Congress on In Vitro Biology. 1997 Meeting of the Society for In Vitro Biology. Washington, DC, June 14-18, 1997. Abstracts.
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Animals↗
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Response of Schwann cells to mitogens in vitro is determined by pre-exposure to serum, time in vitro, and developmental age.
We compared the mitogenic response of Schwann cells freshly isolated from adult, neonatal, and embryonic nerves, and compared these cells with cells that had been cultured in serum for 5 days. DNA synthesis in response to growth factors was measured using bromodeoxyuridine and immunocytochemistry. Freshly isolated adult Schwann cells were unresponsive to growth factors with or without forskolin to elevate intracellular cAMP levels. After 5 days of culture in serum, or alternatively in defined medium containing fibroblast growth factor 2 plus forskolin, or neu-differentiation factor beta2, adult cells were responsive to mitogens, whereas cells cultured in defined medium alone remained unresponsive. Serum also increased expression of type 1 fibroblast growth factor receptor. Freshly isolated embryonic and neonatal Schwann cells in contrast responded to growth factors even in the absence of forskolin. This responsiveness changed with time in culture. Neonatal cells cultured for 5 days in defined medium in the presence or absence of serum no longer responded to FGF alone, but required forskolin for a mitogenic response. Thus, the response of freshly isolated cells to mitogens is developmentally regulated; extrinsic signals are required to render adult cells responsive to mitogens; and with time in culture, neonatal cells develop a requirement for cAMP elevation for mitogenic response.
Animals↗
Use of in vitro assays to predict the efficacy of chemopreventive agents in whole animals.
Five in vitro assays have been applied to screen the efficacy of potential chemopreventive agents. These assays measure a) inhibition of morphological transformation in rat tracheal epithelial (RTE) cells, b) inhibition of anchorage independence in human lung tumor (A427) cells, c) inhibition of hyperplastic alveolar nodule formation in mouse mammary organ cultures (MMOC), d) inhibition of anchorage independence in mouse JB6 epidermal cells, and e) the inhibition of calcium tolerance in human foreskin epithelial cells. The efficacy of many of these same agents in whole animal studies of lung, colon, mammary gland, skin, and urinary bladder carcinogenesis has also been measured. The aim herein is to estimate the positive and negative predictive values of these in vitro assays against whole animal chemopreventive efficacy data using the same chemicals. For three of these assays--using RTE, A427 cells and mouse mammary organ culture (MMOC)-enough data are available to allow the estimate to be made. Such extrapolations of in vitro data to the in vivo situation are difficult at best. There are many dissimilarities between the two assay systems. The in vitro assays use respiratory and mammary epithelial cells, while the in vivo assays use respiratory, mammary, colon, bladder and skin cells. The in vitro assays use the carcinogens benzo(a)pyrene (B(a)P) and 7,12-dimethylbenz(a)anthracene (DMBA), while the in vivo assays use B(a)P, DMBA, N-methyl-N-nitrosourea (MNU), N,N'-diethylnitrosamine (DEN), azoxymethane (AOM), and N-butyl-N-(4-hydroxybutyl)nitrosoamine (OH-BBN). There are vast differences in pharmacodynamics and pharmacokinetics in vitro and in vivo, yet it is possible to rapidly screen chemicals in vitro for efficacy at one-tenth the cost and complete tests in weeks instead of months. A positive in vitro assay was defined as a 20% inhibition (compared with control) for the RTE and A427 assays and a 60% inhibition for the MMOC assay at nontoxic concentrations. For in vivo assays, the criterion for a positive result was a statistically significant inhibition of incidence, multiplicity or a significant increase in latency (mean time to first tumor). For an agent to be considered negative in animals, it required negative results in at least two different organ systems and no positive results. Using the battery of three in vitro tests, the positive predictive value for having one, two, or three positive in vitro assays and at least one positive whole animal test was 76%, 80%, and 83% respectively. The negative predictive values for one, two or all three in vitro assays was 25%, 27%, and 50%. From these data it is observed that in vitro assays give valuable positive predictive values and less valuable negative predictive values. The mechanisms of chemoprevention are not well understood. Seven categories of agents were examined for their cancer preventing both in vitro and in vivo: antiinflammatories, antioxidants, arachadonic acid metabolism inhibitors, GSH inducers, GST inducers, ODC inhibitors, and PKC inhibitors. Three or even five in vitro assays cannot be all-inclusive of the many mechanisms of cancer prevention. However, three assays help to predict whole animal efficacy with reasonable positive predictive values. Much work and development remains to be done to rapidly identify new chemopreventive drugs.
Animals↗
Angiogenesis and morphometry of bovine placentas in late gestation from embryos produced in vivo or in vitro.
