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Biomedical subjects

D Sakkas

Publications and source records attributed to D Sakkas.

At least 19 recordsLinked to original sources
PubMed2004

Reduced senescence and retained nuclear DNA integrity in human spermatozoa prepared by density gradient centrifugation.

PURPOSE: To investigate whether removal of extraneous cells and immotile spermatozoa from a sperm preparation by density gradient centrifugation could help to maintain normal spermatozoa in a viable state and retain their deoxyribonucleic acid integrity. METHODS: Sperm motility was assessed on a daily basis in aliquots of neat semen, extended semen, and spermatozoa prepared on a PureSperm density gradient. At the same time, aliquots of each sperm sample were preserved for TUNEL assay and nick translation. RESULTS: Spermatozoa prepared using density gradient centrifugation survived three times as long as spermatozoa in neat semen or in extended semen. Both deoxyribonucleic acid integrity and sperm motility were retained in the gradient preparations. CONCLUSIONS: Preparing spermatozoa by density gradient centrifugation is advantageous in prolonging sperm survival and maintaining deoxyribonucleic acid integrity, presumably by removing sources of reactive oxygen species. Stored spermatozoa could be used for a second attempt at fertilization if oocyte immaturity was suspected.

Adult↗
PubMed2004

Hypoxia-inducible factor 1alpha/vascular endothelial growth factor axis in astrocytomas. Associations with microvessel morphometry, proliferation and prognosis.

Hypoxia-inducible factor (HIF)-1alpha is a transcription factor that promotes ischaemia-driven angiogenesis. The aim of this study was to determine the relation of HIF-1alpha to vascular endothelial growth factor (VEGF; an important angiogenic molecule in brain tumours), p53 expression, angiogenesis, proliferative potential and clinical outcome in a large series of diffuse astrocytomas. Expression of HIF-1alpha, VEGF, Ki-67 (a proliferation-associated marker) and p53 was determined immunohistochemically in 83 adult patients with supratentorial diffuse astrocytomas. Microvessels, highlighted by means of anti-CD34 immunohistochemistry, were enumerated with computer-assisted image analysis. Although HIF-1alpha and VEGF were expressed in the majority of cases, their levels increased significantly with increasing grade and proliferative potential. HIF-1alpha positively correlated with microvessel counts and VEGF with total vascular area and the presence of rounder vessel sections. There was a positive correlation of VEGF with p53 expression in astrocytomas and anaplastic astrocytomas. In univariate analysis, both VEGF and HIF-1alpha were associated with shortened survival in the entire cohort, but lost significance when grades II/III and grade IV were analysed separately. Multivariate analysis revealed that the combination of HIF-1alpha with grade was a significant prognostic indicator. HIF-1alpha expression may be used to refine the prognostic information provided by grade in patients with diffuse astrocytomas. Its adverse prognostic effect is most likely mediated by hypoxia, the driving force for HIF-1alpha accumulation.

Adolescent↗
PubMed2003

Assessment of embryo viability: the ability to select a single embryo for transfer--a review.

By being able to select the most viable embryo(s) within a given cohort it will be possible to reduce the number of embryos transferred in a given IVF procedure. Several morphological scoring systems have been proposed for the successive stages of human embryo development. Other indicators of embryo viability include rate of cleavage. Finally, non-invasive methods of assessing nutrient uptake and utilization have been developed that can be used to measure the health of individual embryos. A sequential scoring system has therefore been proposed that uses the above parameters in order to create a history for each embryo during the preimplantation period. Such systems will help lead to single embryo transfers for the majority of IVF patients.

Adult↗
PubMed2003

Sequential assessment of individually cultured human embryos as an indicator of subsequent good quality blastocyst development.

