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Biomedical subjects

B E Reubinoff

Publications and source records attributed to B E Reubinoff.

At least 19 recordsLinked to original sources
PubMed2001

Asynchronous replication and allelic exclusion in the immune system.

The development of mature B cells involves a series of molecular decisions which culminate in the expression of a single light-chain and heavy-chain antigen receptor on the cell surface. There are two alleles for each receptor locus, so the ultimate choice of one receptor type must involve a process of allelic exclusion. One way to do this is with a feedback mechanism that downregulates rearrangement after the generation of a productive receptor molecule, but recent work suggests that monoallelic epigenetic changes may also take place even before rearrangement. To better understand the basis for distinguishing between alleles, we have analysed DNA replication timing. Here we show that all of the B-cell-receptor loci (mu, kappa and lambda) and the TCRbeta locus replicate asynchronously. This pattern, which is established randomly in each cell early in development and maintained by cloning, represents an epigenetic mark for allelic exclusion, because it is almost always the early-replicating allele which is initially selected to undergo rearrangement in B cells. These results indicate that allelic exclusion in the immune system may be very similar to the process of X chromosome inactivation.

Alleles↗
PubMed2001

Neural progenitors from human embryonic stem cells.

The derivation of neural progenitor cells from human embryonic stem (ES) cells is of value both in the study of early human neurogenesis and in the creation of an unlimited source of donor cells for neural transplantation therapy. Here we report the generation of enriched and expandable preparations of proliferating neural progenitors from human ES cells. The neural progenitors could differentiate in vitro into the three neural lineages--astrocytes, oligodendrocytes, and mature neurons. When human neural progenitors were transplanted into the ventricles of newborn mouse brains, they incorporated in large numbers into the host brain parenchyma, demonstrated widespread distribution, and differentiated into progeny of the three neural lineages. The transplanted cells migrated along established brain migratory tracks in the host brain and differentiated in a region-specific manner, indicating that they could respond to local cues and participate in the processes of host brain development. Our observations set the stage for future developments that may allow the use of human ES cells for the treatment of neurological disorders.

Animals↗
PubMed2001

Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method.

BACKGROUND: Human embryonic stem (ES) cells originate from the inner cell mass of the blastocyst, and retain in culture the properties of pluripotent cells of the early embryo. The study aim was to determine whether the open pulled straw (OPS) vitrification method, which is highly effective for the cryopreservation of embryos, might be also efficient for human ES cells. METHODS AND RESULTS: All human ES cell clumps that were vitrified by the OPS method could be recovered upon thawing, and gave rise to ES cell colonies after plating. Vitrified colonies were significantly smaller and showed an increased level of background differentiation compared with control colonies. However, these unwanted effects could be overcome by additional cultivation of the colonies for 1 and 2 days respectively. The vitrified human ES cells were cultivated for prolonged periods and retained the properties of pluripotent cells, including a normal karyotype, expression of the transcription factor Oct-4 and surface markers that are characteristic to human ES cells. When grafted into SCID mice, the vitrified cells gave rise to teratomas containing derivatives of all three embryonic germ layers. CONCLUSIONS: Vitrification by the OPS method is reliable and effective for the cryopreservation of human pluripotent embryonic stem cells.

Animals↗
PubMed2000

Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.

We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.

Animals↗
PubMed1999

Asynchronous replication of imprinted genes is established in the gametes and maintained during development.

Genomic imprinting is characterized by allele-specific expression of multiple genes within large chromosomal domains that undergo DNA replication asynchronously during S phase. Here we show, using both fluorescence in situ hybridization analysis and S-phase fractionation techniques, that differential replication timing is associated with imprinted genes in a variety of cell types, and is already present in the pre-implantation embryo soon after fertilization. This pattern is erased before meiosis in the germ line, and parent-specific replication timing is then reset in late gametogenesis in both the male and female. Thus, asynchronous replication timing is established in the gametes and maintained throughout development, indicating that it may function as a primary epigenetic marker for distinguishing between the parental alleles.

Alleles↗
PubMed1998

A birth in non-mosaic Klinefelter's syndrome after testicular fine needle aspiration, intracytoplasmic sperm injection and preimplantation genetic diagnosis.