The objective of this study was to determine the effects of in vitro embryo production on angiogenesis and morphometry of the bovine placenta during late gestation. Blastocysts produced in vivo were recovered from superovulated Holstein cows. Blastocysts produced in vitro were obtained after culture of in vitro-matured and -fertilized Holstein oocytes. Single blastocysts from each production system were transferred into heifers. Fetuses and placentas were recovered on Day 222 of gestation (in vivo, n=12; in vitro, n=12). Cotyledonary and caruncular tissues were obtained for quantification of vascular endothelial growth factor (VEGF) and peroxisome proliferator-activated receptor-gamma (PPARgamma) mRNA and protein. Tissue sections of placentomes were prepared for morphometric analysis. Fetuses and placentas were heavier from embryos produced in vitro than from embryos produced in vivo. More placentas from embryos produced in vitro had an excessive volume of placental fluid. There was no effect of treatment on the expression of mRNA for VEGF and PPARgamma in either cotyledonary or caruncular tissues. The expression of VEGF protein in cotyledons and caruncles as well as the expression of PPARgamma protein in cotyledons were not different between the in vitro and in vivo groups. However, caruncles from the in vitro group had increased expression of PPARgamma protein. The total surface area of endometrium was greater for the in vitro group compared with controls. In contrast, the percentage placentome surface area was decreased in the in vitro group. Fetal villi and binucleate cell volume densities were decreased in placentomes from embryos produced in vitro. The proportional tissue volume of blood vessels in the maternal caruncles was increased in the in vitro group. Furthermore, the ratios of blood vessel volume density-to-placentome surface area were increased in the in vitro group. In conclusion, these findings are consistent with the concept that compensatory mechanisms exist in the vascular beds of placentas from bovine embryos produced in vitro.
Animals↗
Effects of low-frequency ultrasound on the transdermal permeation of mannitol: comparative studies with in vivo and in vitro skin.
The in vivo and the in vitro correlation of the effects of low-frequency ultrasound (low-frequency sonophoresis, LFS) on the percutaneous penetration of mannitol, a model hydrophilic permeant, was investigated using three in vitro skin models (including full-thickness and split-thickness pig skin, and heat-stripped human cadaver skin) and in vivo pig as the animal model. The central objective of this article was to identify the relevant in vitro skin models and ultrasound conditions that may be used in in vitro LFS studies to predict the effects of LFS in vivo on the transdermal delivery of hydrophilic permeants. In this article, by conducting comparative studies of the in vivo pig skin and of the three in vitro skin models under two LFS protocols (a constant ultrasound energy dose protocol, and a constant skin electrical resistance protocol), we demonstrated that: (1) under a constant ultrasound energy dose protocol (protocol A, 5 min LFS), no good correlation was observed between the in vivo skin and the in vitro skin models in terms of the measured skin permeabilities to mannitol. Moreover, the effects of LFS on the barrier functions of the in vivo pig skin, as measured by the enhancement ratio of the skin permeation rate of mannitol and by the reduction of the skin electrical resistance, are much more pronounced than those observed with the excised skin models in vitro; (2) under a constant skin electrical resistance protocol (protocol B) of LFS, a good correlation was found between the skin permeability to mannitol measured using the three in vitro skin models and that of the in vivo pig skin. This result indicates that by utilizing the skin electrical resistance as a quick indicator of the skin permeabilization state due to LFS, the three in vitro skin models can be utilized to predict the transport rate of mannitol across the in vivo skin during LFS; (3) by applying a recently developed skin porous-pathway theory, we demonstrated that within the range of LFS conditions examined, the three in vitro skin models exhibit similar transport properties to mannitol and similar skin effective pore radius values, and hence, represent equivalent skin models for the in vitro LFS studies in the case of hydrophilic permeants; (4) histological studies revealed that the LFS protocol that was shown to be efficacious in enhancing the skin penetration rate of mannitol across the in vivo pig skin, and was also utilized for the in vivo/in vitro skin comparative studies, is safe for the living skin; and (5) through measuring the skin concentration of mannitol in the presence and in the absence of the LFS treatment, we found that the LFS-induced flux enhancement outweighs the enhancement of the skin concentration of mannitol during the LFS studies both in vivo and in vitro. This result suggests that LFS represents a good method of enhancing the systemic absorption of hydrophilic permeants, while it does not significantly alter the vehicle-to-skin partition coefficient for the same class of permeants.
Administration, Cutaneous↗
Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo.
