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Biomedical subjects

D Sakkas

Publications and source records attributed to D Sakkas.

69 records · Page 4Linked to original sources
PubMed1994

Comparison of results after in vitro fertilized human embryos are cultured in routine medium and in coculture on Vero cells: a randomized study.

OBJECTIVE: To investigate whether coculture of human embryos on Vero cells improves embryo viability compared with a routine culture method. SETTING: In vitro fertilization Clinic of the Hôpital Cantonal Universitaire de Genève, Geneva, Switzerland. PATIENT SELECTION: Couples who had given informed consent, had undergone < 3 IVF cycles with ET and where the male had normal semen parameters were selected. Patients who had undergone > or = 3 IVF cycles with ET were allocated directly to coculture. DESIGN: Patients were randomly allocated to have their embryos cultured in a routine embryo culture medium or in coculture with Vero cells. RESULTS: There was no difference in pregnancy rates between the two culture groups. Coculture gave a high (> 50%) rate of blastocyst formation. In 16 cycles where patients had previously undergone > or = 3 IVF cycles 4 patients became pregnant. CONCLUSIONS: Coculture provides no benefit for patients that are performing their initial treatment cycles in IVF.

Adult↗
PubMed1993

Involvement of the plasminogen activator/plasmin proteolytic cascade in fertilization.

At the time of fertilization both murine gametes express plasminogen-dependent proteolytic activity: unfertilized eggs secrete tissue-type plasminogen activator and ejaculated spermatozoa have urokinase-type plasminogen activator bound to their surface. We now report that plasminogen is present in the fertilization environment and that both spermatozoa and eggs are able to specifically bind plasminogen. Furthermore, in vitro fertilization of mouse eggs is inhibited by antibodies which inhibit the catalytic activity of plasmin. Finally, with two different in vitro fertilization protocols, the addition of plasminogen to the fertilization medium increases the yield of fertilized eggs. These results provide evidence for a role of the plasminogen activator/plasmin proteolytic cascade in mammalian fertilization.

Animals↗
PubMed1993

Evidence of sperm entry into assumed unfertilized human oocytes after sub-zonal sperm microinjection.

Sub-zonal sperm microinjection (SUZI) as a treatment for male factor infertility can facilitate fertilization, however, in many cases oocytes remain unfertilized even though the sperm is placed in close contact with the oolemma. In order to improve our understanding of gamete interaction in cases of failed fertilization, we have analysed the failed fertilized oocytes from both SUZI and conventional in-vitro fertilization. The fluorochrome Hoechst 33342 (which binds specifically to DNA) was used to check for the possible presence of paternal chromatin in the unfertilized oocytes. A significantly higher (P < 0.01) number of microinjected oocytes showed signs of fertilization 2-3 days after sperm microinjection compared to normally inseminated oocytes, 30/175 (17.1%) and 2/79 (2.5%) respectively. In addition, four out of eight couples returning for a second treatment by SUZI displayed anomalies in fertilization in both cycles. The semen characteristics of patients with or without anomalies in fertilization was not different. The irregularities observed in the fertilization process infer that certain male factor patients have intrinsic sperm anomalies lying at the sperm membrane and/or chromatin level that could lead to anomalies in the appearance of the pronuclei.

Adult↗
PubMed1993

Mouse embryo cleavage, metabolism and viability: role of medium composition.

The cleavage, metabolism and viability of mouse zygotes were assessed after culture in media of different ionic and metabolite composition. Medium with a high potassium concentration, characteristic of mammalian oviduct fluid, inhibited cleavage and blastocyst formation (P < 0.01). This inhibition was partially alleviated by the removal of phosphate, and subsequently abolished by supplementation with amino acids, vitamins, insulin, epidermal growth factor and transferrin (AVIET). Glucose uptake by cultured blastocysts, measured fluorimetrically, was not affected by the ionic or metabolite composition of the medium, but was significantly reduced by the inclusion of AVIET (P < 0.01). Lactate production was also significantly reduced in the presence of AVIET (P < 0.01). Calculations of metabolic activity revealed that embryos cultured in the presence of AVIET had a glycolytic activity similar to embryos developed in vivo. In contrast, embryos cultured in conventional embryo culture media exhibited an elevated glycolytic activity. Culturing embryos for 4 days in a reduced lactate concentration (4.79 mM), significantly increased fetal development after transfer, compared with embryos cultured in the concentration of lactate present in conventional embryo culture media (23.3 mM; P < 0.01). In contrast, when embryos were transferred on day 3 of culture, significantly more fetuses were obtained from embryos cultured in high levels of lactate (P < 0.01). Supplementation of medium with AVIET significantly increased resultant fetal weights after transfer (P < 0.05). This study demonstrates that different media are required to maintain embryo viability on successive days of culture, and highlights the potential limitations of employing simple salt solutions for the culture of preimplantation mammalian embryos.

