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PubMed2004

Exogenous gamma-glutamyl cycle compounds supplemented to in vitro maturation medium influence in vitro fertilization, culture, and viability parameters of porcine oocytes and embryos.

High concentrations of intracellular glutathione (GSH) enhance in vitro production of porcine embryos. Objectives were: (1) to determine the effects of gamma-glutamyl cycle compound supplements to the IVM medium on IVF and IVC; and (2) to evaluate embryo viability. Porcine oocytes were matured in NCSU 23 medium supplemented with either l-cysteine (3.3 mM), l-cysteamine (150 P < 0.05microM), l-cysteine and l-cystemaine, l-glycine (1, 2.5, or 5 mM), l-glutamate (1, 2.5, or 5 mM), l-alpha-aminobutyrate (3.3mM), beta-mercaptoethanol (BME) (25 microM), l-cysteine and BME, or l-alpha-aminobutyrate and BME. Increases (P < 0.05) in GSH concentrations were observed using l-cysteine, 1.0 mM l-glutamate, l-alpha-aminobutyrate, and l-alpha-aminobutyrate with BME. Oocytes matured with l-alpha-aminobutyrate and BME had a lower (P < 0.05) occurrence of polyspermy during IVF compared to controls and a greater percentage (P < 0.05) of embryos reaching the blastocyst stage compared to other treatment groups. For Objective 2, oocytes were matured in NCSU 23 or NCSU 23 supplemented with l-alpha-aminobutyrate with BME. Embryo cell death was determined using an Annexin V-FITC assay. Supplementation had no effect on the time of cell death. Embryo mortality was increased (P < 0.05) from 24 to 42 h post-IVF, with the greatest occurrence around 36 h. In conclusion, supplementing l-alpha-aminobutyrate and BME into the IVM medium increased intracellular GSH concentrations, decreased the occurrence of polyspermy during IVF, and increased embryo development parameters during IVC, but did not affect cell death during embryo development. The onset of cell death occurred from 24 to 42 h post-IVF, with the greatest occurrence around 36 h post-IVF.

Aminobutyrates↗
PubMed2002

Effect of cumulus cell removal of in vitro matured bovine oocytes prior to in vitro fertilization on subsequent cleavage rate.

The aim of this study is to identify the effect of cumulus cells removal prior to the in vitro fertilization of matured bovine oocytes on cleavage rate. Denuded, matured oocytes were fertilized in presence or absence of loose cumulus cells, cumulus cell conditioned IVF medium (CCCM), charcoal-treated CCCM and charcoal-treated CCCM supplemented with progesterone at a final concentration of 150 ng/ml. After 18 h of incubation with sperm, the presumptive embryos were cultured on a BRL monolayer and the percentage of cleaved embryos was evaluated on Day 4. Removal of cumulus cells prior to IVF significantly reduced the cleavage rate (25% for denuded oocytes versus 56% for cumulus-oocyte complexes (COCs)). The addition of loose cumulus cells partially restored the effect of denudation (cleavage rate: 37% for denuded oocytes supplemented with loose cumulus cells versus 27% for denuded oocytes and 58% for COCs). CCCM also had a positive effect on the cleavage rate of oocytes denuded prior to IVF (36% for denuded oocytes fertilized in CCCM versus 14% for denuded oocytes). Treating the CCCM with charcoal resulted in complete loss of its effect on cleavage rate (18% for denuded oocytes fertilized in charcoal-treated CCCM versus 34% for denuded oocytes fertilized in CCCM). The addition of progesterone to charcoal-treated CCCM partially restored the reduction of the cleavage rate caused by charcoal treatment (27% for denuded oocytes fertilized in charcoal-treated CCCM supplemented with progesterone versus 14% for denuded oocytes fertilized in charcoal-treated CCCM and 36% for denuded oocytes fertilized in CCCM). In conclusion, removal of cumulus cells prior to IVF adversely affects the cleavage rate through loss of a factor secreted by these cells. This factor probably is progesterone.

Animals↗
PubMed1998

Effectiveness of Menuzo's B2 medium with buffalo rat liver cells for development of in vitro matured/in vitro fertilized bovine oocytes.