Non-mosaic Klinefelter patients are generally azoospermic due to primary testicular failure. Nevertheless, in some cases, testicular spermatozoa may be recovered and utilized to fertilize oocytes via intracytoplasmic sperm injection (ICSI). As the risk for an increased number of gonosomes in these spermatozoa is unclear, preimplantation genetic diagnosis (PGD) may be attempted in the resulting embryos. In the present study, we report our experience with the combined approach of sperm retrieval by testicular fine needle aspiration (FNA), ICSI and PGD in seven consecutive non-mosaic Klinefelter individuals. In four patients, between one and five spermatozoa were retrieved in five out of nine consecutive attempts. In a fifth patient, only 10 round spermatids could be isolated. Mature spermatozoa were injected into a total of 16 metaphase-II oocytes, of which 11 (69%) remained intact. Two distinct pronuclei (2PN) were observed in four oocytes (36%) while a single pronucleus (1PN) was documented in two oocytes. Five cleavage stage embryos developed from the oocytes of two couples. Upon the request of one couple, their three embryos (two derived from 1PN oocytes) were transferred without PGD but pregnancy was not achieved. PGD by fluorescence in-situ hybridization (FISH) was performed in the two embryos of the other couple which were derived from normal fertilization. PGD results of one embryo were 18,18,X,X,Y, the embryo was not transferred and FISH analysis of the remaining blastomeres identified variable chromosome numbers in the nuclei. The second embryo was diagnosed as normal and was transferred, resulting in a successful pregnancy and birth. In conclusion, the results of this report indicate that a pregnancy and birth may be attained in azoospermic non-mosaic Klinefelter individuals by testicular FNA combined with ICSI. Due to the unknown risk of gonosomes aneuploidy in embryos from Klinefelter patients, PGD or prenatal diagnosis should be recommended.

Adult↗
PubMed1998

Intracytoplasmic sperm injection allows fertilization and development of a chromosomally balanced embryo from a binovular zona pellucida.

A binovular zona pellucida was found in two in-vitro fertilization (IVF) treatment cycles. In both cases, two oocytes of slightly unequal size were enclosed within a single zona pellucida, the larger oocyte appearing as a metaphase II oocyte while the smaller one as an immature oocyte with a germinal vesicle. Intracytoplasmic sperm injection performed in the mature oocyte of each pair led to normal fertilization and embryonic development in both cases. Results of genetic analysis performed by fluorescence in-situ hybridization in one of the two treatment cycles were consistent with a diploid chromosomal status of both the non-injected immature oocyte as well as the embryo which developed following the microinjection. These results indicate that, in this case, the binovular zona pellucida was most probably created when granulosa cells failed to separate two distinct oocytes during follicular formation. It may also imply that selective fertilization of a single mature oocyte in a binovular zona pellucida by intracytoplasmic sperm injection can lead to the development of a chromosomally balanced embryo and can prevent the undesired consequences that may result if the two oocytes are fertilized in the course of standard IVF.

Adult↗
PubMed1998

Assisted reproduction for the treatment of azoospermia.

Azoospermia, the most severe form of male infertility, is caused by obstructions in the genital tract or by testicular failure. Microsurgical techniques are available for the correction of some of these obstructions but no effective treatment is available for testicular failure. In recent years, methods have been developed for direct surgical sperm sampling from either the epididymis or the testis to be used by intracytoplasmic sperm injection. The main approach proven to be effective for the retrieval of spermatozoa from the epididymis in patients with obstructive azoospermia is microsurgical epididymal sperm aspiration, although recently the retrieval of spermatozoa by fine needle aspiration was shown to be equally effective. Recovery of spermatozoa is also now performed in patients with severely deficient spermatogenesis using testicular open biopsy as well as aspiration by fine needle. The ultimate choice of sperm retrieval method in these patients will depend not only on sperm availability, but also on the physiological consequences of the different techniques on testicular function. This article summarizes the recent advances achieved in the treatment of azoospermic patients using these assisted reproduction surgical techniques.

Humans↗
PubMed1997

Is the obstetric outcome of in vitro fertilized singleton gestations different from natural ones? A controlled study.

