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Biomedical subjects

P M Zavos

Publications and source records attributed to P M Zavos.

At least 19 recordsLinked to original sources
PubMed2006

In-vitro developmental potential of individual mouse blastomeres cultured with and without zona pellucida: future implications for human assisted reproduction.

This study was designed to compare the developmental potential of individual blastomeres derived from 2-, 4-, 6- and 8-cell mouse embryos cultured with and without zona pellucida (ZP). In the first series, one, three, five and seven blastomeres were biopsied from 2-, 4-, 6- and 8-cell embryos respectively, and inserted individually into empty ZP recipients, leaving the remaining blastomere within its original ZP. In the second series, the same protocol was used except that the biopsied blastomeres were cultured without ZP and compared with the remaining blastomere within its original ZP. For the first series, individual blastomeres derived from 2-, 4-, 6- and 8-cell embryos cultured with ZP showed blastocyst development of 82.4, 68.6, 44.4 and 23.1% respectively, with corresponding hatching rates of 70.6, 60.0, 25.9 and 7.7%. For the second series, individual blastomeres cultured without ZP progressed with blastocyst development of 73.3, 64.5, 35.7 and 22.7% respectively. Blastocyst multiplication was achieved most efficiently when using individual blastomeres from 4- and 6-cell embryos. This is the first report on comparative in-vitro propagation of single blastomeres derived from various cleavage stages in a mammalian species. Blastomere cloning with its multiple applications may be envisaged for human assisted reproductive technologies.

Animals↗
PubMed2005

Efficient blastomere biopsy for mouse embryo splitting for future applications in human assisted reproduction.

The objective of the current study was to establish a safe, efficient biopsy procedure for embryo splitting using the mouse model for future applications in human assisted reproduction. From mouse embryos at the 2-, 4-, 6- and 8-cell stage, half the number of blastomeres were microsurgically biopsied and transferred into empty mouse zonae pellucidae. Twin embryonic development was monitored during in-vitro culture. Blastocyst developmental rate using 2-, 4-, 6-, and 8-cell splitting was 74.4, 75.0, 66.7 and 38.4 respectively, with corresponding hatching rates of 94.9, 97.5, 92.7 and 83.8%. Blastocysts from 2-, 4-, and 6-cell splitting resulted in elevated hatching rates compared with non-operated blastocysts (87.5%), due to the Tyrode-assisted hatching effect. Blastocyst morphology was superior from 2- and 4-cell splitting when compared with 6- and 8-cell splitting. Furthermore, outgrowth of twin blastocysts from 2- and 4-cell splitting showed well-developed colonies with trophoblast cells and clusters of ICM cells, whereas those obtained from 6- and 8-cell splitting frequently formed small-sized colonies. Due to the high twinning success rate obtained under the experimental conditions employed in this study, it appears that with further modifications and proper safeguards, such embryo splitting efforts could have potential applications in humans.

Animals↗
PubMed2003

The effect of colloid osmotic pressure in human spermatozoa exposed to hypoosmotic conditions.

The use of a protein source such as serum and albumin had been extensively employed as supplements of culture media for handling and culture of gametes and embryos. Protein molecules behave as colloids in solution and contribute to the osmotic pressure of fluids. The interaction of proteins in solution and spermatozoa needs to be assessed in order to determine their possible role in osmoregulation. The aim of this study was to assess possible osmoregulatory mechanisms of protein supplementation against exposure to hypoosmotic conditions by assessing the sperm's response to those environments. A stock hypoosmotic solution (HOS) was prepared by using a mixture of fructose and sodium citrate and adjusted to an osmotic pressure of 150 mOsm l-1. Another stock solution was prepared by diluting a preparation of synthetic serum supplement [SSS; 6% (v/v) total protein] with distilled water to obtain an osmotic pressure of 150 mOsm l-1 (hypoosmotic SSS or H-SSS). Three additional solutions were prepared by mixing the stock HOS and H-SSS solutions in the following proportions (v/v): (i) 75% H-SSS/25% HOS, (ii) 50% H-SSS/50% HOS and (iii) 25% H-SSS/75% HOS. Aliquots of washed spermatozoa from 18 men were diluted 1 : 10 (v/v) with each of the testing solutions and incubated for 60 min. Specimens were assessed on wet mounts for total and specific swelling patterns. Swelling patterns were classified as maximal (>50% tail length swollen) and minimal (<50% tail length swollen) swelling with or without associated sperm motility. The major finding of this study was that increasing the concentration of protein supplementation resulted in a decrease in the proportion of maximal sperm tail swelling patterns and an increase in the proportion of minimal tail swelling patterns. A proportion of spermatozoa which exhibited minimal swelling patterns were still motile in all solutions tested, and the percentage of those spermatozoa increased as the protein supplementation was also increased in the testing solutions. Incorporation of protein supplementation as described in this study delays the effect of sperm swelling in hypoosmotic conditions.