BACKGROUND: It is of fundamental importance for IVF clinics to determine the most viable embryos for transfer. The challenge for ART clinics is to transfer fewer embryos, thereby minimizing the risk of multiple-infant births, while still maintaining the greatest chance of pregnancy for their patients. In this study, an investigation was made to determine if developmental markers on the day of fertilization (day 1) can predict good subsequent blastocyst development. METHODS AND RESULTS: A total of 1550 individually cultured 2PN embryos from 191 patients undergoing IVF/ICSI treatment at the Yale University Center for Reproductive Medicine and Infertility from February to December 2001 was included. The results showed a significant positive relationship between early-cleaving 2-cell embryos and subsequent good quality > or =4-cell, > or =7-cell and blastocyst development (P < 0.05). PN symmetry (the relative size of the PN to each other), when checked at the time fertilization, is also a significant indictor of good quality > or =4-cell, > or =7-cell stage embryos and blastocysts. Combined, a developing embryo showing PN symmetry with early cleavage and subsequent good > or =4-cell and > or =7-cell cleavage, has a one in two chance of developing into a good-quality blastocyst. CONCLUSION: Early embryo assessment can be used as an indicator of subsequent good blastocyst development.

Adult↗
PubMed2002

Up-regulation of glucose metabolism during male pronucleus formation determines the early onset of the s phase in bovine zygotes.

After in vitro fertilization with spermatozoa from bulls with high in vitro fertility, a beneficial paternal effect is manifested during the G1 phase of the first cell cycle. This benefit determines an earlier onset of the first S phase, and then a successful morula-blastocyst transition 7 days later. We hypothesized that the origin of the paternal effect could be a shift of the metabolism of the fertilized oocyte, because in mice, sperm decondensation is responsible for a dramatic increase in glucose metabolism. In this study we investigated the interaction between both pronuclei and compared glycolysis and pentose phosphate pathway (PPP) activities in bovine oocytes fertilized with spermatozoa from bulls of high or low fertility. Here we demonstrate that male pronucleus formation is necessary for the onset of the S phase in the female pronucleus, and that the component promoting an early S phase in both pronuclei is metabolic and linked to an up-regulation of the PPP during the male pronucleus formation. This long-lasting paternal effect is more evidence of the important role of epigenetic control during early embryo development.

6-Aminonicotinamide↗
PubMed2002

Localization of tyrosine phosphorylated proteins in human sperm and relation to capacitation and zona pellucida binding.

Mammalian sperm must undergo a process known as capacitation before fertilization can take place. A key intracellular event that occurs during capacitation is protein tyrosine phosphorylation. The objective of this study was to investigate and visualize protein tyrosine phosphorylation patterns in human sperm during capacitation and interaction with the zona pellucida. The presence of specific patterns was also assessed in relation to the fertilizing capacity of the spermatozoa after in vitro fertilization. Protein tyrosine phosphorylation was investigated by immunofluorescence. Phosphorylation increased significantly with capacitation and was localized mainly to the principal piece of human sperm. Following binding to the zona pellucida, the percentage of sperm with phosphotyrosine residues localized to both the neck and the principal piece was significantly higher in bound sperm than in capacitated sperm in suspension. When the percentage of principal piece-positive sperm present after capacitation was <7%, fertilization rates after in vitro fertilization were reduced. Different compartments of human spermatozoa undergo a specific sequence of phosphorylation during both capacitation and upon binding to the zona pellucida. Tyrosine phosphorylation in the principal and neck piece may be considered a prerequisite for fertilization in humans.

Female↗
PubMed2001

Assessment of early cleaving in vitro fertilized human embryos at the 2-cell stage before transfer improves embryo selection.

OBJECTIVE: To determine the most viable embryos for transfer. DESIGN: Study 1: Preselection of early-cleaving 2-cell embryos for transfer. Study 2: Alternating weeks during which preselection was performed and not performed. SETTING: ART program, Birmingham Women's Hospital, Birmingham, United Kingdom. PATIENT(S): Patients undergoing IVF or ICSI cycles with transfer on day 2. INTERVENTION(S): Culture of all fertilized embryos. MAIN OUTCOME MEASURE(S): Number of fertilized embryos cleaving to the 2-cell stage on day 1, embryo quality, implantation rates, and pregnancy rates. RESULT(S): Patients with early-cleaving 2-cell embryos had significantly higher pregnancy and implantation rates (45 of 100 [45.0%] and 58 of 219 [25.5%], respectively) than did patients without early-cleaving 2-cell embryos (31 of 130 [23.8%] and 43 of 290 [14.8%], respectively). In weeks during which preselection was used, the overall pregnancy and implantation rates of the clinic improved. CONCLUSION(S): The presence of early-cleaving 2-cell embryos improves a patient's chance of achieving pregnancy. Use of more stringent embryo selection criteria can improve overall pregnancy rates.

