Biomedical subjects

D Sakkas

Publications and source records attributed to D Sakkas.

At least 55 records · Page 3Linked to original sources

PubMed1997

Hoechst staining and exposure to UV laser during flow cytometric sorting does not affect the frequency of detected endogenous DNA nicks in abnormal and normal human spermatozoa.

Controlling the sex of offspring by the separation of X and Y chromosome-bearing spermatozoa using flow cytometry has been reported as a clinical technique aiding prevention of X-linked diseases. Although this technique has resulted in several hundred normal births in animals and at least one human birth, there is still concern over its genetic safety due to the involvement of two potentially mutagenic agents: UV light and the fluorochrome dye, Hoechst 33342 (H33342). Human spermatozoa, particularly those considered abnormal, may be more likely to suffer DNA damage following exposure to mutagenic agents, compared with other mammalian species. The stability of normal fresh and decondensed human spermatozoa were examined after exposure to a range of levels of UV and H33342 staining, using an assay that detects endogenous nicks in the DNA of spermatozoa. The stability of abnormal and normal, fresh and frozen-thawed human spermatozoa was examined following UV laser, H33342 staining and flow cytometry treatments utilizing the same assay. There was an increase in the presence of endogenous nicks when spermatozoa were decondensed compared with fresh spermatozoa. There was no increase in the incidence of nicks in any group of spermatozoa after UV and fluorochrome exposure compared with controls without exposure.

Animals↗

PubMed1997

Development, glycolytic activity, and viability of preimplantation mouse embryos subjected to different periods of glucose starvation.

After compaction, the preimplantation mouse embryo switches to a glucose-based metabolism, whereas for the 2- to 4-cell stage embryo, glucose can be inhibitory. In this study, we investigated the adaptability of preimplantation embryos to different periods of glucose starvation by culturing in vitro fertilized (IVF) and in vivo-fertilized 1-cell OF1 mouse embryos. Blastocysts obtained from exposure to glucose starvation for different periods of time were examined for the number of cells in the trophectoderm and inner cell mass, and for glycolytic activity and viability. A high percentage of blastocysts was obtained when 1-cell embryos fertilized in vitro or in vivo were cultured in M16 until the 2-cell stage, were transferred to M16 without glucose (M16-G) until the 4- or 8-cell stage, and then were transferred to fresh M16-G. When in vivo-fertilized 1-cell embryos were cultured to the 2-cell stage and then left in M16, less than 5% formed blastocysts compared to 26% of those transferred into M16-G. Blastocysts obtained when in vivo-fertilized 1-cell embryos were left in M16-G after the 2-cell stage, however, showed a significantly elevated glycolytic activity compared to those transferred to fresh M16 or M16-G medium at the 4- or 8-cell stage. Interestingly, even though these embryos displayed elevated glycolytic activity, they did not exhibit differences in the numbers of inner cell mass and trophectoderm cells or in viability compared to embryos cultured according to other protocols. Blastocysts from all cultured protocols had a significantly lower total cell number and a lower trophectoderm, but not inner cell mass, cell number compared to blastocysts developed in vivo. This study documents the metabolic adaptability of the preimplantation embryo by highlighting its ability to proceed with development and retain viability when challenged with glucose starvation at different periods.

Animals↗

PubMed1996

The development of preimplantation mouse parthenogenones in vitro in absence of glucose: influence of the maternally inherited components.

Mouse preimplantation embryo development is characterized by a switch from a dependence on the tricarboxylic acid cycle pre-compaction to a metabolism based on glycolysis post-compaction. In-view of this, the role of glucose in embryo culture medium has come under increased analysis and has lead to improved development of outbred mouse embryos in glucose free medium. Another type of embryo that has proven difficult to culture is the parthenogenetic (PN) mouse embryo. With this in mind we have investigated the effect of glucose deprivation on PN embryo development in vitro. Haploid and diploid PN embryos were grown in medium M16 with or without glucose (M16-G) and development, glycolytic rate, and methionine incorporation rates assessed. Haploid PN and normal embryo development to the blastocyst stage did not differ in either M16 or M16-G. In contrast, although diploid PN embryos formed blastocysts in M16 (28.3%), they had difficulty in undergoing the morula/blastocyst transition in M16-G (7.6%). There was no significant difference in mean cell numbers of haploid PN, diploid PN and normal embryos cultured in M16 and M16-G at the morula and blastocyst stage. Transfer of diploid PN embryos from M16-G to M16 at the four- to eight-cell stage dramatically increased blastocyst development. At the morula stage diploid PN embryos grown in M16-G exhibited a higher glucose metabolism and protein synthesis compared to those grown in M16 and to haploid PN embryos. Difficulties of diploid PN embryos in undergoing the morula/blastocyst transition in absence of glucose infer the existence of a link between the maternally inherited components and the preimplantation embryos dependence on glucose.