Porcine embryos produced in vitro have a small number of cells and low viability. The present study was conducted to examine the morphological characteristics and the relationship between actin filament organization and morphology of porcine embryos produced in vitro and in vivo. In vitro-derived embryos were produced by in vitro maturation, in vitro fertilization (IVF), and in vitro development. In vivo-derived embryos were collected from inseminated gilts on Days 2-6 after estrus. In experiment 1, in vitro-derived embryos (</= 8-cell stage) collected 12-48 h after IVF were separately fixed, stained by orcein, and examined under phase contrast microscopy. It was found that 27% of 2-cell, 74% of 3-cell, 51% of 4-cell, and 74% of 5- to 8-cell-stage embryos were abnormal in morphology. Morphological anomalies included fragmentation (no nucleus in one or more than one blastomere) and/or binucleation (two nuclei in one or more than one blastomere). In experiment 2, actin filament distribution of the embryos at 2-cell to blastocyst stages that were produced in vivo and in vitro were stained by rhodamine-phalloidin and examined by confocal microscopy. Actin filaments were distributed in all in vivo-derived embryos at the cell cortex, and at the joints of cells and perinucleus in 2- to 8-cell-stage embryos and in some cells of morulae and blastocysts. Actin filaments were also distributed in the cortex and at the joints of cells of all in vitro-produced embryos. However, only 20% of in vitro-produced embryos at 2- to 8-cell stages had perinuclear actin filaments in all blastomeres. Most in vitro-produced embryos had fewer perinuclear actin filaments or did not have perinuclear actin filaments in some blastomeres. Fragmentation and binucleate blastomeres were not observed in in vivo-derived embryos. In vivo-derived Day 5 (136.5 +/- 60.4 nuclei per blastocyst) and Day 6 (164.5 +/- 51.9 nuclei per blastocyst) blastocysts had significantly (p < 0.001) more cells than in vitro-produced Day 6 blastocysts (37. 3 +/- 11.7 nuclei per blastocyst). In experiment 3, when cytochalasin D, an inhibitor of microfilament polymerization, was included in the culture medium, it prevented 2- to 4-cell-stage embryos from developing to the blastocyst stage. These results indicate that abnormal actin filament distribution is one possible reason for abnormal embryo cleavage and small cell numbers in pig embryos produced in vitro. Culture conditions that mediate normal actin filament distribution may result in an improvement in embryo quality.
Actins↗
T helper cells required for the in vitro primary antibody response to SRBC are neither SRBC-specific nor MHC-restricted.
These studies address the specificity of the T cells which normally function in the in vitro primary immune response. It is generally accepted that SRBC-specific, MHC-restricted T helper cells become activated both in vivo and in vitro by seeing erythrocyte antigens presented in the context of the MHC antigens of an antigen-presenting cell. At this point, the in vivo and in vitro systems differ. In vivo, the interaction between SRBC-specific B cells and the activated T helper cells is itself also MHC-restricted, whereas in vitro, the T cell-B cell interaction is not MHC-restricted, and may be factor-mediated. It is the first phase of the in vitro response, and actually the specificity of the T cells which function in the in vitro primary response that we are questioning, in these studies. The experimental approach was to deplete antigen-specific T helper cell activity by passage of T cells through irradiated mice in the presence of a high dose of antigen, and collecting thoracic duct cells one day later. Using this protocol, we have confirmed that removal of SRBC-specific T cells ablates the adoptive transfer in vivo response to SRBC. However, the same negatively selected T cell populations were just as potent as control T cells in supporting the in vitro response to SRBC. We confirmed that the T cells removed respond to SRBC in an MHC-restricted manner by removing from an F1 T cell population the cells able to respond to SRBC in association with one parental haplotype. These T cells nonetheless provided equal help for both parental B cell populations in vitro. These experiments show that the MHC-restricted antigen-specific T cell which is required for the in vivo response is not required in the primary in vitro response to erythrocytes. The significance of this is twofold. Firstly, this demonstrates clearly that different T cells are functioning in vitro vs. in vivo. Singer and his colleagues have shown that different B cell subpopulations (Lyb5+ vs. Lyb5-), with differing MHC-restriction requirements, are preferentially activated in vivo and in vitro. Our data says that another difference between the in vivo and in vitro SRBC response is that different T cells are working in the two systems. Understanding exactly which B cell and T cell subpopulations are working within the model system (in vivo or in vitro) that an investigator chooses is obviously of critical importance.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Follicle-stimulating hormone and growth hormone act differently on nuclear maturation while both enhance developmental competence of in vitro matured bovine oocytes.