Adult↗
PubMed2001

Blastocyst transfer for patients with multiple assisted reproduction treatment failures: preliminary experience.

This preliminary study reports the results obtained from a patient group in which blastocyst culture and transfer were performed, and discusses the advantages and disadvantages of introducing blastocyst transfer in a clinic. Twenty-six patients who had failed to achieve a pregnancy in previous in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatments were offered the choice of a fresh cycle with culture to the blastocyst stage. Of the 26 patients who elected to attempt blastocyst culture, 11 opted to have transfer on day 2 or day 3 due to low numbers of embryos. Of the 15 patients who proceeded to blastocyst culture, 46.2% of the embryos cultured reached the blastocyst stage or later and eight of the patients achieved a clinical pregnancy. More oocytes were collected in this patient group, hence the chances of obtaining blastocysts were higher. Offering blastocyst culture to patients with a reasonable chance of success who have had previous multiple assisted reproduction failures is an acceptable way of introducing blastocyst culture into practice.

Adult↗
PubMed2001

One-step disposable chambers for sperm concentration and motility assessment: how do they compare with the World Health Organization's recommended methods?

The World Health Organization suggests that 'one-step' type disposable chambers lack the accuracy and precision of the haemocytometer method for assessing sperm concentration. The purpose of this particular study was to compare sperm concentration measurements and motility using the Neubauer((R)) haemocytometer with those obtained using three 'one-step' methods: the Microcell((R)) slide, Leja((R)) slide and a plain glass slide with a 22x22 mm coverslip. A total of 200 sperm concentration measurements and 100 motility assessments were performed on all chambers. Paired comparisons showed enormous discrepancies between the counts, particularly between the Neubauer((R)) and other chambers (P < 0.0001). This discrepancy was less pronounced in oligozoospermic samples, and samples with low (<30% progression) motility but more pronounced in normozoospermic samples and those with good motility (>50% progression). In addition, concentration assessments from a fresh undiluted and unfixed semen sample on the Microcell((R)) slide were found to be significantly lower than both fixed counts on the same slide (P = 0.011) and the initial laboratory reading on the Neubauer((R)) chamber (P = 0.009). No differences were observed in progressive motility between the different chambers and a plain glass slide. There appears to be little comparison between the haemocytometer and either re-useable or disposable one-step chambers. The unfortunate consequence of this is that diagnostic semen analysis and guidelines for allocation of patients to appropriate treatment groups will vary from centre to centre, depending on the method used and may, on occasion, be erroneous.

Humans↗
PubMed2001

Interrelationships between seminal parameters and sperm nuclear DNA damage before and after density gradient centrifugation: implications for assisted conception.

BACKGROUND: With an increase in the use of assisted reproduction technologies the requirements of the diagnostic semen analysis are constantly changing. METHODS: Spermatozoa from patients undergoing IVF were analysed by examining the conventional semen parameters and DNA/chromatin integrity, using in-situ nick translation (NT) and the Chromomycin A(3) fluorochrome, which indirectly demonstrates a decreased presence of protamine. Samples were examined before and after preparation using discontinuous density gradient centrifugation. RESULTS: Density gradient centrifugation enriched samples by improving the percentage of morphologically normal forms by 138% and sperm nuclear integrity by 450%. Sperm nuclear integrity as assessed by in-situ nick translation (NT) demonstrated a very clear relationship with sperm concentration, motility and morphology. Morphology correlated with fertilization rates of patients undergoing IVF, while NT values of the spermatozoa post-preparation were significantly lower in pregnant patients. CONCLUSIONS: We have demonstrated that along with the classical semen parameters, the assessment of nuclear integrity improves the characterization of the semen sample and may be used as a tool for allocating patients to specific assisted reproduction treatments.