Animals↗

PubMed1996

Use of the guanine-cytosine (GC) specific fluorochrome, chromomycin A3, as an indicator of poor sperm morphology.

PURPOSE: We have previously postulated that the chromomycin A3 (CMA3) fluorochrome allows an indirect visualization of sperm chromatin packaging quality and partially denatured sperm DNA. In this study we investigate the relationship between CMA3 positivity and sperm morphology. We also present data on the association between sperm morphology and the presence of endogenous nicks in sperm DNA. METHODS: Semen samples were examined from 81 males of the couples who were consulting for infertility treatment. CMA3 fluorescence was assessed for all samples, while in 24 sperm samples we also examined for the presence of endogenous nicks in the sperm DNA. RESULTS: When sperm morphology was less than 20% normal in a patient, the level of CMA3 fluorescence and presence of endogenous nicks were significantly higher than in patients with a higher incidence of morphologically normal sperm. CONCLUSIONS: CMA3 could be used as an adjunct to the assessment of morphology as an evaluation method for poor sperm. Its value in predicting fertilizing ability when using either SUZI or ICSI awaits to be answered.

Biomarkers↗

PubMed1996

Defects in the allocation of cells to the inner cell mass and trophectoderm of parthenogenetic mouse blastocysts.

Diploid parthenogenetic mouse embryos (which possess two maternally-derived genomes) can develop only as far as the 25-somite stage when transferred in utero and exhibit a substantial reduction in trophoblast tissue. The loss of cultured parthenogenetic embryos during postimplantation indicates that a defect in cell lineage may be evident as early as the blastocyst stage. The possibility that a defect may already be reflected at the preimplantation stage was investigated by examining the allocation of cells to the trophectoderm (trophoblast progenitor cells) and the inner cell mass of haploid and diploid parthenogenetic mouse blastocysts. Utilizing a differential labelling technique for counting cells, diploid parthenogenetic blastocysts were found to have fewer inner cell mass cells and trophectoderm cells than their haploid counterparts and normal blastocysts. In addition, both haploid and diploid parthenogenetic blastocysts had a lower inner cell mass: trophectoderm ratio than normal blastocysts. Thus, the relatively poor development of the trophectoderm lineage at the postimplantation stage is not reflected by a reduction in its allotment of cells at its first appearance. Nevertheless, the findings indicate that parthenogenetic development is already compromised at the blastocyst stage, and provide evidence that the expression of imprinted genes has significance for the development of the embryo at the preimplantation stage.

Animals↗

PubMed1996

Co-culture of 1-cell outbred mouse embryos on bovine kidney epithelial cells: effect on development, glycolytic activity, inner cell mass:trophectoderm ratios and viability.

In an attempt to enhance embryo development, we have co-cultured 1-cell OF1 mouse embryos on bovine kidney epithelia (Madine-Darby bovine kidney; MDBK) cells in a complex medium called complex mouse tubal fluid (cMTF; based on the energy substrate levels found in the mouse oviduct, containing non-essential amino acids, glutamine and EDTA). To determine the quality of the blastocysts obtained, we examined several parameters: morphology, total cell numbers, inner cell mass (ICM):trophectoderm (TE) ratio, glycolytic activity and viability after transfer. A significantly lower number of blastocysts developed on MDBK cells compared with cMTF medium. cMTF blastocysts had a significantly higher glycolytic activity and a lower blastocyst cell number than those grown in co-culture, while both in-vitro groups had higher ICM:TE ratios compared with in vivo. Blastocysts grown on MDBK cells displayed an elevated ICM number compared with those grown in cMTF medium alone. However, the percentage of fetuses after transfer remained drastically low in both culture groups compared with in-vivo blastocysts. In conclusion, co-culture did not increase the number of zygotes reaching the blastocyst stage. Although co-culture blastocysts show some similarities to in-vivo embryos in cell number and glycolytic activity, no enhancement in viability was observed.

Animals↗

PubMed1996

Chromatin packaging and morphology in ejaculated human spermatozoa: evidence of hidden anomalies in normal spermatozoa.