This study was designed to investigate the effect of follicle-stimulating hormone (FSH) on nuclear maturation, fertilization, and early embryonic development of in-vitro-matured bovine oocytes and to find out whether this effect is exerted through a cyclic adenosine monophosphate (cAMP) signal transduction pathway. In addition the effect of the combination of FSH and growth hormone (GH) on subsequent cleavage and embryo development was studied. Therefore cumulus oocyte complexes were cultured in the presence of FSH (0.05 IU/ml) and the nuclear stage of the oocytes was assessed using 4,6-diamino-2-phenyl-indole (DAPI) staining either after 16, 20, or 24 hr of in vitro maturation or 18 hr after the onset of fertilization. To assess the effect of FSH and the combination of FSH and GH added during in vitro maturation on the developmental capacity of the oocytes, cumulus oocyte complexes were incubated in the presence of either FSH (0.05 IU/ml) or FSH (0.05 IU/ml) plus GH (100 ng/ml) for 22 hr, followed by in vitro fertilization and in vitro embryo culture. To investigate whether FSH-induced oocyte maturation is exerted through the cAMP pathway, cumulus oocyte complexes were cultured in M199 supplemented with FSH (0.05 IU/ml) and H-89 (10 microM), a specific inhibitor of cAMP-dependent protein kinase A. After 16 hr of culture, the proportion of oocytes in metaphase II (MII) stage was determined. Cultures with GH and without FSH and H-89 served as controls. The percentage of MII oocytes at 16 hr of incubation was significantly lower (P < 0.001) in the presence of FSH than in the control group, while the number of MII oocytes beyond 20 hr did not differ from the control group. That points to a transient inhibition of nuclear maturation by FSH. Opposite to FSH, addition of GH during in vitro maturation significantly enhanced the number of MII oocytes after 16 hr of culture (P < 0.001), which points to the acceleration of nuclear maturation by GH. Addition of FSH during in vitro maturation significantly enhanced the proportion of normal fertilized oocytes, cleaved embryos and blastocysts (P < 0.001). Similarly, addition of GH during in vitro maturation significantly enhanced the number of cleaved embryos and blastocysts (P < 0.001); however, in vitro maturation in the presence of GH and FSH did not result in an extra enhancement of the embryo development. Both the inhibition of nuclear maturation by FSH and its acceleration by GH was completely abolished by H-89. In conclusion, in vitro maturation of bovine oocytes in the presence of FSH retards nuclear maturation via a cAMP-mediated pathway, while it enhances fertilizability and developmental ability of the oocytes. Supplementation of GH and FSH during in vitro maturation did not result in an extra increase in the number of blastocysts following in vitro fertilization and in vitro embryo culture.
Animals↗
Pharmacokinetic computer simulations of the relationship between in vivo and in vitro neuroreceptor subtype selectivity of radioligands.
Pharmacokinetic computer simulations reveal a discrepancy between the in vivo and in vitro neuroreceptor subtype selectivity of radioligands. For radioligands with an in vitro neuroreceptor subtype selectivity between 0.1 and 10.0, the in vivo neuroreceptor subtype selectivity appears to be constrained to be between 0.1 and 10.0, but, in general, is not equal to the in vitro selectivity. For example, if the in vitro selectivity is 1.0 (that is, the radioligand is nonselective in vitro) the in vivo selectivity may be thought of as a random variable having a significant nonzero probability for values as low as 0.1 or as high as 10.0, with a moderate peak at a value of 1.0. For a radioligand whose in vitro subtype selectivity is greater than 10.0, the in vivo selectivity is bounded above by the in vitro subtype selectivity, but may be several orders of magnitude lower than the in vitro subtype selectivity. Thus, in spite of the discrepancy between the in vivo and in vitro neuroreceptor subtype selectivity of radioligands, there are two useful inferences about the in vivo selectivity that might be drawn from knowledge of the in vitro selectivity: (1) If the in vitro selectivity is between 0.1 and 10.0, then, at best, the in vivo selectivity might be as high as 10.0. (2) If the in vitro selectivity is greater than 10.0, then, at best, the in vivo selectivity might be as high as the in vitro selectivity.
Animals↗
In vitro and in vivo matrix metalloproteinase production by pancreatic cancer cells and by distant organs.
