Cryopreservation of unfertilized human oocytes.

Previous investigations revealed that choline-based freezing media developed in our laboratory were superior to conventional sodium-based media for storing mouse oocytes. This paper examines the ability of the choline-based medium CJ2 and a modified form of this medium, CJ3, to cryopreserve unfertilized human oocytes. Oocytes that were consented for research and matured overnight, as well as freshly collected, donor, mature metaphase II (MII) oocytes, were cryopreserved using choline-based media and an optimized slow-cooling protocol.

Cytoplasmic fragmentation in activated eggs occurs in the cytokinetic phase of the cell cycle, in lieu of normal cytokinesis, and in response to cytoskeletal disorder.

The timing of cytoplasmic fragmentation in relation to the cell cycle was studied in mature oocytes and early cleavage stages using mouse oocytes and embryos as experimental models. The central approach was to remove the nuclear apparatus, in whole or in part, from non-activated and activated oocytes and early embryos, and follow their response during subsequent culture in vitro. Oocytes arrested in metaphase of the second meiotic division did not fragment following complete removal of the meiotic apparatus, provided they were not subsequently activated.

Human blastocysts from aggregated mononucleated cells of two or more non-viable zygote-derived embryos.

This study examined the developmental capacity of aggregates of surviving mono-nucleated cells isolated from several non-viable human embryos on day 3 or day 4 after fertilization. The results clearly demonstrate that some blastomeres from non-viable embryos do indeed maintain their developmental potential and regulatory capacity to the extent of being able to contribute to a normally organized blastocyst, with as many as 90% diploid cells. Although the chimaeric nature of such blastocysts excludes them from use in therapeutic IVF, they are of particular relevance to the discussion of embryonic and trophectodermal stem cell line production.

Fetal development of mouse oocytes and zygotes cryopreserved in a nonconventional freezing medium.

This study (1) analyzed fetal development of mouse embryos after oocyte cryopreservation in CJ2, a choline-based medium, (2) examined the effect of culture duration in vitro on subsequent fetal development, and (3) compared survival and fetal development of zygotes frozen in embryo transfer freeze medium (ETFM; sodium-based medium) or CJ2. Unfertilized oocytes and zygotes were cryopreserved using a slow-cooling protocol. After thawing, oocytes were inseminated after drilling a hole in their zona, cultured in vitro either to the two-cell or blastocyst stage, and transferred to the oviducts or uterine horns of recipient mice.

Cytoplasmic transfer in assisted reproduction.

This report details the use of cytoplasmic transfer in human oocytes. The introduction of a small amount of ooplasm from a donor oocyte or zygote may alter the function of oocytes, with probable deficiencies. Cytoplasmic transfer from fertile donor oocytes or zygotes into compromised oocytes from patients with recurrent implantation failure after assisted reproduction has now led to the birth of nearly 30 healthy babies worldwide.

Spontaneous and artificial changes in human ooplasmic mitochondria.

Our research has focused on promoting the development of compromised embryos by transferring presumably normal ooplasm, including mitochondria, to oocytes during intracytoplasmic insemination. Because of the enigma of mitochondrial heteroplasmy, the mixing of populations of oocyte cytoplasm has provoked considerable debate. We are currently investigating oocyte mitochondrial (mt) DNA mutations and the effects of ooplasmic transplantation on mitochondrial inheritance and mitochondrial functionality.

Mitochondrial DNA heteroplasmy after human ooplasmic transplantation.

Objective: To determine the patterns of mitochondrial inheritance in embryos, fetuses, and infants after ooplasmic transplantation using the technique of mitochondrial DNA (mtDNA) fingerprinting.

Design: Prospective clinical study.

Setting: The IVF program at Saint Barnabas Medical Center, a nonprofit community hospital.

Cryopreservation of mouse oocytes using a medium with low sodium content: effect of plunge temperature.

The effect of various combinations of plunge temperature and thawing protocol on the survival and viability of mouse oocytes was examined. The oocytes were frozen either in a standard freezing medium (ETFM, embryo transfer freezing medium) or in a low-sodium, choline-based freezing medium (CJ2), with 1.5 M 1,2-propanediol and 0.

Genome-wide screen for atopy susceptibility alleles in the Hutterites.

A genome-wide screen for loci influencing positive skin prick tests (SPT) to airborne allergens was conducted in the Hutterites, a founder population of European ancestry. Positive SPT to 14 standardized allergens was measured in 370 subjects in our primary sample and 324 subjects in a replication sample. Evidence for linkage to positive SPT was assessed using the transmission disequilibrium test (TDT) with 337 autosomal markers (average spacing 9.

Determination of Duffy genotypes in three populations of African descent using PCR and sequence-specific oligonucleotides.

The expression of the Duffy Antigen/Receptor for Chemokines (DARC) on red blood cells (RBC) has been commonly determined using hemagglutination tests. Because the vast majority of African individuals are Duffy-negative, screening for DARC expression on RBC is a valuable tool to assess Caucasian admixture in populations of African descent. Furthermore, blood group antigens have been frequently tested as potential risk factors for complex diseases.

Rapid visualization of metaphase chromosomes in single human blastomeres after fusion with in-vitro matured bovine eggs.