This study aimed to investigate the association between anomalies in sperm chromatin packaging, morphology and fertilization in patients undergoing routine in-vitro fertilization (IVF) or subzonal insemination (SUZI). Sperm chromatin packaging was assessed using chromomycin A3 (CMA3), a fluorochrome specific for guanine-cytosine rich sequences of DNA. One hundred to 150 sperm cells were assessed in 55 patients to compare sperm chromatin packaging and morphology to fertilization after IVF or SUZI. When the morphology and CMA3 fluorescence of individual spermatozoa was assessed, > 75% of the macrocephalic sperm fluoresced in all patients. In contrast, a mean of 37% of the spermatozoa with normal morphology fluoresced in IVF patients compared with 58% of the normal spermatozoa in male factor patients treated by SUZI. SUZI patients displaying a high fluorescence (> 70%) in their spermatozoa also had a significantly lower fertilization rate. Lower packaging quality in morphologically normal spermatozoa may represent a major limiting factor in the fertilizing ability of male factor patients. This study confirms that a high percentage of CMA3 positivity is present in certain forms of male factor infertility and that such a test may be used to distinguish separate populations in morphologically normal spermatozoa.

Chromatin↗

PubMed1996

Intrauterine insemination: evaluation of the results according to the woman's age, sperm quality, total sperm count per insemination and life table analysis.

We report on 332 infertile couples who underwent 1115 cycles of intrauterine insemination (IUI) with washed husband's semen. The indication for IUI was an abnormal post-coital test due to either a male or cervical infertility factor. The mean number of IUI cycles per patients was 3.4, the overall pregnancy rate 18.7%, and the pregnancy rate per cycle 5.6%. The cumulative pregnancy rate calculated by life table analysis showed that 16.0% of pregnancies occurred in the first three treatment cycles, while the cumulative pregnancy rate was 26.9% by the sixth cycle. The outcome of the therapy was adversely affected if the woman's age was > 39 years and/or total motile sperm count per insemination was < 1 x 10(6). No pregnancy occurred in women older than 44 years or in cases with a total motile sperm count before semen preparation of < 1 x 10(6).

Adult↗

PubMed1996

Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection.

In this study we investigated whether morphology and chromatin anomalies in human spermatozoa can influence fertilization after intracytoplasmic sperm injection (ICSI). We examined unfertilized oocytes, using the fluorochrome Hoechst 33342, to determine whether a relationship exists between failure of fertilization and sperm chromatin quality. Sperm chromatin packaging quality was assessed using the chromomycin A3 (CMA3) fluorochrome, and the presence of DNA damage in spermatozoa, using in-situ nick translation, Normal males present sperm parameters with a normal morphology of > 20%, CMA3 fluorescence of < 30% and exhibit endogenous nicks in < 10% of their spermatozoa. When patients were separated according to these values no difference was observed in their fertilization rates after ICSI. When the unfertilized ICSI oocytes were examined, we found that patients with CMA3 fluorescence of <30% and nicks in < 10% of their spermatozoa had only 17.5 and 21.6% respectively of their unfertilized oocytes containing spermatozoa that remained condensed. In contrast, patients with higher CMA3 and nick values had a significantly higher number, 41.2 and 48.9%, of their unfertilized oocytes containing condensed spermatozoa. sperm morphology did not show any such pattern. The percentage of spermatozoa which had initiated decondensation in unfertilized oocytes was not influenced by morphology, CMA3 fluorescence or nicks. In light of these results we postulate that poor chromatin packaging and/or damaged DNA may contribute to failure of sperm decondensation after ICSI and result in failure of fertilization.

Adult↗

PubMed1996

Glucose participates in sperm-oocyte fusion in the mouse.

Glucose metabolism is necessary for successful fertilization in the mouse, but the precise step of this process that requires glucose has not been determined. In this study we have investigated the involvement of glucose in the gamete binding and fusion process. The relationship between the metabolism of this carbohydrate in the oocyte and this particular step of fertilization has also been studied. A binding and fusion assay was performed by inseminating zona-free oocytes with capacitated sperm. Sperm binding and the fusion reaction were assessed by counting the sperm bound to the oolemma and the sperm heads decondensed in the ooplasm, respectively. To determine the importance of glucose metabolism, nonmetabolized glucose analogues (L-glucose, 2-deoxyglucose, 3-O-methylglucose) were used instead of glucose. The binding/fusion assay was also performed in the presence of glucose uptake inhibitors (cytochalasin B, phloretin, phlorizin). We have obtained evidence that glucose was specifically required for sperm penetration into zona-free oocytes (gamete fusion) but not for sperm binding to the oolemma. The metabolism of this carbohydrate appears to be essential, since nonmetabolized glucose analogues were unable to support this process. Although glucose metabolism was detectable in the oocyte, it did not appear to be related to the gamete fusion function. We hypothesize that the male gamete, which utilizes glucose as an energy substrate for various functions, may have a specific need to metabolize this hexose during fusion and incorporation within the oocyte.