BACKGROUND: Matrix metalloproteinases (MMP), especially MMP-2 and MMP-9, are thought to play major roles in pancreatic cancer growth and metastasis. MMP inhibition prolongs survival in a murine model of human pancreatic cancer. To establish an in vitro model of human pancreatic cancer, we sought to determine in vitro MMP production by two human pancreatic cancer cell lines, compare in vitro to in vivo production, and, if different, determine if in vitro MMP production could be made to mimic in vivo production by altering the in vitro environment of the cell lines. We also sought to document that in vivo production of MMP was unique to orthotopic pancreatic cancer cells. METHODS: HPAC and PANC-1 cell lines were grown in vitro and in a murine model of orthotopic pancreatic cancer. MMP-2 and MMP-9 production by the cell lines was determined. Noting differences between in vitro and in vivo production, the cell lines were stimulated in vitro with PMA. Orthotopic pancreatic tumor MMP production was compared to splenic, hepatic, and renal production of MMP. MMP-2 and MMP-9 production was sought in both active and latent forms utilizing gelatin zymography. Results were confirmed by Western blot. Densitometry allowed comparisons: scale 0 (none) to ++++ (most). RESULTS: In vitro, HPAC and PANC-1 produce modest amounts of MMP-2 and MMP-9, mostly in latent form. In vivo, both cell lines produce more MMP, particularly in the active forms. In vitro stimulation of the cell lines with the PMA increased MMP production, particularly of active MMP-2 and latent MMP-9, resulting in a profile of MMP production similar to that seen in vivo. Hepatic, renal and splenic MMP levels, especially active forms, were very low relative to the tumors. CONCLUSIONS: Pancreatic cancer cell lines produce less MMP, especially active form, in vitro than in vivo. With in vitro stimulation, the MMP profile of cell lines mimic those of tumors in vivo, suggesting that cancer cells are stimulated in vivo. Both in vitro and in vivo, the poorly differentiated cell line PANC-1 is associated with broadened and enhanced MMP activity. Healthy solid organs produce little MMP-2 or MMP-9, most of which is latent form.
Animals↗
In vitro fertilization-conceived offspring exhibit altered Long Interspersed Nuclear Elements-1 retrotransposition dynamics associated with long-term disease risks.
BACKGROUND: In vitro fertilization has transformed reproductive medicine, yet offspring conceived through in vitro fertilization display elevated risks for diverse long-term health conditions, with underlying mechanisms unclear. Long Interspersed Nuclear Elements-1, a mobile genetic element responsive to environmental stress, represents a potential mediator. OBJECTIVE: This study aimed to test the hypothesis that in vitro fertilization procedures may act as an embryonic stressor that alters Long Interspersed Nuclear Elements-1 dynamics, potentially contributing to genomic instability associated with long-term disease susceptibility. STUDY DESIGN: Umbilical cord blood or peripheral blood from 33 in vitro fertilization and 42 naturally conceived neonates were collected for whole-genome sequencing. Total Long Interspersed Nuclear Elements-1 proportion in individual genome was counted with Bowtie2 software. De novo Long Interspersed Nuclear Elements-1 insertion and Long Interspersed Nuclear Elements-1 deletion were detected with Mobile Element Locator Tool. Three parent-matched in vitro fertilization-naturally conceived sibling pairs were included to control for genetic background. Disease association analysis was performed for genes within 500 kb of differential Long Interspersed Nuclear Elements-1 sites in The Database for Annotation, Visualization and Integrated Discovery (DAVID). Statistical analysis was performed using the R language. RESULTS: In vitro fertilization offspring demonstrate elevated global Long Interspersed Nuclear Elements-1 content compared to naturally conceived controls (P=.04). This finding was corroborated in 3 sibling pairs from identical genetic backgrounds, where in vitro fertilization-conceived children consistently exhibited higher Long Interspersed Nuclear Elements-1 levels than their naturally conceived siblings. Eleven genomic loci with differential Long Interspersed Nuclear Elements-1 insertion frequencies and 14 loci with differential Long Interspersed Nuclear Elements-1 deletion frequencies between in vitro fertilization offspring and naturally conceived controls were identified. Notably, these differential Long Interspersed Nuclear Elements-1 sites demonstrated significant enrichment near genes implicated in metabolic, cardiovascular, neuropsychiatric, and neoplastic diseases, conditions associated with in vitro fertilization conception. CONCLUSION: These findings provide preliminary evidence that in vitro fertilization conception is associated with increased Long Interspersed Nuclear Elements-1 content and altered genomic distribution of Long Interspersed Nuclear Elements-1 elements. The proximity of these differential Long Interspersed Nuclear Elements-1 sites to disease-associated genes suggests a plausible genomic mechanism linking in vitro fertilization-associated embryonic stress to elevated disease risk. This work provides valuable molecular insights that may inform the ongoing discussion about assisted reproductive technology safety and suggests that continued attention to genomic integrity in in vitro fertilization-conceived individuals would be beneficial.
Humans↗
Effects of cryopreservation on glucose metabolism and survival of bovine morulae and blastocysts derived in vitro or in vivo.