OBJECTIVE: To determine whether singleton IVF pregnancies carry adverse maternal or fetal outcome when compared with naturally conceived gestations. DESIGN: An analysis of the obstetric outcome of singleton IVF pregnancies in comparison with matched, naturally conceived singleton controls. SETTING: In vitro fertilization unit and obstetric service at a tertiary medical center. PATIENT(S): Two hundred sixty consecutive singleton IVF pregnancies and 260 naturally conceived singleton controls matched 1:1 for maternal age, parity, ethnic origin, and location and date of delivery. INTERVENTION(S): In vitro fertilization-ET. MAIN OUTCOME MEASURE(S): The rate of antenatal obstetric complications, nonvertex presentation, cesarean section, preterm labor, low birth weight, small and very small for gestational age, neonatal intensive care unit admissions, and perinatal mortality. RESULT(S): The rates of most antenatal complications were similar in both groups. Urinary tract infection was the only complication diagnosed significantly more frequently after IVF (7.3% versus 1.2%); however, the rates of severe urinary tract infection necessitating hospitalization were similar. The incidence of nonvertex presentation was also similar. The cesarean section rate was significantly higher among IVF patients (41.9% versus 15.5%). The rates of preterm labor, low birth weight, small and very small for gestational age, neonatal intensive care unit admissions, and perinatal mortality were comparable. CONCLUSION(S): When controlling for maternal age, parity, ethnic origin, and location and date of delivery, singleton IVF pregnancies do not carry an increased risk for prematurity, low birth weight, or maternal or fetal complications. Still, these pregnancies are associated with a high rate of cesarean sections.

Adult↗
PubMed1997

Intracytoplasmic sperm injection combined with preimplantation genetic diagnosis for the prevention of recurrent gestational trophoblastic disease.

A strategy for the prevention of repeated molar pregnancies by using intracytoplasmic sperm injection (ICSI) coupled with preimplantation genetic diagnosis (PGD) with fluorescence in-situ hybridization (FISH) was developed. In this approach, complete moles which arise from dispermic fertilization are avoided by the use of ICSI. ICSI is followed by preimplantation selection against the transfer of 46,XX embryos, thus preventing complete moles resulting from a fertilization of an inactive oocyte, by a haploid X-bearing spermatozoon which subsequently duplicates. Triploid partial moles which arise mainly from dispermic fertilization may also be prevented by ICSI. The preimplantation confirmation of diploidy by FISH guards against triploid partial moles which may result from mechanisms other than dispermic fertilization. The employment of this strategy in an attempt to prevent a repeated event of molar pregnancy in a patient with a history of two previous episodes of gestational trophoblastic disease is reported.

Adult↗
PubMed1996

Increased levels of redox-active iron in follicular fluid: a possible cause of free radical-mediated infertility in beta-thalassemia major.

OBJECTIVE: Our purpose was to investigate the follicular fluid parameters associated with redox activity and the consequent production of the deleterious hydroxyl radical in beta-thalassemia major. STUDY DESIGN: The levels of ferritin, total iron, total copper, and redox-active iron were measured in follicular fluid aspirated from three follicles during three consecutive ovum pickups from a patient with beta-thalassemia major and were compared with the levels in nine follicles aspirates from nine healthy control patients. The redox activity in the follicular fluid samples was monitored by the extent of follicular fluid-mediated deoxyribonucleic acid degradation and salicylate hydroxylation. RESULTS: Total iron and ferritin concentrations were elevated in thalassemic follicular fluid samples compared with control samples (6.7 fold, and 53.3-fold, respectively), whereas the total copper concentration was similar. Thalassemic follicular fluid samples exhibited a marked increase of redox activity, indicating a higher potential of free radical production leading to deoxyribonucleic acid degradation. Likewise, free radical-induced conversion of salicylate to dihydroxybenzoic acid derivatives was enhanced in the thalassemic follicular fluid samples compared with controls (2,3-dihydroxybenzoic acid: 67.7 +/- 22 vs 20.3 +/- 12.9 ng/mg protein; 2,5-dihydroxybenzoic acid: 101.6 +/- 25.9 vs 4.42 +/- 2.7 ng/mg protein). CONCLUSIONS: The increased level of redox activity found in the follicular fluid from a patient with beta-thalassemia major focuses the attention on the small fraction of redox-active iron ions as mediators of free radical production, inducing tissue injury and possibly contributing to impairment of reproduction in these patients.

