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PubMed2003

Legal considerations for cryopreservation of sperm and embryos.

OBJECTIVE: To summarize the case law for cryopreservation of sperm and embryos and make recommendations for desirable characteristics that should be included in a cryopreservation clinic's disposition agreements. DESIGN: A literature review of case law and legal review articles was performed. SETTING: Academic research center. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): None. RESULT(S): Two court cases involving cryopreserved semen and five cases involving cryopreserved embryos are reviewed. CONCLUSION(S): The state of the law surrounding cryopreservation is recent and unsettled. Disposition agreements can provide cryopreservation clinics some degree of certainty in this unsettled area of law. Those drafting cryopreservation disposition agreements are assisted by lessons learned from cryopreservation cases and insight gained from law review articles. It is evident that cryopreservation agreements will be most successful and most likely to be enforced if they are unambiguous, consistent with public policy, and include: a duration provision, each individual's contact information, the individual's provisions for use of their gametes in case of death, and responsibilities of cryopreservation clinic and individuals. Individuals must enter into agreements with a true understanding of the contained provisions.

Contracts↗
PubMed2004

Ethical aspects of cryobiology: responsible applications in biomedicine and in clinical practice.

This paper focuses on ethical issues in applications of cryobiology to humans, more particularly in the field of human reproduction and cryosurgery. The paper also provides essential ingredients for the interface of bioethics and cryobiology. For instance, since the 1970s bioethicists have developed four principles to guide the moral evaluation of the 'new medicine.' These are: respect for persons, non-maleficence, beneficence, and justice. In the field of human reproduction major progress was made by the cryopreservation of reproductive material. Still, ethical issues arise whenever partnerships cease to exist (death) or deteriorate (divorce), and decisions have to be made about the disposition of frozen gametes and embryos. Policy-making becomes, then, a prime concern. Examples of regulation in the United States of America, in the United Kingdom, and across Europe are being offered. Cryosurgery remains a field where cryobiologists struggle in their quest for an optimal technique, thus illustrating the need for assessment of safety, efficacy, and benefit to patients. Increasingly, cryobiologists have been joining in the ethical reflection on the use of cryo-technologies. They may further do so by perfecting their ability to identify ethical aspects, by analysing the norms and values at stake, by learning the skill of making the appropriate choices, and by showing their willingness to justify the choices made be it in the inner circle of pairs or publicly.

Bioethical Issues↗
PubMed2000

A follow-up study with oocyte donors exploring their experiences, knowledge, and attitudes about the use of their oocytes and the outcome of the donation.

UNLABELLED: OBJECTIVE To learn what information oocyte donors were given and wanted to have about the use of their oocytes and the outcome of the donation. DESIGN: In-depth interviews. SETTING: Participants recruited through IVF clinics, matching agency, the Internet, word of mouth, and newspaper ads. PARTICIPANT(S): Thirty-three former oocyte donors and six women preparing to donate. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): None. RESULT(S): Thirty-three former donors completed 66 donation cycles; 48 donation cycles were anonymous. Only 41% (16 of 39) of all participants were comfortable giving the recipient couple complete dispositional authority over the resulting embryos; the remainder wanted some control. One quarter did not want embryos used for research. Fifty-four percent (21 of 39) thought donation of excess embryos to another couple was acceptable, but one third wanted to be informed. Of the 25 anonymous donors, 6 learned the outcome of the donation; 14 others wanted to know. All hoped the donation was successful. CONCLUSION(S): Because oocyte donors' need for information varies, clinics should consider being more flexible in their disclosure policies. Disclosure about the possible uses of donor oocytes or embryos should be mandatory. Findings have implications for the informed consent and counseling processes.

Adult↗
PubMed1980

Metabolic disposition of glucose carbon by sensory ganglia of 15-day-old chicken embryos, with new dynamic models of carbohydrate metabolism.