The present study was aimed to facilitate karyotyping of human blastomeres using the metaphase-inducing factors present in unfertilized eggs. A rapid technique for karyotyping would have wide application in the field of preimplantation genetic diagnosis. When cryopreserved in-vitro matured bovine oocytes were fused with human blastomeres, the transferred human nuclei were forced into metaphase within a few hours.

Cryopreservation of activated mouse oocytes and zygote reconstitution after thaw.

A new approach to cryopreservation of unfertilized oocytes is proposed using techniques of artificial egg activation combined with nuclear transplantation. Matured mouse oocytes were released from metaphase II arrest by brief exposure to alcohol, allowed to progress to the pronuclear stage and then frozen according to a standard freezing protocol in propandiol. After thaw the female pronuclei were enucleated and fused with a male karyoplasts that were divided from in-vivo fertilized zygotes.

Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium.

Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stage in vitro.

Culture and quality control of embryos.

Improvement of embryo quality during in-vitro culture can be achieved by understanding and controlling the requirements of gametes and embryos. The most obvious route is to alter culture media, but standardization could be influenced by diverse environmental factors. Abnormal embryos from patients with multiple failures probably do not benefit from standardization and require specialized therapy, that is if their physiology is not already irreversibly jeopardized during gametogenesis.

Genome-wide search for asthma susceptibility loci in a founder population. The Collaborative Study on the Genetics of Asthma.

Founder populations offer many advantages for mapping genetic traits, particularly complex traits that are likely to be genetically heterogeneous. To identify genes that influence asthma and asthma-associated phenotypes, we conducted a genome-wide screen in the Hutterites, a religious isolate of European ancestry. A primary sample of 361 individuals and a replication sample of 292 individuals were evaluated for asthma phenotypes according to a standardized protocol.

Detrimental effects of sodium during mouse oocyte cryopreservation.

Cryopreservation is an established way of storing embryos, but effective methods are not available for freezing eggs. Most freezing damage is caused by high solute concentration (solution effects) and intracellular ice. Sodium salts are the major components of cryopreservation media, and the main contributor to the solution effects.

Expression and cytogenetic localization of the human SM22 gene (TAGLN).

SM22 is a 22-kDa protein identified variously as SM22, transgelin, WS3-10, or mouse p27. Though its precise function is unknown, it is abundant in smooth muscle and so may contribute to the physiology of this widespread tissue. We found that cosmid 16b6 contains the entire 5.

Ooplasmic transfer in mature human oocytes.

Ooplasmic transplantation aimed at restoring normal growth in developmentally compromised oocytes and embryos was evaluated in seven couples (eight cycles) with multiple implantation failures. Two approaches were investigated to transfer ooplasm from donor eggs at metaphase II (MII) stage into patient MII eggs: (i) electrofusion of a ooplasmic donor fragment into each patient egg (three cycles), and (ii) direct injection of a small amount of ooplasm from a donor egg into each patient egg (five cycles). Some donor eggs were used multiple times.

HLA-G1 protein expression is not essential for fetal survival.

HLA-G is a nonclassical, class I HLA gene that is primarily expressed by fetal cells at the maternal-fetal interface and is thought to play a key role in the induction of tolerance in pregnancy. This paper reports the identification of a single base pair deletion at position 1597 (1597delC) in exon 3 (encoding the alpha2-domain) of HLA-G on 20 of 272 (7.4 per cent) African American chromosomes, three of 102 (2.

The development of mouse zygotes after fusion with synchronous and asynchronous cytoplasm.

Cytoplasts with diameters of 40-45, 50-55 and 70-75 microm, derived from mouse oocytes at the germinal vesicle, metaphase II and zygote stages were incorporated into zygotes by electrofusion. Manipulated (n = 867) and culture-control (n = 1114) embryos were cultured in vitro and transferred to pseudo-pregnant recipients at the blastocyst stage. When synchronous cytoplasts measuring 40-45 and 50-55 microm in diameter were incorporated into 138 and 86 zygotes respectively, only one embryo in each group (not significant) became arrested at the 1-cell stage.

Formation of male pronuclei in partitioned human oocytes.

Ninety-five metaphase II human oocytes, aged in vitro for either one day or for two days, and five fresh immature oocytes with no visible germinal vesicle nucleus were partitioned into small cytoplasts after removal of the zona pellucida and exposure to cytochalasin B. Seventy-one metaphase II and four immature oocytes were used as intact zona-free controls. The cytoplasts derived from each partitioned oocyte and all the zona-free whole oocytes were exposed to normal or subfertile donor sperm and later assessed for signs of male pronucleus development.

Highly effective method of human oocyte activation.

Fresh and aged unfertilised human oocytes were activated by electroporation and by exposure to isotonic solution of mannitol supplemented with low concentrations of calcium, magnesium and chloride. Over 95% of the fresh oocytes were activated, all showing formation of one pronucleus and extrusion of the second polar body. Oocytes activated 1 and 2 days post-collection showed activation rates of 66.

Male and female genomes associated in a single pronucleus in human zygotes.

The ploidy of single-pronucleated human zygotes obtained after conventional in vitro fertilization was determined by fluorescent in situ hybridization (FISH) using multiple simultaneous probes for gonosomes and autosomes. After zona removal the single-pronucleated zygotes were exposed to cytochalasin B, and the pronucleus, surrounded by scant cytoplasm and the plasma membrane (karyoplast), was divided from the rest of the egg (cytoplast). The karyoplasts and the corresponding cytoplasts were analyzed separately by FISH.