Adult↗
PubMed1996

RhD genotype determination by single sperm cell analysis.

OBJECTIVE: An Rh-negative woman with preexisting anti-D antibodies may affect some or all subsequent fetuses, depending on the genotype of her Rh-positive partner. Currently, a reliable technique for an absolute determination of RhD genotype is not available. This study was initiated to develop an accurate method for RhD genotyping in men. STUDY DESIGN: RhD genotype was determined by deoxyribonucleic acid amplification of a D-specific sequence in single sperm cell samples. Micromanipulation techniques were used for sampling of single sperm cells, which were further amplified by multiplex nested polymerase chain reaction at the RhD locus. A RhD sequence amplification product was expected in all of the successfully amplified samples from Rh-positive homozygotes, in some of the samples from heterozygotes, and in none of the samples form Rh-negative subjects. RESULTS: RhD genotype was accurately determined in 10 of 10 donors. A total of 132 single sperm cells were analyzed (8 to 17 samples per donor), of which 96 were successfully amplified as assessed by an internal control. As expected, the specific region of the RhD gene was amplified in all, some, and none of the signal-positive sperm samples from Rh-positive homozygotes, heterozygotes, and Rh-negative subjects, respectively, allowing accurate determination of the genotype. CONCLUSION: An accurate diagnosis of the RhD genotype can be attained from single sperm cell analysis by means of polymerase chain reaction and may have major clinical applications in the management of Rh isoimmunization.

Base Sequence↗
PubMed1996

New advances in sex preselection.

OBJECTIVE: To review the current developments in the field of preconceptual sex selection and to discuss the moral dilemmas that accompany the scientific progress. DESIGN: A survey of the major publications on sex preselection. RESULTS: Examination of current methods of preconceptual gender selection revealed that in vivo methods such as timing of intercourse, the use of ovulation induction medications, and artificial insemination do not appear to affect the sex ratio to a clinically significant degree. In vitro separation of X- and Y-bearing spermatozoa by gradient techniques have been reported to alter significantly the sex ratio at birth. However, these trials were noncontrolled, and molecular biological techniques could not validate that these methods indeed change the Y- to X-bearing spermatozoa ratio sufficiently for clinical use. Nevertheless recent scientific advances have made highly reliable preconceptual sex selection possible by using preimplantation diagnosis or sperm separation by flow cytometry combined with IVF. At present, these methods have been used to avoid sex-linked disorders. Both involve the invasive procedure of IVF and thus are held by most as inappropriate for nonmedical indications. However, improvement in flow cytometry output of sexed spermatozoa might provide in the near future sufficient sorted gametes for artificial insemination. This technique then will provide an available noninvasive method of sexing for social purposes. CONCLUSIONS: Reliable preconceptual sex selection is currently possible only by preimplantation diagnosis, or sperm separation by flow cytometry combined with IVF. Both methods involve invasive procedures and are at present exclusively used for medical indications. It may be that in the near future, an improvement in flow cytometry output of sexed spermatozoa will provide sufficient sorted gametes for artificial insemination. In such a case, the medical community will be forced to take a stand, whether this reliable noninvasive method of sexing will be allowed for social purposes.

Cell Separation↗
PubMed1996

Management of rhesus isoimmunization by preimplantation genetic diagnosis.

A genetic assay by single blastomere analysis was developed for rhesus (RhD) blood group typing of early cleavage stage embryos. The method, which is based on the simultaneous amplification of an RhD-specific sequence and an internal control in single cells, was applied for the selective transfer of RhD-negative embryos in a family of an RhD sensitized woman and a heterozygote partner. The RhD status of two out of three biopsied embryos was determined. According to their amplified products, both were typed as RhD-negative and transferred to the uterus. Pregnancy was not achieved.

Adult↗
PubMed1995

Effects of low-dose estrogen oral contraceptives on weight, body composition, and fat distribution in young women.