Dorsal root ganglia from the lumbar region of chicken embryos 14--16 days old were incubated at 37 degrees in modified McCoy's culture medium containing [1-(14)C]glucose or [6-(14)C]glucose and sometimes 32Pi. A volume of 10 microliters of medium was used for four ganglia (dry weight approx. 90 micrograms). The output of 14CO2 was measured continuously. Incorporation of 14C into tissue constituents and into products released to the medium was measured after incubation for 3--17 h. Among nine radioactive components resolved in paper chromatograms of ganglion constituents, the most 14C was found in lipids and on materials remaining at the origin. Only the lipids and the origin materials were detectably labeled by 32Pi. Only relatively small amounts of 14C from [6-(14)]glucose were found in chromatographic regions that should contain intermediates of the pentose cycle. At least five labeled products were released to the bathing medium. Among these, the largest amount of 14C was found in lactic acid. A second component released, possibly alanine, also received considerable 14C. No 32P was detected in products in the medium. The rate of glucose uptake remained relatively constant as the concentration of glucose in the medium declined nearly 10-fold during prolonged experiments. Two new dynamic models of glucose metabolism successfully explained the time courses and magnitudes of previously reported 14CO2 outputs from [1-(14)C]glucose, [2-(14)C]-glucose, and [6-(14)C]glucose. These models are based on the assumption that glucose carbon was delayed on its way to CO2 in a pool of intermediates early in the metabolic chain and in a second pool either in or before the citric acid cycle. Both models assigned the pentose cycle to one cellular compartment, and incorporation into slowly-turning-over substances to another cellular compartment. According to both models, not more than one-half of the glyceraldehyde-P produced by the pentose cycle was converted to fructose-6-P, while at least half of this and other fructose-6-P from the pentose cycle was recycled into it. These conclusions differ from those from a previous model, which assumed that glucose carbon was delayed in a pool related to the pentose cycle; that model had suggested full recycling of both the glyceraldehyde-P and the fructose-6-P produced by the pentose cycle. In the citric acid cycle the efficiency of recycling was over 80%, according to all models. All models demonstrated the large differences that can occur in the metabolic handling of carbons 1, 2, and 6 of glucose. These differences need consideration in any description of the partitioning of glucose metabolism between alternative pathways.

Animals↗
PubMedDate not supplied

[The legal question of the obtention of human stem cells for biomedical research. Legislation policy considerations].

The future Law on Biomedical Research, whose draft bill has been approved by the Council of Ministers and that will soon begin its parliamentary process of approval, will regulate, among other matters, the research with embryos. Likewise, it will make a pronouncement on the so-called therapeutic cloning. This report makes a detailed analysis of different matters that must be borne in mind by the legislator in order to face the process of evaluation and approval of said Law in relation with the aforementioned matters. It makes a special analysis of the legal texts of an international nature to which Spain is unavoidably subjected to, in such a way that the legislative text that will finally be approved is not contrary to the dispositions that are within such.

Biomedical Research↗
PubMed1987

Hydrocortisone-induced embryotoxicity and embryonic drug disposition in H-2 congenic mice.

Congenic mouse strains C57BL/10Sn (B10) and B10.A/SgSn (B10A), genetically different only in the region of the H-2 complex, were compared for sensitivity to hydrocortisone-induced embryotoxicity and embryonic drug disposition. Pregnant B10A mice dosed intramuscularly with 0, 100, 150, and 200 mg hydrocortisone/kg body weight and B10 mice injected with 0, 200, 400, 600, and 800 mg/kg, both on gestational day (GD) 12, were evaluated on GD 18 for reproductive toxicity. The induction of cleft palate demonstrated a linear dose-response by probit analysis: The ED50s were 143.6 mg/kg and 512.0 mg/kg for B10A and B10 mice, respectively. Comparison of fetal weight revealed statistically significant intrauterine growth retardation at all doses administered to B10 mice. However, growth retardation was shown only in the high-dose group in the B10A strain. Embryonic drug concentrations were evaluated by administration of hydrocortisone to mice of both strains on GD 12, at the ED50 for cleft palate production in the B10A strain, with 3H-hydrocortisone (5 muci/mouse) added as a tracer. Maternal serum and embryos were analyzed for steroid content. Disposition and pharmacokinetics of 3H-hydrocortisone were similar in both strains, with the majority of serum radioactivity recovered as hydrocortisone and the major radioactive peak in embryos comigrating with cortisone. The results indicate that H-2 haplotype does not influence hydrocortisone-induced cleft palate sensitivity through an alteration of embryonic drug exposure.