PURPOSE: The effectiveness of two culture media on the development of bovine embryos in a buffalo rat liver (BRL) coculture system was investigated. METHODS: Bovine oocytes were matured and fertilized in vitro, then cocultured, 25 per well, for 7 days in 500 microliters of modified M199 or modified Menuzo's B2 medium over a BRL cell monolayer at 39 degrees C in an atmosphere of 5% CO2 in air. Medium 199 was modified by the addition of 10% of (v/v) fetal bovine serum (FBS), 9 mg/ml bovine serum albumin, 2 mM glycine, 1 mM alanine, and 0.1 mM nonessential amino acids (NEAA). Menuzo's B2 medium was modified by the addition of 10% (v/v) FBS, 1 mM alanine, and 0.1 mM NEAA. RESULTS: Modified Menuzo's B2 medium improved embryo development to the morula or blastocyst stage compared to modified M199 (121/353, 34.3%, versus 99/362, 27.3%, P < 0.05). CONCLUSIONS: These results suggest that Menuzo's B2 medium with modifications in a BRL coculture system can provide a significant benefit for culture of early bovine embryos over the traditional use of medium 199.

Animals↗
PubMed2003

Parents of 5-year-old in vitro fertilization children: psychological adjustment, parenting stress, and the influence of subsequent in vitro fertilization treatment.

Sixty-six parents who had conceived by in vitro fertilization (IVF) were compared with 46 matched naturally conceiving control parents regarding psychosocial adjustment and parenting stress 5 years after the birth of their first child. IVF mothers reported a more external locus of control than did control mothers but did not differ on other measures. Within the IVF group, higher levels of treatment predicted lower parenting stress and more defensive responding on the Parenting Stress Index (R. Abidin, 1990). The more positive reporting of high-treatment IVF parents may be attributable to either defensive responding or the fact that they are particularly highly motivated and competent parents. The study findings confirm a growing body of research regarding overall positive adjustment in IVF parents but also highlight the importance of considering individual differences among IVF mothers with respect to treatment experience.

Adaptation, Psychological↗
PubMed1995

Coculture of ovine zygotes fertilized in vivo or in vitro and positive effect of CZB medium on the development of in vitro fertilized zygotes.

The development and quality of ovine zygotes derived from in vivo (IVOF) or in vitro fertilization (IVF) were compared after coculture on sheep oviductal cells. The same criteria were used to evaluate the coculture of IVF zygotes in CZB medium for 2 d followed by 199 medium for 6 d (CZB-199 coculture) or in 199 medium for 8 d (199 coculture). A higher overall developmental rate to blastocyst stages was obtained with IVOF (65.7%) than with IVF (23.2%) zygotes. More IVF zygotes reached blastocyst stages in CZB-199 (36.7%) than in 199 coculture (22.9%). The morphological aspect did not differ significantly between IVOF and IVF or between 199 and CZB-199 blastocysts. Histological examination revealed no significant difference in the pyknotic and mitotic indices and mean number of cells in the trophoblast and in the inner cell mass of hatched blastocysts between IVOF and IVF or between CZB-199 and 199 cocultures. According to criteria used in this study, the quality of blastocysts was equivalent, independently of fertilization or coculture systems. The use of CZB medium during the first cleavages increases the proportion of blastocysts.

Animals↗
PubMed1988

In vitro proteolytic processing of a diubiquitin and a truncated diubiquitin formed from in vitro-generated mRNAs.

Ubiquitin, a highly conserved protein of 76 amino acids found in all eukaryotes, is translated from mRNAs that contain either multiple, contiguous coding sequences of the protein or a single ubiquitin coding sequence fused to sequences coding for 52 or 76 amino acids. We describe here formation of monoubiquitin from in vitro translation of mRNAs containing either two complete sequences or one complete ubiquitin and 60% of a second ubiquitin. No diubiquitin precursor was found with the complete diubiquitin mRNA, but the truncated mRNA formed proteins with apparent molecular masses of 30, 24, 7, and 4 kDa. The latter two are the expected products from truncated ubiquitin mRNA. The 30-kDa protein was immunoprecipitated by anti-ubiquitin antibodies and was converted to ubiquitin and the 4-kDa form by a ubiquitin isopeptidase-like activity in wheat germ. Other data indicated that the 30-kDa protein had multiple ubiquitins, all linked by isopeptide bonds to the truncated ubiquitin. One of these was the radiolabeled translation product, which should have been linked to the truncated protein by a normal peptide bond. A model is proposed in which ubiquitin itself participates in a transpeptidase activity.