Adult↗
PubMed2001

Facilitated glucose transporters play a crucial role throughout mouse preimplantation embryo development.

The role of glucose fluctuates during preimplantation mouse embryo development, indicating that a specific interplay exists between glucose metabolism and uptake. In this study, attempts were made to characterize the role of the Na(+)-coupled active and the facilitated glucose transporters (GLUT) during preimplantation development by using specific glucose analogues and transport inhibitors and by examining the expression of GLUT1. One-cell outbred mouse embryos were cultured in medium M16 (5.5 mmol/l glucose), M16 without glucose (M16-G), M16-G + 2-deoxyglucose, M16-G + 3-O-methylglucose, M16 + phlorizin and M16 + phloretin and development to the blastocyst stage assessed. The absence of glucose, or the presence of 3-O-methylglucose, which is taken up but not metabolized, did not inhibit blastocyst development. 2-Deoxyglucose, which is phosphorylated but not metabolized, inhibited blastocyst development. Culture in M16 supplemented with phlorizin, an inhibitor of Na(+)-coupled active glucose transport did not inhibit blastocyst formation. Phloretin had no effect on the cleavage of two-cell embryos to the four-cell stage, but inhibited the morula/blastocyst transition. Both phloretin and phlorizin inhibited glucose uptake in two-cell embryos. Finally, GLUT1 expression was 10-fold less in blastocysts cultured in M16 compared to in-vivo blastocysts and those cultured in M16-G. The results show that both types of glucose transporters influence preimplantation embryo development and that the embryo has an innate ability to control the uptake of glucose by regulating the expression of GLUT1.

3-O-Methylglucose↗
PubMed2001

Protein tyrosine phosphorylation in sperm during gamete interaction in the mouse: the influence of glucose.

A key intracellular event during capacitation is protein tyrosine phosphorylation, but its involvement during sperm interaction with the oocyte has not been investigated. Glucose is necessary to achieve fertilization and thus may have an influence on sperm protein tyrosine phosphorylation. The objectives of this study were to 1) visualize protein tyrosine phosphorylation patterns in sperm during capacitation and interaction with the oocyte and 2) determine the influence of glucose. Protein tyrosine phosphorylation was investigated by Western analysis and immunofluorescence. Protein tyrosine phosphorylation was increased during capacitation, and immunofluorescence revealed that zona binding and gamete fusion were correlated with an increase in tyrosine phosphorylation of proteins in the midpiece. During capacitation, the absence of glucose led to a delay in the appearance of protein tyrosine phosphorylation. Following binding to the zona pellucida and the oolemma, tyrosine phosphorylation in the flagellum was also delayed in the absence of glucose and resulted in a significant inhibition of the midpiece phosphorylation. The correlation between successful gamete fusion and the tyrosine phosphorylation of midpiece proteins suggests that the effect of glucose on sperm-oocyte interaction is mediated through regulation of protein tyrosine phosphorylation in a specific area of the fertilizing sperm.

Animals↗
PubMed2001

Reflections on the number of embryos to replace at the dawn of the 21st century.

The present review examines recent publications concerned with the issue of the number of embryos to replace in an in-vitro fertilization cycle, and concentrates on the proposed selection methods that may allow us to transfer one or two embryos. A number of techniques that improve our selection criteria for deciding which is the most viable embryo are discussed, including blastocyst culture. It is proposed that the next step in assessing which embryos to transfer is to combine these techniques by performing assessment at the pronuclear stage, cleavage stages, and then after development to the blastocyst stage, when the number of embryos is sufficient. In so doing our embryo selection processes will be more critical, allowing us to limit the number of embryos replaced without detriment to pregnancy rates.

Embryo Disposition↗
PubMed2000

Sperm decondensation during fertilisation in the mouse: presence of DNase I hypersensitive sites in situ and a putative role for topoisomerase II.