Animals↗
PubMed1981

Cephalic flexure formation in the chick embryo.

The cephalic flexure, found in all vertebrate brains, is a ventrally directed bend through the mesencephalon, and a ventral bulging and elongation of the prosencephalon. Most sources say the cephalic flexure is caused by differential growth. We have measured the changing angle of flexure through time and find that flexure occurs between chick embryo stages 10 to 15. We measured, during these stages, the lengths, thicknesses, and volumes of the floor and roof of the mesencephalon and of the prosencephalon. As expected, during flexure the mesencephalic roof elongates much more than the floor. Both roof and floor increase in thickness, and mesencephalic roof volume increases twice as much as floor volume. However, prosencephalon, which does not bend, also has differential growth between roof and floor, but the growth is taken up in complex changes of shape other than flexure. There are sufficient numbers of mitoses in the brain to account for the observed tissue growth, assuming accompanying cell enlargement. We deleted brain parts adjacent to the mesencephalon before flexure and the mesencephalon bent, so migration of cells from or to these adjacent parts to contribute to the differential growth of the mesencephalon is unlikely. We reduced cerebrospinal fluid pressure during flexure by explanting heads to the chorioallantoic membrane, or into New cultures. The mesencephalon of explanted heads bends, but the prosencephalon fails to elongate. We conclude that differential growth may be necessary for mesencephalic flexure in the chick embryo, but other factors that decide the disposition of the products of growth in space must determine the shape.

Animals↗
PubMed1995

Xenopus laevis: a model system for the study of embryonic retinoid metabolism. III. Isomerization and metabolism of all-trans-retinoic acid and 9-cis-retinoic acid and their dysmorphogenic effects in embryos during neurulation.

These investigations provide data pertaining to the metabolism and disposition of exogenous 9-cis-retinoic acid and all-trans-retinoic acid during neurulation in Xenopus embryos. Each isomer elicited malformations of the heart, eye, and brain, but approximately 2-fold higher concentrations of all-trans-retinoic acid than 9-cis-retinoic acid were required to produce qualitatively and quantitatively similar dysmorphogenic effects. The dymorphogenic effects of all-trans-retinoic acid could not be attributed to the isomerization of all-trans-retinoic acid to 9-cis-retinoic acid. Evidence is provided that all-trans-retinoic acid and 9-cis-retinoic acid are both direct-acting dysmorphogens. After Xenopus embryos were exposed to all-trans-retinoic acid, elevated levels of 4-oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid, all-trans-retinoyl-beta-glucuronide, and 13-cis-retinoic acid were detected in the embryos, whereas embryonic levels of 9-cis-retinoic acid were actually slightly lower than endogenous levels during early neurulation. After embryos were exposed to 9-cis-retinoic acid during neurulation, elevated levels of 4-oxo metabolites, glucuronides and 9,13-di-cis-retinoic acid were observed in the embryos. At equivalent concentrations, 4-oxo-13-cis-retinoic acid and 13-cis-retinoic acid elicited fewer severe multiple malformations than all-trans isomers 9,13-di-cis isomers, or 9-cis isomers. The dysmorphogenic effect of 9,13-di-cis-retinoic acid may be caused by its isomerization to 9-cis-retinoic acid. All-trans retinoyl-beta-glucuronide was only marginally teratogenic at the highest concentrations tested.

Abnormalities, Drug-Induced↗
PubMed1988

Radial arrangement of clonally related cells in the chicken optic tectum: lineage analysis with a recombinant retrovirus.

A recently described method for lineage analysis in rodents uses a recombinant retrovirus to insert a foreign gene into the genome of a precursor cell; the gene's product is later detected histochemically in the infected cell's progeny. We have adapted this method for use in chicken embryos and used it to study the disposition of clonally related cells in the optic tectum. We report that descendants of a single precursor form narrow, radially oriented columns that span the thickness of the developing tectum. Analysis of embryos injected with virus at different stages suggests a developmental scheme in which early-born progeny are displaced laterally, late-born progeny are displaced radially, and cell mixing is limited. Many clones remain radially arrayed as laminae form and contribute neurons of diverse types of several laminae. In light of previous studies showing physiological relationships among vertically arrayed neurons, our results suggest that neurons descended from a single precursor are richly interconnected and functionally related.