Animals↗
PubMed1993

IVF treatment of moderate male factor infertility: a comparison of mini-Percoll, partial zona dissection and sub-zonal sperm insertion techniques.

In this study we examined various techniques of in-vitro fertilization (IVF) for treating couples in whom the male had subnormal semen parameters. We compared two sperm preparation methods (mini-Percoll and conventional swim-up) for efficiency of recovery after preparation and for fertilization rates after IVF, and compared the suitability of partial zona dissection (PZD) and sub-zonal sperm insertion (SUZI) to patients with different types of male factor infertility. The mini-Percoll technique allowed the recovery of significantly more motile spermatozoa from the same semen sample compared to the swim-up method. More oocytes were fertilized after spermatozoa were prepared by the mini-Percoll technique. An increased number of spermatozoa recovered from an ejaculate led to an improvement in the quality of spermatozoa in the insemination droplet. Subsequently, when using the PZD technique, the fertilization rate increased when there was a higher number of spermatozoa in the patient's ejaculate. When comparing the two micromanipulation techniques, SUZI provided patients with oligoasthenzoospermia (i.e. < 10 x 10(6) spermatozoa/ml and 40% motility) with a higher chance of obtaining 2-pronculeate eggs.

Colloids↗
PubMed1993

Effect of deoxyribonucleic acid protamination on fluorochrome staining and in situ nick-translation of murine and human mature spermatozoa.

A major event in enhancing sperm chromatin stability is the replacement of the histones by protamines during spermiogenesis. In this study, we present results indicating that chromomycin A3 (CMA3) can be used to show protamine deficiency in sperm chromatin. Fixed chromatin of mature mouse spermatozoa showed high fluorescence after treatment with ethidium bromide (EB), but was completely unstained after treatment with CMA3. The same chromatin was found to be highly resistant to in situ nick-translation. In contrast, a substantial fraction of human spermatozoa were positive for CMA3. The accessibility of CMA3 to the DNA of human sperm was eliminated if the slides were previously treated with protamine in situ. This treatment did not affect the accessibility of EB to the chromatin. Individual human sperm samples revealed a substantial frequency of spermatozoa with endogenous nicks, which was found to be the same as the frequency of spermatozoa responding positively to CMA3 staining. Treatment of preparations with protamines prevented the identification of the endogenous nicks. These data as a whole suggest that CMA3 could represent a useful tool for the detection of protamine deficiency in sperm chromatin. Furthermore, confirmation of experiments relating sensitivity to nick translation and positivity to CMA3 may allow an indirect in situ visualization of nicked and partially denatured DNA, which could correlate with certain forms of male factor infertility.

Animals↗
PubMed1993

Effects of glucose and fructose on fertilization, cleavage, and viability of mouse embryos in vitro.

In this study we examined the role of glucose, and the use of fructose as a replacement, in embryo culture medium. Three embryo culture media were used: a routine embryo culture medium (M16), M16 without glucose (M16-G) and M16-G supplemented with fructose (M16F-G). Their effect on fertilization, rate of cleavage, and embryo viability were examined in both an outbred (OF1) and an inbred (C57Bl) mouse strain. Of the three media, only M16 was found to support fertilization. In vitro-fertilized embryos from OF1 oocytes and C57Bl oocytes and OF1 sperm were placed in the different media at the early 2-cell stage. In 65% of OF1 embryos cultured in M16, development was blocked at the 2-cell stage, whereas in M16-G and M16F-G embryos, only 22% and 32%, respectively, were blocked. M16F-G medium also produced morulae and blastocysts with higher cell numbers than M16-G. In vitro-fertilized C57Bl 2-cell embryos cultured in M16 displayed retarded cleavage to the 4-cell stage compared to embryos cultured in M16-G and M16F-G. In contrast, the morulae and blastocyst cell numbers were significantly lower in M16-G compared to M16 and M16F-G. The viability of morulae and blastocysts obtained from OF1 and C57Bl embryos cultured in M16-G and M16F-G was lower compared to control C57Bl morulae and blastocysts cultured in M16. The results show that although morphologically normal embryos could be obtained in M16-G and M16F-G, inherent anomalies existed that limited viability.