Colloids↗
PubMed2001

Comparison between the quality and function of sperm after semen processing with two different methods.

AIM: To compare the recovery rate of morphologically normal and chromatin condensed spermatozoa from native semen samples using the SpermPrep filtration columns and Percoll gradient centrifugation and to determine the influence of the two processing techniques on fertilization and pregnancy rates in an IVF-ET program. METHODS: Sixteen semen samples obtained from patient's husband were included in this study. Each was divided into two aliquots. The first aliquot was processed with SpermPrep filtration columns and the second, Percoll gradient centrifugation. Smears were made before and after semen processing with both methods for the evaluation of chromatin condensation (chromomycine CMA3) as well as morphology (strict criteria) of spermatozoa. One hundred and seventy oocytes were retrieved from the patients and the oocytes from each patient were subdivided into two sets: one set was inseminated using spermatozoa processed with SpermPrep and the other inseminated after semen processing with Percoll gradient centrifugation. RESULTS: The Percoll method yielded a significantly higher percentage of chromatin condensed (90.8 +/- 6.5% vs 82.3 +/- 8.8%, P = 0.017) and morphologically normal spermatozoa (12.9 +/- 7.4% vs 6.9 +/- 4.8%, P = 0.001) in comparison to SpermPrep. Whereas, sperm count recovery rate was significantly higher after the use of SpermPrep than after the Percoll gradient centrifugation. The fertilization rate was similar between the two methods. CONCLUSION: Semen processing with Percoll should be recommended for intracytoplasmic sperm injection as the natural selection is bypassed and the SpermPrep technique could be recommended for IVF and IUI programs as the sperm concentration plays a more significant role in these procedures.

Centrifugation, Density Gradient↗
PubMed1999

Novel vaginal controlled-delivery systems incorporating coprecipitates of nonoxynol-9.

The purpose of this study was to formulate Nonoxynol-9 (N-9) into a solid coprecipitate form which can be used in preparing pharmaceutically attractive and nonirritating vaginal controlled-release delivery systems (DDSs) such as gelatin capsules (HGC) and tablets. N-9 was coprecipitated with polyvinylpyrrolidone (PVP) with or without iodine to produce solid powders which were incorporated into either (a) bilayer tablet DDSs which possess a fast- (outer) and slow- (inner core) releasing compartment, and (b) HGC DDSs (named Triad HGC) composed of fast- (outer), intermediate- (granules), and slow- (pellets) releasing compartments. The rates of release of iodine and/or [14C]N-9 from the two DDSs were studied in vitro in phosphate buffer at pH 5.0, in human seminal plasma and in vivo after intravaginal administration in rabbits. In all of the above-described release studies, the DDSs were shown to release their N-9 or iodine content rapidly, reaching spermicidal levels within 3 min. This was further substantiated by experiments in which the DDSs were introduced in whole human semen containing live spermatozoa. Complete spermicidal kill was obtained in less than 1 min and in less than 3 min from the bilayer tablet and the Triad HGC, respectively. Furthermore, the release of N-9 from the two DDSs was shown to continue for at least 4 hr in buffers (pH 5.0), human seminal fluid, and after intravaginal administration in rabbits. The resulting powder from the coprecipitation of N-9 and PVP (K-30) can be appropriately formulated into a controlled-released HGC or bilayer tablet to produce vaginal controlled-release DDSs which are nonirritating and have the potential to become effective spermicidal products.