Bovine morulae and blastocysts were either produced in vitro through maturation, fertilization and culture of immature oocytes recovered from slaughterhouse-derived ovaries, collected in vivo or obtained after 24 h in vitro culture of in vivo collected embryos. The morulae and blastocysts were classified into four categories of embryo quality and two stages of embryonic development. Embryos were frozen by a controlled freezing method using 10% glycerol as a cryoprotectant. The ability of individual embryos to withstand freeze/thawing was measured immediately before and after cryopreservation by changes in CO2 production from (U-14C)glucose during a 2 h incubation period in a non-invasive closed system immediately before and after cryopreservation. Post-thaw survival was assessed by development in vitro during a 48 h culture period. Survival rates and oxidative metabolism after freeze/thawing were similar in embryos of the two developmental stages. However, after freeze/thawing, the rate of CO2 production of in vitro produced embryos was reduced to one half of their pre-freeze levels and was associated with poor survival rates. In vivo collected embryos had a significantly better tolerance to freezing and higher survival rates. However, when in vivo embryos were exposed to in vitro culture conditions, the rates of CO2 production and survival were significantly reduced. Pre-freeze embryo quality affected post-thaw in vitro development and metabolic activity markedly in embryos produced in vitro or pre-exposed to in vitro culture conditions. While there was no relationship between pre-freeze levels of CO2 production and post-thaw in vitro embryo development, all embryos which developed in vitro after freezing/thawing retained at least 58% of the pre-freeze levels of CO2 production regardless of their origin. Results of the present study indicate that embryos produced in vitro or pre-exposed to in vitro culture conditions are more sensitive to cryo-injury. This sensitivity is affected by embryo quality and is similarly reflected at the biochemical level. Determination of oxidative metabolism offers a feasibility for selection of viable morulae/blastocysts after freezing/thawing.
Animals↗
State of the art in in-vitro oocyte maturation.
PURPOSE OF REVIEW: The recovery of immature oocytes followed by in-vitro maturation (IVM) and in-vitro fertilization is an attractive alternative to conventional in-vitro fertilization treatment in which controlled ovarian stimulation with gonadotropins is used to increase the number of available oocytes and embryos. Significant progress has been made to improve pregnancy and implantation rates from in-vitro matured oocytes. This review summarizes current knowledge and achievements in human oocyte in-vitro maturation for clinical application, and will highlight recent advances reported in in-vitro maturation treatment. RECENT FINDINGS: It has been demonstrated that priming of ovarian immature oocytes with follicle-stimulating hormone or human chorionic gonadotropin prior to immature oocyte retrieval improves oocyte maturation rates and embryo quality as well as pregnancy rates in infertile women with polycystic ovaries or polycystic ovary syndrome. The size of follicles may be important for the subsequent embryonic development, but the developmental competence of oocytes derived from the small antral follicles is not adversely affected by the presence of a dominant follicle. However oocyte maturation in vitro is profoundly affected by culture conditions. Currently more than 300 healthy infants have been born following immature oocyte retrieval and in-vitro maturation. In general, the clinical pregnancy and implantation rates have reached 30-35% and 10-15% respectively in infertile women with polycystic ovaries or polycystic ovary syndrome. SUMMARY: In-vitro maturation treatment can now be offered as a successful option to infertile women with polycystic ovaries or polycystic ovary syndrome. It is possible to combine natural cycle in-vitro fertilization with immature oocyte retrieval followed by in-vitro maturation, and thus offer women with various causes of infertility reasonable pregnancy and implantation rates without recourse to ovarian stimulation. Further research remains to be done to address the mechanism of oocyte maturation in order to refine culture conditions and improve the implantation rate of oocytes matured in vitro.
Female↗
Refining in vitro digestibility assays: fractionation of digestible and indigestible peptides.
Typically, in vitro methods used for estimating the amount of ileal digestible AA do not exhaustively digest samples, and arbitrary methods for separating digestible from indigestible protein are used. This may lead to over- or underestimation of digestibility coefficients. A method that exhaustively digests proteins using pepsin and pancreatin was developed, and the first objective of this research was to confirm that exhaustive digestion was indeed appropriate and to determine the fractionation method for separating digestible from indigestible proteins. For this, three homoarginine-labeled animal proteins were prepared. Samples were subsequently digested in vivo and in vitro to determine which fraction should be considered indigestible, and in vitro followed by in vivo to determine whether the extent of digestion in vivo was improved by predigestion. In vivo, soluble but unabsorbed peptides were smaller than 1 kDa, suggesting that the size of soluble peptides is not what prevents their absorption. Thus, all in vitro-soluble proteins should be considered digestible. In vitro, 88 +/- 3% of the soluble peptides were smaller than 1 kDa, with the remainder between 1 and 5 kDa, suggesting that in vitro digestion is less complete. Predigested samples were digested in vivo to the same size distribution as the nonpredigested samples. The second objective was to test whether in vitro digestibility assays based on these principles equaled in vivo digestibility. For this, digestibility data for 25 animal proteins were compared. Results showed a lack of correlation between lysine digestibility coefficients; however, across samples, the extent of digestion did not differ for lysine (P = 0.71), threonine (P = 0.26), methionine (P = 0.18), or valine (P = 0.66), whereas in vitro digestibility coefficients were lower for (the less water-soluble) histidine (P = 0.05), isoleucine (P < 0.01), leucine (P < 0.01), and phenylalanine (P = 0.05). In conclusion, in vitro digestibility assays should exhaustively digest proteins to mimic in vivo digestibility. All in vitro-soluble peptides could be considered digestible, because in vivo, no large soluble peptides were observed whose size prevented them from being absorbed. However, an in vitro assay based on these principles lacked precision for highly water-soluble AA, and underestimated digestibility for other AA. Better solubilization of the digesta and more replicates may improve the in vitro assay further.