Cloning, Molecular↗
PubMed1998

Short-term in-vitro culture and cryopreservation of spermatogenic cells used for human in-vitro conception.

Testicular cell suspensions were prepared from obstructive and non-obstructive azoospermic men and were cultured in vitro for 96 h as (i) mixed cell populations and (ii) isolated homogeneous populations of primary spermatocytes, round spermatids and elongating spermatids. The cells lost their viability gradually during the first 24 h period. By 72 h almost 90% of the cells were non-viable. Isolated pure fractions showed better viability at each time interval (P < 0.0005). Throughout the culture period primary spermatocytes, elongating spermatids and other non-spermatogenic cells showed no change in their morphology, but almost 22% of round spermatids showed growth of flagella. Most of the round spermatids developed their flagella during the first 4-8 h period of culture. Isolated pure round spermatids showed better flagellar growth compared with mixed cell suspensions (P < 0.0005). The spermatogenic cells were successfully cryopreserved. However, when mixed spermatogenic cell suspensions were cryopreserved, more cells lost their viability compared with when isolated pure fractions were cryopreserved (P < 0.0005).

Cell Survival↗
PubMed1998

Embryos cultured in vitro with multinucleated blastomeres have poor implantation potential in human in-vitro fertilization and intracytoplasmic sperm injection.

The aim of the present study was to investigate pregnancy rates ensuing from transfer of embryos with multinucleated blastomeres. In our in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) programme, 1735 embryo transfers were performed from January 1, 1995 to August 31, 1996. In 136 of these transfers at least one embryo with one or more multinucleated blastomeres was present per transfer (study group). For each of these 136 transfers, two matched controls with transfer of exclusively mononucleated embryos were selected (control group). Matching was carried out according to age, method of fertilization (IVF or ICSI), number of transferred embryos and quality score of transferred embryos. In the study group, there were eight transfers of exclusively multinucleated embryos from which one pregnancy ensued and 128 transfers in which multinucleated and mononucleated embryos were transferred together leading to 23 pregnancies. The overall clinical pregnancy rate per transfer was 16.9% in the study group versus 28.7% in the control group (P = 0.01). The ongoing pregnancy rate per transfer was 13.2% in the study group versus 23.2% in the control group (P = 0.03). The implantation rate per transferred embryo was 6.0% in the study group versus 11.3% in the control group (P = 0.003). This study shows that embryos with one or more multinucleated blastomeres have a poorer implantation potential than embryos with mononucleated blastomeres. Transfer of embryos with multinucleated blastomeres should hence only be considered when insufficient numbers of embryos with only mononucleated blastomeres are present.

Adult↗
PubMed2000

Effects of gonadotrophin-releasing hormone agonists on human ovarian steroid secretion in vivo and in vitro-results of a prospective, randomized in-vitro fertilization study.

The aim of this prospective randomized study was to compare the effects of two gonadotrophin-releasing hormone (GnRH) agonists, buserelin and triptorelin, on human ovarian follicular steroidogenesis, oocyte fertilization and IVF treatment outcome. Ovulatory, healthy women undergoing IVF were treated either with human menopausal gonadotrophin (HMG) alone or with HMG and one of the two GnRH agonists. Serum and follicular fluid hormonal concentrations and cultures of luteinizing granulosa cells obtained during follicular aspiration were analysed. GnRH agonist treatment significantly affected steroidogenesis both in serum and follicular fluid. In follicular fluid, progesterone and oestradiol concentrations were significantly elevated while testosterone concentrations were significantly lower in the triptorelin group. The ratios of testosterone/progesterone, oestradiol/progesterone but not oestradiol/testosterone concentrations were significantly affected by GnRH agonist administration. Similarly, the steroidogenic activity of luteinizing granulosa cells in vitro was significantly decreased in women treated with GnRH agonists. Women treated with GnRH agonists had significantly more fertilized oocytes and cleaving embryos. The results indicate a marked effect of GnRH agonists on the pattern of ovarian follicular steroidogenesis that cannot be explained solely by changes in gonadotrophin concentrations.