Animals↗
PubMed1993

A cytochemical study of mature mouse spermatozoa after C-banding treatment.

Heterochromatin in mature mouse spermatozoa has been investigated by using C-banding treatment followed by either i) Giemsa staining, ii) staining with DAPI, a fluorochrome specific for AT rich DNA and iii) by using quantitative DNA measurements [Acriflavine-Feulgen, DAPI and Ethidium Bromide (EB)]. We have shown that a fraction of the mature sperm chromatin is affected by C-banding treatment. The sperm chromatin treated with Ba(OH)2 and stained with EB doubled fluorescence emission values found in untreated control preparations. This experimental result clearly shows that the use of intercalating fluorochromes, such as EB, is unsuitable for quantitative evaluation of highly condensed DNAs.

Animals↗
PubMed1992

Subzonal sperm microinjection in cases of severe male factor infertility and repeated in vitro fertilization failure.

OBJECTIVE: To examine (1) fertilization rates obtained with subzonal sperm microinjection when different numbers of sperm are injected into the perivitelline space; (2) when subzonal sperm microinjection is combined with dilute insemination; and (3) the association of semen quality characteristics with fertilization. DESIGN: Subzonal sperm microinjection and subzonal sperm microinjection combined with dilute insemination was performed in 109 and 41 cycles on patients in two clinical trials in Melbourne, Australia, and Bologna, Italy, respectively. PATIENT PARTICIPANTS: Couples who have experienced repeated in vitro fertilization failure or in whom the husband has severe male factor infertility. PRIMARY OUTCOME MEASURES: The number of oocytes fertilized after injection of different numbers of sperm into the perivitelline space, the number of patients transferred, and pregnancy outcome. RESULTS: The injection of multiple numbers of sperm into the perivitelline space failed to improve monospermic fertilization rates but caused an increase in polyspermic fertilization. In patients with initial semen parameters exhibiting greater than 50% motility or greater than 50% normal morphology fertilization rates were improved when subzonal sperm microinjection-treated eggs were incubated in a dilute insemination medium. Six pregnancies were obtained, two of which have progressed to term. CONCLUSIONS: When applied to male factor patients, the subzonal sperm microinjection technique results in a 14% to 15% fertilization rate. However, of the 102 embryos transferred only three (2.9%) fetal heart beats were obtained.

Female↗
PubMed1991

Formulation of a complex serum-free medium (CSM) for use in the co-culture of mouse embryos with cells of the female reproductive tract.

Co-culture of pre-implantation embryos with cells of the reproductive tract requires a medium that is beneficial to both embryos and cells. However, many studies in this area utilize media originally formulated for specific cell lines. In the present study, a complex serum-free medium (CSM) was formulated on the basis of the ionic compositions of existing embryo culture media and mouse oviductal fluid as well as the concentrations of growth factors that appear to benefit mouse embryo development. The study began by investigating the effect of altering the concentrations of K+ ions (0-40 mM) and sulfate ions (0-10 mM) in embryo culture media on the development of 2-cell mouse embryos. Mouse embryos showed improved cell numbers at the blastocyst stage when cultured in 10 mM K+ compared with Whittingham's T6 medium. Embryos were also cultured in T6 supplemented with bovine serum albumin (BSA) containing various concentrations of insulin, insulin-like growth factors I and II, fibroblast growth factor, and epidermal growth factor. Insulin concentrations of 100 ng mL-1 significantly (P less than 0.05) improved the cell numbers of 2-cell embryos cultured to the morulae and blastocyst stages compared with those cultured in T6 + BSA alone. CSM was formulated on the basis of the results of these experiments and was found to support both improved development of 2-cell mouse embryos and the culture of mouse fibroblast and mouse oviduct cells.

Animals↗
PubMed1990

Co-culture of mouse embryos with oviduct and uterine cells prepared from mice at different days of pseudopregnancy.

Oviduct and uterine cell cultures were prepared from mice at different days of pseudopregnancy and their effects on the development of 1- and 8-cell mouse embryos in co-culture were examined. One-cell mouse embryos in co-culture with oviduct cells from 20 h to 120 h after hCG had a mean (+/- s.e.) cell number of 70.1 +/- 3.6, significantly (P less than 0.001) higher compared with those cultured in Whittingham's T6 medium supplemented with 5% fetal calf serum (T6 + 5% FCS) (30.4 +/- 1.6). Transfer of embryos, at 96 h after hCG, to synchronous pseudopregnant recipients showed that more embryos in oviduct co-culture formed fetuses than those cultured in T6 + 5% FCS. Co-culture of 1-cell embryos with uterine cells did not confer an advantage in cell numbers over T6 + 5% FCS. However, more 8-cell embryos formed blastocyst outgrowths after 100 h in co-culture with uterine cells prepared from mice at Day 3 of pseudopregnancy than with uterine cultures prepared from mice at Day 1 of pseudopregnancy or oviduct cells. In addition, there was further improvement when the Day 3 uterine co-cultures were supplemented with 1 or 10 ng progesterone/ml. These results highlight the importance of the oviduct and uterine cells during the different stages of preimplantation embryo development.