Amino Acids↗
Differences in IL-10 and IL-12 production patterns and differences in the effects of acute ethanol treatment on macrophages in vivo and in vitro.
Several recent studies have documented that signaling can be fundamentally different in vivo and in vitro. However, studies of signaling and cytokine production by macrophages are often conducted in vitro, without confirmation in vivo. In addition, the direct effects of drugs and chemicals, including ethanol, on these processes are also often investigated in vitro. The purpose of the present study was to compare production of interleukin-6 (IL-6), IL-10, and IL-12 by macrophages in response to two different ligands for toll-like receptors and the effects of acute ethanol exposure on these responses in vivo and in vitro. The macrophage-like cell line RAW 264.7 is also widely used in cytokine and signaling studies, so these cells were also evaluated in this study. The results indicate that IL-6 production and the effects of Ethanol on IL-6 were similar in vivo and in vitro. In contrast, IL-10 was produced to a much greater extent in vitro than in vivo, and IL-12 was often undetectable in vitro even though it was produced at greater concentrations than IL-10 in vivo. To determine the role of altered secretion of preformed IL-10 as compared to new synthesis, cells were treated in vitro with protein and mRNA synthesis inhibitors. The results suggest that preformed IL-10 is released in vivo, but almost all IL-10 secreted in vitro is newly synthesized. Ethanol suppressed IL-12 and enhanced or had no effect on IL-10 production in vivo, whereas it decreased IL-10 production in vitro. These effects were similar at different times and using different concentrations of toll-like receptor ligands. In general, RAW 264.7 cells responded similarly to peritoneal macrophages in vitro. This suggests that results for cytokine studies and probably signaling studies as well that are conducted in vitro should be interpreted with caution and confirmed in vivo, particularly if they involve IL-10 and IL-12.
Animals↗
Use of in vitro and in vivo data in the design, development, and quality control of sustained-release decongestant dosage forms.
STUDY OBJECTIVES: To investigate the use of in vitro and in vivo data in the development of a sustained-release, carbomer-based dosage form (Entex LA tablets); and to compare the in vitro dissolution of pseudoephedrine from a sustained-release, hydroxypropylcellulose-based dosage form (Entex PSE tablets) and four branded competitors with different sustained-release matrixes. DESIGN: Entex LA: In vitro testing by rotating bottle method and in vivo testing as double-blind, randomized, crossover, 24-hour study. Entex PSE and four competitors: in vitro testing by paddle method. SETTING: A pharmaceutical research and development facility. PATIENTS: Fifteen health, adult, volunteer Caucasian men between ages 18 and 40 years. MAIN RESULTS: Three formulations of Entex LA, varying in carbomer content by 3-5%, were studied. As carbomer content increased, in vitro dissolution rate directionally decreased. Plasma concentrations of active ingredients guaifenesin and phenylpropanolamine were also slightly although directionally decreased. The in vitro method was sensitive to small changes in carbomer content. Larger changes in carbomer content would be required to establish an in vitro-in vivo correlation. The in vitro comparison of Entex PSE and four similar branded products showed important differences in dissolution profiles. The mean cumulative release of pseudoephedrine from Entex PSE was 39% at 1.5 hours, 62% at 4.0 hours, and 80% at 8.0 hours. The other products released pseudoephedrine more rapidly, with the two fastest-dissolving products releasing 61-62% in the first 1.5 hours. CONCLUSIONS: Sustained-release, polymer-based dosage forms such as Entex LA and Entex PSE can be complex and pose special challenges in design, development, and reformulation. For Entex LA, changes in polymer concentration and dye system influenced the in vitro (dissolution) performance. In vivo (plasma) data helped establish a defined range in which carbomer concentration could be varied to achieve the best manufacturing performance without affecting product performance. For Entex PSE and four branded competitor products, the cumulative in vitro release (dissolution) of pseudoephedrine varied widely. Release from Entex PSE was more consistent and more gradual than that from some of the comparison products. Because the absorption rate of the active ingredients pseudoephedrine and gauifenesin is governed by the dissolution rate, the observed differences suggest that the products tested may differ in biologic performance. Although in vitro dissolution data may not necessarily correlate with in vivo differences in clinical safety or efficacy, the potential for unexpected product performance may be signaled by inconsistent in vitro dissolution characteristics, especially for sustained-release dosage forms.