Animals↗
PubMed1985

[Psychological studies of sterile couples within the scope of an in vitro fertilization program].

The results of a psychological individual and process diagnostic programme at sterility patients undergoing an in-vitro-fertilization and embryo transfer are demonstrated. The women showed an optimistic emotional disposition and negation of conflict or trouble experience during their stay in the hospital. From the day of laparoscopy till embryo transfer a short labilization sets in, i.e. a slight deterioration of the emotional disposition as an expression of a phase of stress compensation. Questions of motivation, partner constellation and special personality characteristics are discussed.

Adaptation, Psychological↗
PubMed1985

Inhibition of cell spreading on the band of extracellular fibres in early chick and quail embryos.

The ventral surface of the upper layer shows a band of extracellular fibrils around the anterior and lateral border of the area pellucida during gastrulation of the chick embryo. Using scanning electron microscopy, we found that this disposition is correlated with the motility of the middle-layer cells of gastrulating chick and quail embryos. Outside the fibrous band, single middle-layer cells and a sheet of mesoblast cells were spread out and possessed lamellae. Single cells on the fibrous band did not form lamellae. The same cell behaviour was obtained with the explants of deep layer on the fibrous band. The fibrous band is assumed to operate as a barrier that inhibits cell motility during gastrulation.

Animals↗
PubMed2002

Posthumous reproduction: current and future status.

Posthumous reproduction became possible with the technologies of sperm cryopreservation and ART. The legal and social status of children born as a result of these technologies continue to evolve. The proper disposition of unwanted stored gametes and embryos remains unknown. Physicians are increasingly asked to make quick judgments on posthumous gamete retrieval. The procedures for gamete harvest are technically simple; however, one must carefully select cases with definitive prior intent to have children. There is a need for standardized legal protocols to protect the physician and the patient. The physician must use sound judgment and comply with accepted standards, when present, before performing any service for posthumous sperm retrieval and reproduction.

Female↗
PubMed1998

Prenatal exposure to amphetamines. Risks and adverse outcomes in pregnancy.

Based on findings in humans and the confirmation of prenatal exposures in animals, amphetamines and methamphetamines increase the risk of an adverse outcome when abused during pregnancy. Clefting, cardiac anomalies, and fetal growth reduction deficits that have been seen in infants exposed to amphetamines during pregnancy have all been reproduced in animal studies involving prenatal exposures to amphetamines. The differential effects of amphetamines between genetic strains of mice and between species demonstrate that pharmacokinetics and the genetic disposition of the mother and developing embryo can have an enormous influence on enhancing or reducing these potential risks. The effects of prenatal exposure to amphetamines in producing altered behavior in humans appear less compelling when one considers other confounding variables of human environment, genetics, and polydrug abuse. In view of the animal data concerning altered behavior and learning tasks in comparison with learning deficits observed in humans, the influence of the confounding variables in humans may serve to increase the sensitivity of the developing embryo/fetus to prenatal exposure to amphetamines. These factors and others may predispose the developing conceptus to the damaging effects of amphetamines by actually lowering the threshold of susceptibility at the sites where damage occurs. Knowledge of the effects of prenatal exposure of the fetus and the mother to designer amphetamines is lacking. Based on the few studies in which designer drugs have been examined in animal models, more questions are raised than answered. Possible reasons why no malformations or significant fetal effects were found in the study by St. Omer include the genetic strain of rat used, the conservative exposure profile, or the fact that the placenta metabolized MDMA before reaching the embryo. These questions underscore the need for further investigations concerning the prenatal exposure effects of designer compounds and the effects of amphetamine and methamphetamine in general.

Amphetamines↗
PubMed1982

Acid and alkaline phosphatase activity in migrating primordial germ cells of the early chick embryo.