Administration, Intravaginal↗
PubMed1998

Effects of seminal plasma from cigarette smokers on sperm viability and longevity.

OBJECTIVE: To evaluate the effects of cigarette smoking on the ability of seminal plasma (SP) to maintain sperm viability. DESIGN: Clinical randomized study. Spermatozoa from cigarette smoking or nonsmoking subjects were reconstituted in SP from smokers and nonsmokers and in modified Ham's F-10 medium, followed by sperm quality assessment during a 48-hour incubation period. SETTING: Andrology Institute of Lexington, Lexington, Kentucky. PATIENT(S): Twenty men who had been smoking cigarettes for longer than 3 years (30 cigarettes per day or more) and 20 nonsmokers participated in this study. MAIN OUTCOME MEASURE(S): Improvement in sperm viability by removal of SP--and associated detrimental factors present in the SP--from smoker subjects. RESULT(S): The results obtained indicate that the quality of spermatozoa obtained from nonsmokers was superior to that of smokers. The SP from the two patient groups had a definite effect on their respective sperm quality, i.e., beneficial effects for the nonsmokers, detrimental effects for the smokers. Exposure of spermatozoa from the nonsmokers to SP from the smokers resulted in a significant reduction in sperm viability. However, exposure of spermatozoa from the smokers to SP from the nonsmokers or to Ham's F-10 medium yielded significant improvements in sperm viability. CONCLUSION(S): The detrimental effects of smokers' SP on nonsmokers' spermatozoa was prominent and a rather unique phenomenon. The results generated in this study could be of clinical significance since removal of smokers' SP and subsequent reconstitution and incubation in physiological media seems to enhance the viability, longevity, and possibly the fertilizing ability of these spermatozoa for use in various assisted reproductive technologies.

Adult↗
PubMed1998

Antisperm antibody treatment mode: levels of antisperm antibodies after incubation with TEST-yolk buffer and filtration using the SpermPrep II method.

OBJECTIVE: To assess whether incubation in TEST-yolk buffer (TYB) or human tubal fluid (HTF) could alter the sperm membrane characteristics and its relationship to antisperm antibodies (ASA) and/or antigen detachment from the sperm membrane and to evaluate the filtration of those specimens and possible recovery of ASA-free spermatozoa. DESIGN: A prospective clinical study. SETTING: Andrology Institute of Lexington, Lexington, Kentucky. PATIENT(S): Twenty patients undergoing infertility treatment. MAIN OUTCOME MEASURE(S): Recovery of spermatozoa with reduced levels or antisperm antibody-free sperm after treatment with TYB or HTF, followed by filtration using the SpermPrepII method (Sephadex based). RESULT(S): Assessment of ASA using the direct immunobead test showed no significant differences between specimens incubated for 2 hours in seminal plasma (fresh) or HTF with regard to levels of IgA and IgG. The percentage binding of anti-IgA and anti-IgG immunobeads was significantly reduced in specimens incubated for 2 hours in TYB compared with specimens incubated in seminal plasma or HTF. Furthermore, selection of spermatozoa using the SpermPrepII filtration method significantly reduced the percentage binding of anti-IgA and anti-IgG immunobeads compared with specimens incubated in HTF. CONCLUSION(S): The results suggest that TYB either altered the sperm membrane properties so that there was a decreased affinity at the antibody and/or antigen sites or that the egg yolk proteins were absorbing the antibodies and/or antigens complexes from the sperm membrane surface. Incubation of spermatozoa in TYB followed by filtration with the SpermPrepII method improved the recovery of ASA-free spermatozoa by selectively entrapping spermatozoa with ASA bound to its surface.

Antibodies, Anti-Idiotypic↗
PubMed1998

An electron microscope study of the axonemal ultrastructure in human spermatozoa from male smokers and nonsmokers.