Animals↗
PubMed1989

In vivo cleavage rates and viability obtained for early cleavage mouse embryos in co-culture with oviduct cells.

The cleavage rate and development of two-cell mouse embryos to the morulae stage in co-culture with mouse oviduct cells was studied in vitro and compared with those achieved in vivo. Embryos were cultured in Whittingham's T6 (T6), T6 supplemented with fetal calf serum (FCS) and in co-culture with either Dulbecco's Modified Eagles Medium supplemented with sodium lactate (DMEM + 1a) or a modification of T6 medium containing vitamins and amino acids (T6 + v + aa). Co-culture of oviductal cells with DMEM + la medium supported two-cell mouse embryo development to eight cells at a rate significantly better (P less than 0.001) than T6, but the rate of embryo development was not equivalent to that in vivo. DMEM + la alone was inadequate as an embryo culture medium. Co-cultures using T6 + v + aa with mouse oviductal cells were prepared from mice at days 1, 2 or 3 of pseudopregnancy. Day 2 and 3 co-cultures allowed two-cell embryos to develop at a rate comparable to that in vivo up to the mid eight-cell stage (68 h after hCG), but by 76 h after hCG embryos were retarded. Transfer to pseudopregnant recipients of embryos co-cultured with day 2 oviductal cells until 68 h after hCG resulted in a rate of fetal development equivalent to that of embryos grown in vivo. Our results show that co-culture of early cleavage-stage embryos with mouse oviductal cells allows embryos to retain cleavage rates and viability comparable to in vivo development.

Animals↗
PubMed1989

Development of preimplantation goat (Capra hircus) embryos in vivo and in vitro.

Preimplantation goat embryos were cultured with or without goat oviduct epithelial cells in Earle's 199 medium + 10% goat serum (E199 + 10%GS), and in three different simple chemically defined media. In-vivo development was characterized by an extended 8- to 16-cell stage followed by a rapid cleavage rate in the next 3 cell cycles. Culture of 1-8-cell embryos in Medium E199 + 10%GS led to cleavage arrest at the 8-16-cell stage, while in the chemically defined media embryos developed poorly and a high percentage failed to pass the 8-16-cell stage. In co-culture, however, a high percentage (77% and 96%) of 1-2-cell and 4-8-cell embryos, respectively, developed beyond the 16-cell stage. In co-culture, 1-2-cell embryos maintained cleavage rates equivalent to those in vivo until the 8-cell stage, but thereafter cell numbers lagged behind those in vivo, and by 168 h after ovulation, cell numbers (+/- s.e.) in vitro were 47.6 +/- 7.9 compared to 238 +/- 27.2 in vivo (t = 6.93, P less than 0.001). The results demonstrate that co-culture of embryos with oviduct cells allows a high percentage of embryos to develop through the period of cleavage arrest, providing a favourable environment for development through the 1-16-cell stages but a less adequate environment for development to the blastocyst stage.

Animals↗
PubMedDate not supplied

Biochemical markers of sperm function: male fertility and sperm selection for ICSI.

The expression of a 70 kDa chaperone protein, HspA2 (formerly called CK-M), has been identified in mature human spermatozoa. The central role of HspA2 has been established, as the expression level of this protein is related to sperm cellular maturity, DNA integrity, chromatin maturity, chromosomal aneuploidy frequency and sperm function, including fertilizing potential. The spermiogenetic events of cytoplasmic extrusion and remodelling of the plasma membrane, which facilitate the formation of zona pellucida binding site(s) in human spermatozoa, are related. Finally, the presence of the hyaluronic acid (HA) receptor on the plasma membrane of mature sperm coupled with the HA-coated slide sperm-binding assay, facilitates the testing of infertile men and the selection of single mature spermatozoa for ICSI. Because mature spermatozoa have no residual cytoplasm, the HA-bound sperm fraction is also enriched in spermatozoa that are normal by the Kruger strict morphology method.

Aneuploidy↗