Adult↗
PubMed1996

Mimicking the high levels of activity of a large in-vitro fertilization unit leads to early success at the commencement of an in-vitro fertilization and embryo transfer programme.

Infertile couples seeking treatment will endeavour to obtain the highest standards of care at a conveniently located centre. The success rated of a centre would also be an important concern. The busier in-vitro fertilization (IVF) units (> 400 cycles per year) have substantially higher pregnancy rates than those carrying out < 300 cycles per year. We report our experiences at the IVF centre, Maternity Hospital, Kuwait of mimicking the high level of activity of a large IVF unit. Infertile couples were treated in groups of 30-35 every 4-6 weeks. Patients in each group had menstrual cycles synchronized to their luteal phase using progestogens. Thereafter they were down-regulated with gonadotrophin-releasing hormone agonist (long protocol). Oocyte retrieval and embryology activities were concentrated and completed in 7-10 days in each group. This episodic high level of activity was aimed at mimicking that of a busy large IVF centre, although continuous in the latter. The overall results in terms of fertilization cleavage, implantation and clinical pregnancy rates were similar to those obtained in large IVF units. The clinical pregnancy rate per cycle was 25.8-39.1% in IVF and 46.1% in intracytoplasmic sperm injection.

Adult↗
PubMed1996

Application of image analysis cytometry in follicular fluid cells obtained from in-vitro fertilization cycles: relationships to patient's age, oocyte maturity, fertilizability and in-vitro fertilization outcome.

In an in-vitro fertilization (IVF)/embryo transfer programme granulosa cells obtained from 59 individual preovulatory follicles were analysed using multiparameter image analysis cytometry, in an attempt to determine whether their morphometric and DNA-cytometric parameters could prove useful in assessing follicle and oocyte maturity and in predicting fertilizability and outcome of these IVF cycles. Almost all morphometric and DNA-cytometric parameters were not correlated with either the patient's age or oocyte maturity, and did not predict oocyte fertilization or occurrence of a clinical pregnancy. The only possible relevant parameter which, despite its inverse correlation to total luteinizing hormone administration, also proved to be inversely correlated to pregnancy outcome (in the seven cases in which a pregnancy occurred), was the percentage of granulosa cell nuclei with increased DNA content (> 5c). Finally, if granulosa cells do not reveal euploid polyploidization in spontaneous or induced ovulatory cycles, the detected cells with increased DNA content should be interpreted as aneuploid, i.e. with chromosomal aberrations, and so their presence could also be discussed in connection with the hypothetical risk of prospective neoplastic transformation of the tissue.

Adult↗
PubMed1989

In-vitro processing of sperm with autoantibodies and in-vitro fertilization results.

In-vitro fertilization and embryo transfer were carried out in twenty infertile couples with significant levels of anti-sperm antibodies on the ejaculated spermatozoa. Over 21 cycles (15 couples), 95 mature oocytes were collected and inseminated with spermatozoa obtained by swim-up migration after rapid dilution and washing of the ejaculates. An overall fertilization rate of 38.9% was obtained with these post-migration (PM) preparations. When greater than 70% of the inseminated spermatozoa were covered with both IgG and IgA antibodies, only 14% of the 43 oocytes were fertilized. A higher fertilization rate was obtained with the PM preparations containing less than 70% of spermatozoa coated with one or both classes of antibodies. Under these conditions 60% of the 52 oocytes were fertilized. Fertility rate correlated better with IgG than with IgA antibody levels. In order to decrease the proportion of antibody-coated spermatozoa in the inseminated populations, washed spermatozoa were immuno-adsorbed on Mage's plates before swim-up migration. Over 11 cycles (10 couples) 52 mature oocytes were inseminated with these post-migration immuno-depleted sperm preparations (PMP) containing less than 65% of antibody-coated spermatozoa: 31% of the oocytes were fertilized. This rate compares favourably to IVF results obtained with the PM preparations with greater than 70% of spermatozoa coated with both classes of antibodies. In five couples who had different IVF attempts with the two sperm preparations, the immuno-depletion resulted in a slight increase in the fertilization rates: 10% for the PM preparations versus 26% for the PMP preparations. Sperm binding to the zona pellucida was decreased in the majority of unsuccessful attempts.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Human follicular fluid from pre-ovulatory follicles in patients undergoing in-vitro fertilization inhibits the in-vitro growth of gram-positive micro-organisms.

