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ABSTRACT: Human embryonic stem cells (hESC) face ethical sensitivities and the problem of teratoma formation. Although Wharton's jelly stem cells (WJSC), also of embryonic origin, may not face such ethical concerns, it is not definitely known whether under hESC culture conditions they would be as pluripotent as hESC. WJSC grown on plastic showed two types of morphology (epithelioid and short fibroblastic) in primary culture depending on the culture medium used, and only fibroblastic morphology when passaged. When grown in the presence of hESC medium on mouse feeder cells, they produced atypical colonies containing hESC-like cells with high-nuclear cytoplasmic ratios and prominent nucleoli. They were positive for the hESC markers Tra-1-60, Tra-1-81, SSEA-1, SSEA-4, Oct-4 and alkaline phosphatase, negative for SSEA-3, showed normal karyotypes, developed embryoid body (EB)-like structures, did not produce teratomas in SCID mice and differentiated into neuronal derivatives. They were also positive for the mesenchymal CD markers (CD105, CD90, CD44), negative for CD34 and HLA, and although nine out of 10 embryonic stem cell genomic markers were detectable, these were expressed at low levels. WJSC are thus not as pluripotent as hESC but widely multipotent, and have the advantages of being able to be scaled up easily and not inducing teratomas.
Reproductive biomedicine online 01/2008; 15(6):708-18. · 2.04 Impact Factor
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Annals of the New York Academy of Sciences 12/2006; 626(1):438 - 444. · 3.15 Impact Factor
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ABSTRACT: The aims of this study were to investigate whether the human embryo could sustain development beyond the blastocyst stage in vitro and to identify the precise origins of embryonic stem cells (ES cells) from the embryoblast. A frozen-thawed 4-cell embryo was cultured to the post-blastocyst stage. This 9-day-old embryo presented a solid mass of inner cells (resembling a tumour) surrounded by surface trophoblast cells. Clumps of multinucleated syncytiotrophoblast cells were evident at one pole. Most cells resembled those of blastocysts. However, there were groups of comparatively undifferentiated cells within the inner cell mass somewhat resembling ES cells documented previously, that might give a clue as to their origins. The embryo attempted to form an amnion with a cavity, but did not present a bilaminar, discoidal structure as expected in week 2 of development, and hence was abnormal.
Reproductive biomedicine online 10/2004; 9(3):321-5. · 2.04 Impact Factor
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ABSTRACT: Enzymatic treatment of the zona pellucida to either soften or remove totally the zona before blastocyst transfer has resulted in high implantation rates. The zona is usually completely dissolved after 1.5 min exposure with 10 IU pronase at 37 degrees C. Since there may be concerns that pronase treatment for periods of 1.5 min or longer may cause adverse effects on the trophectoderm (TE) and inner cell mass (ICM), the changes to human blastocysts exposed to different time intervals of pronase were investigated. Of 18 blastocysts exposed to pronase for 1.5 min, the zona was completely dissolved and no changes were observed by light microscopy (LM) or transmission electron microscopy (TEM), compared with 11 naturally hatched untreated blastocysts (controls). In another five blastocysts exposed to pronase for 2 min, no LM changes were observed but subtle TEM changes such as fewer bundles of tonofibrils attached to desmosomes were observed. When three other blastocysts were exposed to pronase for 5 min, the blastocoele collapsed, and the TE cells started to show blebbing under LM. Under TEM, the cytoplasm of TE cells was extensively vacuolated and many TE cells showed cytoplasmic blebbing towards the blastocoele. However, the epithelium was uninterrupted with intact tight junctions and desmosomes. Of a separate group of 44 blastocysts cultured in vitro, 54.5% had hatching difficulties when monitored from day 5 to day 8 and 80% of these could be rescued by removal of the zona with pronase for 1.5 min prior to extensive degeneration taking place. The results confirm that the optimal time for softening or complete removal of the zona before transfer was around 1.5 min and that enzymatic treatment was a safe, non-invasive procedure to remove the zona of blastocysts. The human embryonic TE is a very hardy, robust epithelium that withstands pronase treatment.
Human Reproduction 04/2001; 16(3):540-6. · 4.47 Impact Factor
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ABSTRACT: 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.