Animals↗

PubMed1996

Glucose is not essential for the occurrence of sperm binding and zona pellucida-induced acrosome reaction in the mouse.

Glucose is necessary, in some species, for successful fertilization, but its role in the different gamete functions has only been documented partially. In this study we have examined the progression of mouse spermatozoa through the oocyte investments in order to determine which steps require glucose. Evidence is presented of glucose involvement during this sequence of events, since in-vitro fertilization was inhibited in glucose-free medium even when spermatozoa were capacitated in the presence of glucose. Access to the zona pellucida was not inhibited in the absence of glucose, suggesting that capacitation, progressive motility and zona binding of spermatozoa do not depend on the presence of an extracellular glycolysable substrate. However, zona penetration was prevented in the absence of glucose. Most importantly, the zona pellucida-induced acrosome reaction was found to occur in glucose-free medium. It is proposed that hyperactivated motility, which has been described to require glucose in the literature, may be the precise process responsible for the failure of spermatozoa to enter the perivitelline space.

Acrosome↗

PubMed1995

Co-culture of the early human embryo: factors affecting human blastocyst formation in vitro.

Co-culture systems have been designed to overcome the embryonic developmental arrest observed in vitro in conventional culture media. Oviduct and uterine epithelial cells can sustain embryonic development, as can trophoblastic tissue and transport epithelia of non-genital origin. Its benefits involve neither hormone dependency nor histo-specificity. Fibroblasts do not overcome the developmental arrest in most mammalian species, but whether they do in humans is still unsure. In all systems, the quality of the feeder cells and the co-culture medium are very important. Using the Vero cell line, 60% of human IVF embryos reach the blastocyst stage. The quality of the sperm seems to affect results. We have observed: For 10% of the patients with unexplained fertility, blastocyst stage is not attained; this probably involves a maternal (ovarian) problem. When at least one blastocyst is transferred, the pregnancy rate per transfer is 31%. The implantation rate in pregnant women is higher than after transfer at day 2. After repeated failures of transfer at early stages (2-6 cells), transfer at the blastocyst stage gives high pregnancy rates (40%). This indicates an in vitro selection. There is a strong paternal effect on blastocyst formation: poor quality sperm give lower rates of blastocyst. Co-culture helps to understand treatment failures related to male factors. Around 60% of the patients having spare embryos have had blastocysts frozen. Transfers of frozen-thawed blastocysts give a 20% pregnancy rate and an implantation rate per embryo of 11%. Co-culture is a new tool which has to be carefully evaluated in human IVF programs. It does not impair "a minima" embryo viability and it allows in vitro selection.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

[Treatment of male sterility using intra-oocytic sperm injection: critical evaluation].

Intracytoplasmic injection of a single spermatozoon into an oocyte (ICSI) is capable of achieving fertilization of oocytes in cases of severe male sterility resistant to classical andrological treatments. We report 3 cases of long-standing male sterility in which fertilization of oocytes using ICSI in 3 cases resulted in clinical pregnancies in 2. Indications for ICSI are discussed emphasizing the genetic risk in the case of bilateral absence of the vas deferens, frequently associated with mutation of the gene responsible for cystic fibrosis. To date, follow-up of 339 babies has revealed 2.9% congenital abnormalities.

Adult↗

PubMed1995

Differential effect of epithelial cell-conditioned medium fractions on preimplantation mouse embryo development.

Coculture studies using preimplantation embryos have led to a number of conflicting studies. In the human, ethical considerations have led to the preferential use of epithelial cell lines as distinct from human Fallopian tube cells. In an attempt to isolate factors influencing embryo development we have cultured 2-cell OF1 mouse embryos in media [Ménézo's B2 and Whittingham's T6 supplemented with vitamins and amino acids (T6VA)] conditioned on two types of kidney epithelial cells (MBDK and Vero). Different molecular weight fractions of conditioned medium were used to show the absence or presence of specific embryotrophic factors. With MDBK cells, B2 conditioned medium enhanced embryo development up to the blastocyst stage, while no blastocysts developed in B2 alone. When using T6VA medium, both the control and conditioned media showed a high percentage of blastocyst formation (57.0 and 54.0% respectively), while the different molecular weight fractions showed no added improvement. With Vero cells, B2 alone, B2 conditioned medium and fractions were all detrimental to embryo development. A high percentage of blastocyst formation (between 64.7 and 75.8%) was observed in T6VA alone. T6VA conditioned medium and fractions. Low blastocyst formation in a control medium can show strong positive results when medium is conditioned by cells. In contrast, a good base medium, such as T6VA, can equal the results using conditioned medium. Different cells in contact with different types of medium show variability in the pattern of responses, highlighting the presence of false positives in coculture studies.

