-time quantitative polymerase chain reaction analysis, we found a reduction in the levels of PRM1 and PRM2 transcripts in spermatozoa from asthenozoospermic men, as compared to controls (

Cytogenetic and molecular analyses of de novo translocation dic(9;13)(p11.2;p12) in an infertile male

BACKGROUND: Whole arm t(9;13)(p11;p12) translocations are rare and have been described only a few times; all of the previously reported cases were familial. RESULTS: We present here an infertile male carrier with a whole-arm reciprocal translocation dic(9;13)(p11.2;p12) revealed by GTG-, C-, and NOR-banding karyotypes with no mature sperm cells in his ejaculate. FISH and genome-wide 400 K CGH microarray (Agilent) analyses demonstrated a balanced chromosome complement and further characterised the abnormality as a dicentric chromosome (9;13): dic(9;13)(pter→p11.2::p12→qter),neo(9)(pter→p12→neo→p11.2). An analysis of the patient’s ejaculated cells identified immature germ cells at different phases of spermatogenesis but no mature spermatozoa. Most (82.5%) of the germ cells were recognised as spermatocytes at stage I, and the cell nuclei were most frequently found in pachytene I (41.8%). We have also undertaken FISH analysis and documented an increased rate of aneuploidy of chromosomes 15, 18, X and Y in the peripheral blood leukocytes of our patient. To study the aneuploidy risk in leukocytes, we have additionally included 9 patients with non-obstructive azoospermia with normal karyotypes. CONCLUSIONS: We propose that the azoospermia observed in the patient with the dic(9;13)(p11.2;p12) translocation was most likely a consequence of a very high proportion (90%) of association between XY bivalents and quadrivalent formations in prophase I

Hematopoietic stem cell mobilization with the reversible CXCR4 receptor inhibitor plerixafor (AMD3100)—Polish compassionate use experience

Recent developments in the field of targeted therapy have led to the discovery of a new drug, plerixafor, that is a specific inhibitor of the CXCR4 receptor. Plerixafor acts in concert with granulocyte colony-stimulating factor (G-CSF) to increase the number of stem cells circulating in the peripheral blood (PB). Therefore, it has been applied in the field of hematopoietic stem cell mobilization. We analyzed retrospectively data regarding stem cell mobilization with plerixafor in a cohort of 61 patients suffering from multiple myeloma (N = 23), non-Hodgkin’s lymphoma (N = 20), or Hodgkin’s lymphoma (N = 18). At least one previous mobilization attempt had failed in 83.6% of these patients, whereas 16.4% were predicted to be poor mobilizers. The median number of CD34+ cells in the PB after the first administration of plerixafor was 22/μL (range of 0–121). In total, 85.2% of the patients proceeded to cell collection, and a median of two (range of 0–4) aphereses were performed. A minimum of 2.0 × 106 CD34+ cells per kilogram of the patient’s body weight (cells/kg b.w.) was collected from 65.6% of patients, and the median number of cells collected was 2.67 × 106 CD34+ cells/kg b.w. (0–8.0). Of the patients, 55.7% had already undergone autologous stem cell transplantation, and the median time to neutrophil and platelet reconstitution was 12 and 14 days, respectively. Cases of late graft failure were not observed. We identified the diagnosis of non-Hodgkin’s lymphoma and previous radiotherapy as independent factors that contributed to failure of mobilization. The current report demonstrates the satisfactory efficacy of plerixafor plus G-CSF for stem cell mobilization in heavily pre-treated poor or predicted poor mobilizers

Standards in semen examination:publishing reproducible and reliable data based on high-quality methodology

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.Peer reviewe