Little information is available concerning enzyme activity in primordial germ cells (PGCs) of the early chick embryo. The present study is designed to examine the disposition of alkaline and acid phosphatase activity in the PGCs during their migration into the developing gonads of the early chick embryo. White Leghorn chick embryos were sacrificed at daily intervals from 1 to 6 days of incubation. Following sacrifice the embryos were fixed, dehydrated, and embedded in glycol methacrylate (GMA). Alkaline and acid phosphatases were demonstrated by the simultaneous diazo-coupling method. The embryonic tissues at the different ages were examined for PGCs and the histochemical reactions for alkaline and acid phosphatases in these cells evaluated. Acid phosphatase activity did not appear within PGCs until 3 days of incubation, and then in only a few PGCs in the active phase of their migration in the dorsal mesentery, suggesting that there is no large wave of degeneration of these cells during migration. Alkaline phosphatase activity was observed as early as 2 days of incubation in PGCs during the passive phase of their migration in extraembryonic blood vessels. Alkaline phosphatase-positive PGCs in the active phase of migration were also found in the dorsal mesentery; however, the cellular localization of this enzyme differed from that observed in the passively migrating PCGs, indicating that there are alterations in the metabolic activities of these cells during the active and passive phases of migration.

Acid Phosphatase↗
PubMed2000

Development of a physiologically based pharmacokinetic model of 2-methoxyethanol and 2-methoxyacetic acid disposition in pregnant rats.

An accurate description of developing embryos' exposure to a xenobiotic is a desirable component of mechanism-based risk assessments for humans exposed to potential developmental toxicants during pregnancy. 2-Methoxyethanol (2-ME), a solvent used in the manufacture of semiconductors, is embryotoxic and teratogenic in all species tested including nonhuman primates. 2-Methoxyacetic acid (2-MAA) is the primary metabolite of 2-ME and the proximate embryotoxic agent. The objective of the work described here was to adapt an existing physiologically based pharmacokinetic (PBPK) model for 2-ME and 2-MAA kinetics during midorganogenesis in mice to rats on gestation days (GD) 13 and 15. Blood and tissue data were analyzed using the extrapolated PBPK model that was modified to simulate 2-ME and 2-MAA kinetics in maternal plasma and total embryo tissues in pregnant rats. The original mouse model was simplified by combining the embryos and placenta with the richly perfused tissue compartment. The model includes a description of the growth of the developing embryo and changes in the physiology of the dam during pregnancy. Biotransformation pathways of 2-ME to either ethylene glycol (EG) or to 2-MAA were described as first-order processes based on the data collected from rats by Green et al., (Occup. Hyg. 2, 67-75, 1996). Tissue partition coefficients (PCs) for 2-ME and 2-MAA were determined for a variety of maternal tissues and the embryos. Model simulations closely reflected the biological measurement of 2-ME and 2-MAA concentrations in blood and embryo tissue following gavage or iv administration of 2-ME or 2-MAA. The PBPK model for rats as described here is well suited for extrapolation to pregnant women and for assessment of 2-MAA dosimetry under various conditions of possible human exposure to 2-ME.

Acetates↗
PubMed1998

Late specification of Veg1 lineages to endodermal fate in the sea urchin embryo.