Adolescent↗
Developmental competence of in vivo and in vitro matured porcine oocytes after subzonal sperm injection.
In vivo and in vitro matured porcine oocytes were fertilized by subzonal sperm injection (SUZI), and their subsequent development in vitro was examined to determine whether ooplasmic incompetence is the major cause of limited developmental ability of in vitro matured/ fertilized porcine oocytes (Experiment 1). There was no significant difference in rates of fertilization (61% vs. 70%), monospermy (37% vs. 45%), and male pronuclear formation (77% vs. 61%) between in vivo and in vitro matured oocytes. Blastocyst formation rate was significantly lower for in vitro matured oocytes (11% vs. 42%; P < 0.001). Forty-six percent of in vivo matured oocytes cleaved to the 2-4 cell stage by 24 hr in culture after SUZI, compared with 3% of in vitro matured oocytes (P < 0.01). In experiment 2, in vitro development of in vitro matured oocytes with evenly and unevenly granulated cytoplasm were compared after SUZI to examine whether developmentally competent in vitro matured oocytes can be identified on the basis of morphological appearance. Most of the blastocysts obtained developed from oocytes with unevenly granulated cytoplasm (7/56 vs. 1/45; P > 0.05). Experiment 3 revealed that the proportion of oocytes with evenly granulated cytoplasm was originally low (11%) in the population of oocytes used for in vitro maturation, and it increased approximately 3-fold (36%; P < 0.001) after maturation. These results suggest that ooplasmic incompetence in porcine in vitro matured oocytes is the major cause of their limited developmental competence. Cytoplasmic maturation measured by male pronucleus formation does not directly reflect developmental competence of the oocytes. It was also shown that evenness of granulation of the cytoplasm is not a useful morphological indicator of developmental competence.
Animals↗
Are cytokine gene polymorphisms related to in vitro cytokine production profiles?
Currently, there is much interest in the genetic basis for diseases or disease manifestations and, in particular, in whether they are related to cytokine gene polymorphisms. It has become accepted to denote such single-nucleotide polymorphisms of cytokine genes by their presumed association with high or low in vitro cytokine production. In this article, we analyze the relationship between cytokine gene polymorphisms and in vitro tumor necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma), and interleukin (IL)-10 and IL-13 production, both in liver transplant recipients and in healthy volunteers. The evaluated cytokine gene polymorphisms involved TNF-A-308; TNF-d3; IFN-G+874; IL-10-1082, -819, and -592; and IL-13+2043, and -1055. For healthy volunteers, we observed a relationship between polymorphisms of TNF-d3 and IL-10-1082 with in vitro production of TNFalpha and IL-10, respectively, whereas no significant associations were found for the other tested cytokine gene polymorphisms. For liver transplant recipients, no significant relationship could be established between any of the cytokine gene polymorphisms and in vitro production of corresponding cytokines. Also, we reviewed the literature for the association between cytokine gene polymorphisms and in vitro cytokine production in various patient groups and healthy volunteers. We found that the cellular sources, from which the cytokines were released into the culture supernatant, were different between studies. They were either whole blood, isolated monocytes, or peripheral blood mononuclear cells (PBMC). Also, the in vitro incubation protocol varied to a great extent between studies. This applied for the used in vitro stimulant, the concentration of a particular stimulant, and the length of the incubation period. Moreover, the study populations were either healthy individuals or very diverse patient groups. Therefore, it was impossible to evaluate whether in vitro cytokine production profiles really can be deduced from a particular cytokine gene polymorphism. Given the inconclusive findings, we propose to set up a multicenter workshop in which the relationship between certain cytokine gene polymorphisms and in vitro cytokine production is analyzed, using an identical in vitro cell culture system and study population. Furthermore, we suggest that cytokine gene polymorphisms be described by their localization within the gene or gene-promoter, rather than by their presumed in vitro cytokine production profile, to properly evaluate the relationship between cytokine gene polymorphisms and disease manifestations.
Adult↗