OBJECTIVE: To investigate possible abnormalities or deterioration of the sperm axonemal ultrastructure in men who have smoked a large quantity of cigarettes (> 20 per day) for a prolonged period. DESIGN: Semen specimens were collected by patients via masturbation; qualitative characteristics of the sperm were assessed and ultrastructural analysis of the sperm axoneme was performed using standard operating procedures for electron transmission microscopy. SETTING: The Andrology Institute of Lexington, Lexington, Kentucky, and the Department of Histology and Embryology, University of Salonika, Greece (collaborative effort). PATIENT(S): Twenty-nine men (mean age +/- SD, 30.7 +/- 2.1 years) who smoked a mean (+/- SD) of 30.7 +/- 2.1 cigarettes per day for 10.7 +/- 0.7 years and 15 men who never smoked (mean age +/- SD, 30.4 +/- 2.2 years) participated in this study. MAIN OUTCOME MEASURE(S): Ultrastructural organization of the sperm axoneme in male smokers and nonsmokers. RESULT(S): Changes in the number and the arrangement of axonemal microtubules were noted in the smoker group when compared to the nonsmoker group. The incidence of axonemal abnormalities was higher in spermatozoa from smokers compared with that in spermatozoa from nonsmokers. CONCLUSION(S): Smoking a large quantity of cigarettes per day, under the conditions of the current study, severely affected the ultrastructure of the flagellum and, more specifically, it affected the axoneme of the human spermatozoon.

Humans↗
PubMed1998

Assessment of a tablet drug delivery system incorporating nonoxynol-9 coprecipitated with polyvinylpyrrolidone in preventing the onset of pregnancy in rabbits.

OBJECTIVE: To assess the in vivo efficacy of the tablet drug delivery system containing nonoxynol-9 coprecipitated with polyvinylpyrrolidone by delivering the spermicidal agents vaginally and evaluating their ability to prevent the onset of pregnancy in rabbits. DESIGN: Controlled clinical study. SETTING: Division of Laboratory and Animal Resources, College of Pharmacy, University of Kentucky. ANIMAL(S): Forty-two New Zealand White female rabbits. INTERVENTION(S): The rabbits were artificially inseminated at various intervals after vaginal insertion of the tablet drug delivery system containing either polyvinylpyrrolidone only (0 minutes) or nonoxynol-9 coprecipitated with polyvinylpyrrolidone (polyvinylpyrrolidone/nonoxynol-9; 0, 3, 30, 180, and 360 minutes). The rabbits were induced to ovulate 6 hours before insemination by i.m. injection of hCG (200 IU). MAIN OUTCOME MEASURE(S): The onset of pregnancy in the rabbits was evaluated after insertion of the tablet drug delivery system containing polyvinylpyrrolidone only or polyvinylpyrrolidone/nonoxynol-9 at various intervals, followed by artificial insemination. RESULT(S): The onset of pregnancy was not reduced significantly when the tablet drug delivery system containing polyvinylpyrrolidone or polyvinylpyrrolidone/nonoxynol-9 was used and insemination was performed immediately after tablet insertion (time 0). However, pregnancy rates (PRs) were reduced significantly in the rabbits that received the tablet drug delivery system containing polyvinylpyrrolidone/nonoxynol-9 and were inseminated at 3, 30, 180, and 360 minutes after tablet insertion. The highest PR reduction occurred between 30 and 180 minutes after insertion of the tablet drug delivery system containing polyvinylpyrrolidone/nonoxynol-9. CONCLUSION(S): The tablet drug delivery system is an efficient method of delivering the tested spermicidal agents vaginally. The design and dosage used in preparing the tablet drug delivery system provide short- and long-term release of the spermicidal agents, which results in almost immediate and extended enhancement of their contraceptive properties.

Administration, Intravaginal↗
PubMed1998

Fertilization potential and qualitative characteristics of human spermatozoa after short-term cryostorage at 5 degrees C in two different TEST-yolk buffer preparations.