Single blastomeres of the sixth-cleavage veg1 and veg2 tiers of Strongylocentrotus purpuratus embryos were labeled with DiI lineage tracer, and the disposition of the progeny was followed through the blastula and gastrula stages in order to determine their respective endodermal and ectodermal contributions. In the endoderm of postgastrula embryos, veg1-derived cells constituted nearly all of the prospective hindgut and about half of the prospective midgut, while veg2-derived cells made up the prospective foregut and half the midgut. Oral veg1 clones consistently contributed more cells to endoderm than aboral veg1 clones. Oral veg1 clones extended along the archenteron up to the foregut region, while aboral veg1 clones contributed only small numbers of hindgut cells but large patches of ectoderm cells that extended out to the prospective larval vertex. The oral/aboral asymmetry in veg1 allocations was also demonstrated using chimeric embryos, the animal halves of which were labeled with a rhodamine-dextran. Lineages expressing the vegetal plate marker Endo16 were more precisely determined by combining lineage tracer injection with whole-mount in situ hybridization. Endo16 expression was found in all cells that are going to participate in gastrulation. Recruitment of new cells to the Endo16 domain occurs in advance of the actual invagination of those cells. During the blastula stages Endo16 expression expands radially until all cells in the veg2 lineages express this gene. The first phase of gastrulation, including the normal buckling of the vegetal plate and primary invagination of the archenteron, involves only the Endo16-expressing cells of the veg2 lineages. As the archenteron begins to elongate, marking the onset of the second phase of gastrulation, there is an asymmetric expansion of Endo16 into the veg1-derived cells that will contribute to the hindgut and midgut in accordance with lineage tracing observations. The results indicate a relatively late specification of veg1-derived cells, resulting in late recruitment to the periphery of the vegetal plate territory as gastrulation proceeds. Differential recruitment of veg1-derived cells on the oral side of the embryo introduces an oral bias to gastrulation by disproportionately increasing the number of cells on the oral side that are competent to participate in gastrulation.

Animals↗
PubMed2002

High rates of embryonic loss, yet high incidence of multiple births in human ART: is this paradoxical?

Humans have low natural fecundity, as the probability of establishing a viable conception in any one menstrual cycle is 20-25% for a healthy, fertile couple. There are numerous underlying causes for this low rate of human fertility, not the least of which are intrinsic abnormalities within the oocyte and/or embryo, which likely account for greater than 50% of failed conceptions. During assisted reproduction technology (ART) interventions, controlled ovarian stimulation is used to obtain several oocytes in attempts to increase the likelihood of having at least one developmentally competent embryo available for transfer. However, current techniques for identifying the competent embryo(s) are by no means perfect. These limitations, coupled with pressures to maximize the chance of pregnancy, typically result in the transfer of multiple embryos. Not surprisingly, this practice has resulted in an unacceptably high rate of multiple pregnancies arising from ART. During the last few years, concerted efforts have focused on reducing these rates. Programs for ART are developing patient-specific policies, restricting the number of embryos to transfer. In addition, strategies are being adopted to improve the accuracy for selecting viable embryos for transfer. One such strategy involves further refinement of morphological criteria associated with improved viability by considering, for example, pronuclei disposition, nucleolar organization, and identification of the fast-cleaving embryos with only mononucleate blastomeres. Another strategy employs pre-implantation genetic diagnosis (PGD) whereby a biopsied blastomere is tested for ploidy using fluorescence in situ hybridization (FISH). A final strategy involves extending the duration of culture to the blastocyst stage, thereby allowing self-selection of those embryos capable of proceeding to blastulation and exclusion of those less viable embryos that succumb to developmental arrest. Together, these strategies are enabling fewer embryos of higher quality to be transferred. Accordingly, the overall pregnancy rate from ART continues to increase, while the rate of triplet and higher order multiple births continues to decline. Nevertheless, the high incidence of intrinsic developmental anomalies in human oocytes inevitably will continue to result in a high degree of embryonic loss in ART.

Adult↗
PubMed2004

Morphological aspects of in vivo cleavage in Myocastor coypus (coypu).

The objective of the present work was to characterize the in vivo cleavage stage of Myocastor coypus embryos. For this purpose a colpocytological follow-up and controlled mating of 18 females were performed. Specimens from the beginning of the first cleavage to the acquisition of a morula appearance were considered to be in cleavage stage. Embryos in cleavage were collected between days 3 and 6 post-coitus. Of the collected embryos, 80% presented an even number of blastomeres and the remaining 20% an odd number. Embryos from 3 to 7 cells were blastomere associations in a spherical disposition within the zona pellucida. Blastomeres were spherical or ovoid, presenting slight flattening in areas contacting with other blastomeres. Embryos of 8 and 9 cells were as a group of blastomeres slightly elongated, surrounded by a spherical zona pellucida. The percentage of peri-vitelline space occupied by the embryonic mass ranged from 74.1 to 95.8% for all the substages. The cleavage pattern, developed in the oviduct, was of a rotational holoblastic type and asynchronic.

Animals↗