OBJECTIVE: To determine prospectively whether the use of low-dose estrogen oral contraceptives (OC) is associated with changes in weight, body composition, or fat distribution. DESIGN: Anthropometric measurements were performed in 49 healthy young (16 to 21 years old) women before commencement of OC use (30 micrograms ethinyl estradiol [EF2] plus 75 micrograms gestodene) and after three and six treatment cycles. Thirty one age- and weight-matched women who were not using OC served as controls. SETTING: Outpatient gynecological clinic of Hadassah Medical Center, a tertiary level hospital, and the "Shilo" voluntary service for the prevention of unwanted pregnancy. MAIN OUTCOME MEASURES: Anthropometric measurements included body mass index (BMI), waist-to-hip girth ratio, and body composition (the percentage of body fat and water), estimated by mean of infrared interactance. RESULTS: In the group of OC users, baseline BMI, percent fat, percent water, and waist-to-hip girth ratio were 21.1 +/- 0.32 (kg/m2), 23.8% +/- 0.63%, 57.4% +/- 0.39%, and 0.73 +/- 0.01, respectively, and did not change significantly after six cycles (20.6 +/- 0.41 [kg/m2], 23.9% +/- 0.57%, 58.1% +/- 0.49%, and 0.72 +/- 0.03, respectively). These measurements were not significantly different when compared with the nonusers. Fifteen OC users (30.6%) gained weight (> 0.5 kg). Weight gain was due to a significant accumulation of fat (from 22.5% +/- 1.1% to 25.6% +/- 0.74%), whereas the percentage of body water remained stable. The waist-to-hip girth ratio also was not changed significantly. Similarly, 11 nonusers (35.4%) gained weight because of similar nonabdominal fat accumulation. Ten OC users (20.4%) lost weight (57 kg +/- 1.51 to 55.4 +/- 1.47 [mean +/- SEM]) and 6 nonusers (19.3%) also lost weight (59 kg +/- 1.42 to 57.3 +/- 1.92). In both groups the loss of weight was not associated with significant change in body composition. CONCLUSIONS: The use of low-dose OC (EE2 plus gestodene) was not associated with overall impact on weight, body composition, or fat distribution. However, when weight gain did occur during OC use, it was due to increase in body fat and not in volume of body water, and it was not associated with changes in fat distribution.

Adipose Tissue↗
PubMed1995

Effects of hormone replacement therapy on weight, body composition, fat distribution, and food intake in early postmenopausal women: a prospective study.

OBJECTIVE: To evaluate the effects of hormone replacement therapy (HRT) on body weight and composition, fat distribution, and food intake in women entering the climacteric. DESIGN: Prospective clinical study. SETTING: Outpatient menopause clinic at a tertiary medical center. PARTICIPANTS: Sixty-three early postmenopausal women (44 to 54 years old) were prospectively studied for 1 year. They consisted of two groups: group A, 34 subjects who initiated continuous estrogen and progestin treatment (daily oral conjugated estrogen 0.625 mg and medroxyprogesterone acetate 2.5 mg), and group B, 29 women who refused hormonal therapy and served as controls. The age, menopausal status, initial anthropometric measurements (weight, body mass index [BMI], fat mass, and waist-to-hip girth ratio), and daily food intake (total caloric intake and food composition) were similar in both groups. INTERVENTIONS: Anthropometric measurements were performed before commencement of HRT use and after 12 months. MAIN OUTCOME MEASURES: Anthropometric measurements included BMI, waist-to-hip girth ratio, and body composition (the percentage of body fat and water) estimated by means of infrared interactance. Daily food intake was also recorded. RESULTS: The body weight and fat mass increased significantly in both the treatment (73.22 +/- 2.01 [mean +/- SE] to 75.57 +/- 1.12 kg) and the control group (71.45 +/- 3.11 to 73.51 +/- 1.23 kg). However, a significant shift from gynoid to android fat distribution was observed only in the control group (waist-to-hip ratio shifted from 0.80 +/- 0.01 to 0.85 +/- 0.01), whereas no significant change was observed in the treatment group (0.81 +/- 0.01 to 0.82 +/- 0.01). Caloric and macronutrient intake did not change in either group. CONCLUSIONS: These results indicate that continuous daily estrogen and progestin replacement therapy neither prevents nor increases early postmenopausal weight gain and fat accumulation. However, it does minimize the shift from gynoid to android fat distribution.

Adult↗