Use of the media TEST-yolk buffer (TYB) in semenology today enables the short-term incubation and cryostorage of spermatozoa and its subsequent use in the various assisted reproductive technologies (ART). Preparation of TYB media involves the addition of egg yolk (20% v/v) to a physiological solution of the zwitterion buffers TES and Tris. The TYB is usually thermoprecipitated to remove the majority of the egg yolk globules and other macromolecules from the medium. However, removal of these egg yolk constituents could possibly eliminate or reduce essential factors that could enhance the sperm viability and fertilization potential after short-term dilution and storage. Improvements in the quality of the TYB could add greater benefits to those techniques employed in the various forms of ART. The objectives of the investigation were 1) to study the sperm qualitative characteristics following short-term cryostorage at 5 degrees C in either thermoprecipitated (T-TYB) or non-thermoprecipitated (NT-TYB), and 2) to compare the fertilizing potential of spermatozoa stored for 24 hours at 5 degrees C in the two TYB preparations. In Experiment 1, semen specimens from 15 patients were collected, assessed and split into two aliquots. Sperm specimens were processed by diluting 1:1 (v/v) with the T-TYB or NT-TYB, followed by centrifugation and reconstitution of the specimen to its initial volume with the corresponding TYB medium. Sperm specimens were cryostored for 1, 2, 24, 48 and 72 hours. Samples were taken at each interval and placed in a 37 degrees C water bath and allowed to warm for 15 minutes after each cryostorage interval. Semen specimens were assessed for percentage and grade of motility. The results of this study indicated that, although the NT-TYB yielded better results than the T-TYB, overall those differences were not statistically significant. In Experiment 2, the fertilization potential of spermatozoa recovered after 24 hours of cryostorage in the two TYB preparations and further prepared via filtration, was assessed by the sperm penetration assay (SPA) using zona-free hamster oocytes. The average penetration rate (PR) and penetration index (PI) were significantly better for the NT-TYB than for the T-TYB. The PR was 54% vs. 25%, and the PI 0.78 and 0.27 for spermatozoa incubated in the NT-TYB vs. T-TYB. The range of penetration was also much lower for the T-TYB (6 to 100%) preparation when compared to the NT-TYB (22 to 100%). The highest penetrator showed 100% for both preparations. However, the lowest penetrator showed 6% for the T-TYB and 22% for the NT-TYB. The data obtained in this study suggest that both TYB preparations can be employed in short-term cryostorage (5 degrees C) of human spermatozoa and can adequately maintain the qualitative characteristics of those spermatozoa. The data also showed that the NT-TYB preparation yielded sperm samples of higher fertilization potential, thus possibly establishing the superior usefulness of the NT-TYB in an ART program.

Animals↗
PubMed1998

Evaluation of techniques for the cryopreservation of washed spermatozoa: comparisons between Ham's F-10 and TEST-yolk media.

The objective of this study was to develop new techniques for the cryopreservation of washed spermatozoa. Two media (Ham's F-10 and nonthermoprecipitated TEST-yolk buffer [NT-TYB]) containing 7% (v/v) glycerol were compared to semen cryopreservation by adding glycerol directly to the semen. Twenty four men collected a semen specimen each after 4 days of sexual abstinence via the use of a semen collection device at intercourse. Specimens were assessed for volume (ml), count (x 10(6)), percentage and grade of motility, morphology (% normal) and acrosomal status (% intact acrosomes). Each ejaculate was split into 3 aliquots (Aliquots 1 to 3) and processed for freezing. Aliquot 1 was prepared for cryopreservation by adding glycerol (7% [v/v] final concentration) directly via a dropwise mode. Aliquot 2 and 3 were diluted 1:1 (v/v) with Ham's F-10 and NT-TYB, respectively. Aliquots 2 and 3 were then centrifuged (400 x g for 10 minutes) and resuspended into the corresponding media containing 7% (v/v) glycerol to complete the sperm wash procedure. All aliquots were frozen in 0.5 ml french straws. Sperm specimens were frozen in liquid nitrogen (LN2) vapor from +23 degrees C to -68 degrees C at a slow rate (2.3 degrees C/minute), after which the specimens were plunged directly into LN2 and stored for 30 days. The quality of the spermatozoa were monitored throughout each step of the overall procedure by measuring the motility characteristics of the spermatozoa. Straws corresponding to each aliquot were thawed in a water bath at 37 degrees C for 2 minutes, followed by assessment of sperm motility and acrosomal status. The percentage of motility after thawing was 31.6 +/- 5.6%, 32.8 +/- 1.8% and 37.3 +/- 1.9% in Aliquots 1 to 3, respectively. Similarly, the grade of motility was 2.4 +/- 0.2, 2.6 +/- 0.1 and 3.0 +/- 0.1 in Aliquots 1 to 3, respectively. The acrosomal status (% intact acrosomes) in Aliquots 1 to 3 was 41.2 +/- 2.6, 43.1 +/- 3.6 and 51.6 +/- 4.5, respectively. The results suggest that the characteristics of spermatozoa washed and frozen in NT-TYB (Aliquot 3) were improved over those spermatozoa prepared via direct addition of glycerol to the semen (Aliquot 1) or by using Ham's F-10 (Aliquot 2). The most significant reduction noted during freezing was in the loss of acrosomal integrity. The results obtained in this study point out that washed spermatozoa can be cryopreserved with some success and that the recovered spermatozoa could be used for intrauterine insemination in an artificial insemination program using husband's or donor sperm, or for the various assisted reproductive technology procedures. It is the opinion of the authors that the information generated in this study is of importance for those scientists and clinicians involved in the handling and manipulation of cryopreserved spermatozoa.