Animals↗

PubMed1995

Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility.

During spermiogenesis, mammalian chromatin undergoes replacement of nuclear histones by protamines, resulting in a DNA that is highly condensed in the mature sperm. We have previously demonstrated that a percentage of human spermatozoa exhibit 1) positivity to the guanine-cylosine-specific chromomycin A3 (CMA3) fluorochrome and 2) the presence of endogenous nicks in their DNA. In situ protamination of mature human sperm limits the percentage of sperm positive to CMA3 and exhibiting endogenous nicks. In this study, we report further investigations that aim to clarify the relationship existing between levels of CMA3 stainability and the presence of endogenous nicks in the DNA of mature human spermatozoa. Human spermatozoa from 25 different samples showed values of sensitivity to the CMA3 fluorochrome ranging from 13% to 75%. The same samples showed a percentage of sensitivity to endogenous nick translation ranging from 1% to 38%. A strong correlation (r = 0.86) was evident between these two parameters. Prior staining of sperm with the CMA3 fluorochrome drastically reduced sensitivity to nick translation. In contrast, previously nick-translated sperm stained with CMA3 showed very little difference from samples that had not been pretreated. The presence of nicked sperm in the ejaculate may indicate anomalies during spermiogenesis and be an indicator of male infertility.

Biotin↗

PubMed1995

Relationship between the presence of endogenous nicks and sperm chromatin packaging in maturing and fertilizing mouse spermatozoa.

Mammalian spermiogenesis involves the replacement of histones by protamines, resulting in a highly compacted chromatin. Upon fertilization, the reverse process occurs. We have previously shown that the chromomycin A3 (CMA3) fluorochrome represents a useful tool for detecting protamine deficiency in spermatozoa. In this study we investigated CMA3 fluorochrome accessibility and the presence of endogenous nicks in maturing and fertilizing mouse sperm. Testicular sperm of stages 1-7 and 8-14 showed high positivity (> 96%) to CMA3, decreasing to 63% in stage 15-16 spermatids. In situ protamination of stage 15-16 spermatids saw an inhibition of CMA3 accessibility. Only 8% of the mature spermatozoa in the efferent ducts were CMA3-positive; this value decreased to 0% in the caput epididymidis. At fertilization, CMA, fluorescence reappears in decondensing sperm. Fluorescein isothiocyanate (FITC) fluorescence, identifying endogenous nicks, was evident in 6% of stage 1-7 spermatids, increased to 22% in stage 8-14 spermatids, and disappeared in stage 15-16 spermatids. During fertilization, endogenous nicks were not observed in decondensing sperm. We propose that 1) the presence of nicks in mouse testicular spermatids suggests that DNA cutting and ligating occurs prior to completion of protamination and 2) the absence of nicks during fertilization indicates that decondensation is not simply the reversal of the initial chromatin packaging process.

Animals↗

PubMed1994

Use of lasers in assisted fertilization and hatching.

The erbium-yttrium-aluminium-garnet (Er:YAG) laser has been applied to micromanipulation in humans. It was used in the fertilization process for both subzonal insemination (SUZI) and for partial zona dissection (PZD). Laser-assisted micromanipulation achieved significantly higher fertilization rates (34.8%) when compared to mechanical SUZI (16.1%), but use of the laser did not improve the PZD results (laser 14.8% versus mechanical 14%). The Er:YAG laser was used to assist hatching. In the mouse it significantly improved the hatching rate (80 versus 29.3%) 110 h after administration of human chorionic gonadotrophin. This technique was applied in two different centres to patients with previous in vitro fertilization (IVF) failures. the implantation rate per embryo (14.4% laser-assisted hatching versus 6% control group) and the pregnancy rate per transfer (40 versus 16.2%) were improved.

Adult↗

PubMed1994

[Assisted hatching and micro-fertilization].

Erbium-YAG laser has been applied for micromanipulation in human beings. In a first series of attempts it was used in the fertilization process for subzonal insemination (SUZI): laser assisted micromanipulation achieved significant higher fertilization rates (34.8%) when compared to mechanical SUZI (16.1%), and for partial zona dissection (PZD): in this case laser did not improve the results (laser 14.8% vs mechanical 14%). Erbium-YAG laser was used to assist hatching. In the mouse it significantly improved the hatching rate (80% vs 29.3%) 110 hours post hCG. This technique was applied in two different centres to patients with previous IVF failures. The implantation rate per embryo (14.4% laser assisted hatching vs 6% control group) and the pregnancy rate per transfer (40% vs 16.2%) were improved.

Animals↗