In this study, we assessed the antibacterial activity of human follicular fluid obtained from patients undergoing oocyte aspiration for in-vitro fertilization and embryo transfer. Gram-positive and Gram-negative microorganisms and Candida albicans were added to the follicular fluid and broth cultures which served as controls. Follicular fluid was shown to be inhibitory against Gram-positive microorganisms; this inhibition was probably due to lysosyme and progesterone. Growth of Gram-negative micro-organisms and C. albicans were, however, supported by follicular fluid incubation.

Candida albicans↗
PubMed1993

Effects of phosphate, energy substrates, and amino acids on development of in vitro-matured, in vitro-fertilized bovine oocytes in a chemically defined, protein-free culture medium.

Bovine oocytes that had been matured and fertilized in vitro were cultured in a simple, chemically defined, protein-free medium (mTLP-PVA). When the medium was supplemented with 19 amino acids, development to the 8-cell (14-20% vs. 38-46%), morula (0-6% vs. 27-32%), and blastocyst (0-1% vs. 9-13%) stages 96, 144, and 192 h after insemination, respectively, was significantly greater in the absence than in the presence of glucose (5.56 mM) regardless of the presence of phosphate (1.05 mM). However, blastocyst development was difficult in medium with any combination of glucose and phosphate without amino acids. In mTLP-PVA with amino acids and different concentrations of phosphate, the highest proportions of embryos reaching the > or = 8-cell (56%), morula (44%), and blastocyst (24%) stages were obtained at a 0.35 mM concentration. When lactate and pyruvate were omitted from mTLP-PVA (mT-PVA) supplemented with amino acids and 0.35 mM phosphate, the first cleavage was completely inhibited. Although lactate or pyruvate alone could support blastocyst development to a limited extent (10-15%), a significantly higher proportion (22%) of blastocysts was obtained in medium with both lactate (10 mM) and pyruvate (0.5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1996

Effect of hypotaurine on in vitro fertilization and production of term offspring from in vitro-fertilized ova of the Japanese field vole, Microtus montebelli.

The present study was proposed to establish new in vitro fertilization (IVF)-embryo transfer techniques for wild mammals. We demonstrated that IVF of the Japanese field vole, Microtus montebelli, is improved by hypotaurine, and that IVF vole embryos can develop to normal offspring after transfer to recipient females. The rate of IVF was significantly increased when 0.1 or 1 mM of hypotaurine was added during sperm preincubation and fertilization (p < 0.05). Preincubation of spermatozoa with 1 mM hypotaurine prior to oocyte insemination did not have a significant effect on fertilization rate (p < 0.01). At 96 h after insemination, most cultured embryos developed to the 2-cell stage (68-83%), but development to blastocysts was very low (0-3%) for all treatments. Although 43 living offspring were produced after transfer of IVF pronuclear embryos to recipients, the delivery rate of recipients was very low (21%). The litter size of pseudopregnant recipients that produced a litter was 4-11, and the rate of in vivo development of transferred embryos in the pregnant recipients was 41%. These results suggest that hypotaurine affects IVF rather than sperm preincubation and that IVF embryos can develop to normal offspring.

Animals↗
PubMed1979

In vitro studies of poison oak immunity. I. In vitro reaction of human lymphocytes to urushiol.

Poison oak, ivy, and sumac dermatitis is a T-cell-mediated reaction against urushiol, the oil found in the leaf of the plants. This hapten is extremely lipophilic and concentrates in cell membranes. A blastogenesis assay employing peripheral blood lymphocytes obtained from humans sensitized to urushiol is described. The reactivity appears 1--3 wk after exposure and persists from 6 wk to 2 mon. The dose-response range is narrow, with inhibition occurring at higher antigen concentrations. Urushiol introduced into the in vitro culture on autologous lymphocytes, erythrocytes and heterologous erythrocytes produces equal results as measured by the optimal urushiol dose, the intensity of reaction, and the frequency of positive reactors. This suggests that the urushiol is passed from introducer to some other presenter cell. Although the blastogenically reactive cell is a T cell, there is also a requirement for an accessory cell, found in the non-T-cell population, for reactivity. Evidence is presented that this cell is a macrophage.

Adolescent↗