Buffers↗
PubMed1997

Relationships among frozen-thawed sperm characteristics assessed via the routine semen analysis, sperm functional tests and fertility of bulls in an artificial insemination program.

Frozen semen specimens from 22 Holstein bulls representing a wide range of field fertility levels or nonreturn rates (NRR) were used in this study. Semen specimens were thawed at 37 degrees C for a minimum of 30 sec, followed by assessment via a routine semen analysis (RSA) and other sperm functional tests. The RSA was performed by assessing sperm count, motility and morphological characteristics. Other sperm functional tests were performed by assessing the acrosomal membrane integrity, sperm penetration into the cervical mucus and the sperm membrane functional integrity. Following assessment of sperm characteristics, the fertility data of the various bulls were compared to the RSA and the functional tests results. Bulls of high and low fertility were similar in terms of sperm count and progressive motility (P > 0.05). Other characteristics measured by the RSA and functional tests were significantly higher in high fertility bulls (P < 0.05). Correlation coefficients among the various sperm characteristics and fertility of bulls were highly significant (P < 0.01). The highest correlation coefficients between sperm characteristics and fertility were obtained for motility (r = 0.53; P < 0.01), normal morphology (r = 0.59; P < 0.01) and swollen spermatozoa (r = 0.57; P < 0.01). Analysis of specific sperm swelling patterns showed that those patterns considered to reflect maximal sperm swelling were indicative of high fertility.

Journal Article↗
PubMed1997

Vaginal delivery of new formulations of nonoxynol-9 coprecipitated with polyvinylpyrrolidone in rabbits. Comparisons between two formulation delivery systems.

The objective of this study was to assess the efficacy of two formulation delivery systems (FDS) in preventing the onset of pregnancy in rabbits. Nonoxynol-9 (N-9) was coprecipitated with polyvinylpyrrolidone (PVP), which yields PVP/ N-9, and prepared as capsules or tablets. Semen specimens were collected (from eight male rabbits), pooled, and used for in vitro spermicidal assessment or artificial insemination (AI). In vitro spermicidal assessment was performed by introducing and mixing the FDS containing PVP or PVP/N-9 with 1.0 mL of semen, followed by incubation at 37 degrees C for 6 h. Semen samples were taken at various time intervals to determine killing of spermatozoa and dissolution of the FDS. The efficacy of the FDS in preventing the onset of pregnancy was assessed by inserting the FDS vaginally. The does were artificially inseminated at 0, 0.5, and 6 h after insertion of the FDS vaginally. The number of pregnant does and newborn rabbits was recorded. In the in vitro spermicidal trial, semen specimens exposed to both FDS containing PVP/N-9 were killed within 10 to 15 min of incubation. Tablets containing PVP only or PVP/N-9 dissolved completely after 3 h of incubation. However, capsules did not dissolve completely by 6 h of incubation. The results obtained in the in vivo trial showed that both FDS exhibited some variations in preventing the onset of pregnancy over the various time intervals following the insertion of tablets or capsules and AI. The tablet seemed to be a more efficient delivery system than the capsule, yielding significantly lower pregnancy rates at all three time intervals assessed. The tablet FDS, as applied in this study, was found to be the most efficient mode of delivery of the tested spermicidal formulations.

Administration, Intravaginal↗