In this study our aim was to characterise the presence and the role of DNA alterations during sperm decondensation in the mouse. To visualise the changes during decondensation we investigated for the presence of DNase I hypersensitive sites in situ and for a putative role for topoisomerase II by examining the effect of teniposide, a topoisomerase II inhibitor, during fertilisation. In situ nick translation without the previous addition of DNase I failed to reveal the presence of endogenous nicks in decondensing sperm and pronuclei whereas preincubation of fixed oocytes with DNase I indicated that decondensing sperm were sensitive to this enzyme. Addition of 100 microM teniposide did not completely inhibit pronuclei formation but its addition to the fertilisation medium did lead to the presence of endogenous DNA nicks in decondensing sperm. These observations suggest that DNase I hypersensitivity during sperm decondensation is related to the dramatic conformational changes that the chromatin undergoes during the decondensation process, in which topoisomerase II may be implicated.

Animals↗
PubMed2000

Assessment of sperm competence.

The predictive values of four categories of established sperm function assays--computer-assisted semen analysis (CASA), induced-acrosome reaction testing, sperm penetration assay, and sperm-zona pellucida binding assays--are still unsure. In this article we examine the evaluation of sperm competence. We propose that assessment of sperm competence should include investigations at the nuclear, organelle, and cytoskeletal levels. In light of this, we discuss the assessment of sperm nuclear integrity as an alternative new method of analysis. We also question the merit of having such tests, whereby it may be an easier choice to direct these patients straight to an assisted reproduction treatment.

Acrosome Reaction↗
PubMed2000

Is a review of standard procedures for cryopreservation needed?: safe and effective cryopreservation-should sperm banks and fertility centres move toward storage in nitrogen vapour?

A recent consultation documentation by the Human Fertilisation and Embryology Authority (HFEA) which focused on the safe cryopreservation of gametes and embryos highlighted the need for a review of the way that fertility clinics in the UK store potentially infective material. The main points for consideration were to: (i) ensure containers used for cryopreservation are guaranteed by manufacturers to withstand low temperatures; (ii) use secondary containers, i.e. 'double bagging' of samples if stored in the liquid phase; and (iii) store in nitrogen vapour as a 'safer' alternative. In this article we examine a number of issues related to vapour storage which need careful consideration, including safety, cost and the effectiveness of various storage techniques in maintaining gamete and embryo viability. We also discuss the effectiveness of vapour storage in comparison with current liquid nitrogen storage techniques. In conclusion, we propose that fertility clinics should be compelled to review their cryopreservation procedures, not just because of new legislation or indeed fear of litigation but by a moral obligation.

Bacterial Infections↗
PubMed2000

The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies.

Human semen is heterogeneous in quality, not only between males but also within a single ejaculate. Differences in quality are evident, both when examining the classical parameters of sperm number, motility and morphology and in the integrity of the sperm nucleus. The aim of this study was to determine the efficiency of the PureSperm((R)), Percoll((R)) and swim-up preparation techniques to eliminate spermatozoa with nuclear anomalies. Semen samples were collected, washed and one part of the semen spread on a slide, the remainder was prepared using the swim-up, PureSperm((R)) or Percoll((R)) techniques. Spermatozoa from different fractions were fixed on slides and assessed. Sperm samples (n) from different men were stained using the chromomycin A(3) (CMA(3)) fluorochrome, which indirectly demonstrates a decreased presence of protamine (n = 31 for swim-up; n = 45 for PureSperm((R)); n = 39 for Percoll((R))). Spermatozoa prepared using PureSperm((R)) (n = 35) and Percoll((R)) (n = 37) were also examined for the presence of endogenous DNA nicks. Good quality spermatozoa should not possess DNA nicks and not stain (i.e. fluoresce) with CMA(3). When prepared using the swim-up technique the spermatozoa recovered showed no significant improvement with the CMA(3) staining. When spermatozoa were prepared using the PureSperm((R)) and Percoll((R)) techniques, a significant (P < 0.001) decrease in both CMA(3) positivity and DNA strand breakage was observed. These results indicate that both the PureSperm((R)) and Percoll((R)) techniques can enrich the sperm population by separating out those with nicked DNA and with poorly condensed chromatin.

Centrifugation, Density Gradient↗