Nature Biotechnology 05/2000; 18(4):399-404. · 23.27 Impact Factor
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ABSTRACT: It has been postulated that premature shortening of the oocyte growth phase due to the recovery of oocytes from small diameter follicles may be responsible for the developmental anomalies associated with in-vitro maturation. 6-Dimethylaminopurine (DMAP) was used to artificially lengthen the pre-maturation period of oocyte growth, in vitro, by inhibiting germinal vesicle breakdown in mouse and human oocytes. DMAP inhibited the meiotic maturation of mouse and human oocytes and the inhibition was fully reversible. The timing of polar body extrusion was accelerated in mouse oocytes following the withdrawal of DMAP; however, the kinetics of nuclear maturation in human oocytes was unaffected by exposure to DMAP. All mouse and human DMAP-treated oocytes that matured to metaphase II expressed histone H1 kinase activity. Fertilization rates in both DMAP-treated and control mouse and human oocytes were comparable, and human embryonic development was similar in control and DMAP-treated oocytes. However, blastocyst development was significantly reduced in DMAP-treated mouse oocytes (P < 0.05). It is concluded that lengthening the prematuration growth phase, by temporarily inhibiting kinase activity with DMAP, does not directly improve oocyte developmental competence but provides a useful tool for further investigating meiotic and developmentally related events in vitro by manipulating meiotic resumption.
Human Reproduction 03/2000; 15(2):379-88. · 4.47 Impact Factor
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ABSTRACT: To study the effects of peritoneal macrophages on endometrial cellular proliferation in an in vitro coculture model and to compare the magnitude of these effects between macrophages from women with endometriosis and normal women.
Controlled study of peritoneal macrophage function.
University hospital.
Patients with a normal peritoneal cavity (n = 15) and with pelvic endometriosis (n = 20) undergoing laparoscopy.
Peritoneal macrophages were cocultured with endometrial epithelial and stromal cells; endometrial cell cultures without macrophage coculture acted as controls.
Endometrial cellular proliferation measured by 3H-thymidine incorporation.
Endometrial epithelial cells cocultured with peritoneal macrophages from women with endometriosis showed significantly increased proliferation compared with cocultures using macrophages from normal women when assessed at 24 hours (1.56 versus 1.03 times, respectively, over control) and at 72 hours (1.55 versus 1.10 times over control). Endometrial stromal cells cocultured with peritoneal macrophages from women with endometriosis similarly exhibited increased proliferation compared with cocultures using macrophages from normal women when assessed at 24 hours (1.65 versus 1.17 times over control) and at 72 hours (1.65 versus 1.21 times over control).
Peritoneal macrophages of patients with endometriosis stimulate cellular proliferation of endometrial epithelial and stromal cells in vitro.
Fertility and Sterility 10/1999; 72(3):533-8. · 3.56 Impact Factor
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ABSTRACT: Recent interest in delayed embryo transfers necessitated the evaluation of two improved in-vitro systems that could generate viable blastocysts. A total of 178 two-pronucleated embryos (entire cohorts) from 19 patients was cultured in IVF50 medium (100 microl) under oil for 24 h until day 2. Each patient's day 2 embryos were then equally allotted to two in-vitro systems. Embryos in system A were grown until the morning of day 3 on Vero cells covered with IVF50 medium (100 microl) under oil. The medium was then replaced on day 3 with a 1:1 mixture (100 microl) of IVF50:S2 medium and on day 4 with S2 medium only. The same culture protocol was used for system B without Vero cells. Throughout the 5 days all dishes were housed in sealed humidified modular chambers containing a triple gas atmosphere. Separately, 175 spare embryos from 80 patients were grown in system A and B up to days 6 and 7 for total cell number (TCN) analysis. Blastulation rates were not significantly different between system A and B (67.4 versus 68.5%; P > 0.01) although co-cultured embryos cleaved slightly faster by day 4. The overall pregnancy and implantation rates were 52.0% and 32.1% for the 19 patients each of whom received a mixed cohort of three day 5 embryos from both systems. TCN values for the day 6 and 7 blastocysts from both systems were high and increased steadily from days 6-7 and from expanded to hatching stages. There were no significant differences in TCN for day 6 expanded blastocysts between the two systems although day 6 hatching and hatched co-cultured blastocysts had greater values than non-co-cultured blastocysts (246.0 +/- 18.5 and 236.7 +/- 17.8 versus 173.0 +/- 13.5 and 166.5 +/- 16.0; P < 0.01). The results demonstrated that the culture protocol using the sequential IVF50-S2 media combination was a good substitute for Vero cell co-culture for the transfer of viable day 3-6 embryos.
Human Reproduction 04/1999; 14(3):774-81. · 4.47 Impact Factor
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ABSTRACT: It has been shown recently that delayed transfers improve implantation rates in assisted reproductive technology programmes. In a prospective study, the pregnancy rates and safety of outcome were evaluated in a group of patients after the transfer of day 5 blastocysts with enzymatic treatment of the zona pellucida. Nineteen women with a mean age of 32.6+/-5.2 years and mean 2.1+/-2.2 repeated attempts had blastocyst transfers with a mean number of 2.5+/-0.7 embryos replaced per patient. The clinical pregnancy rates per cycle/transfer and implantation rate were 53% and 33%, respectively. The multiple pregnancy rate was 40% (two pregnancies were triplets). The pregnancy and implantation rates were very much higher than observed for most assisted reproduction technology centres. The 'in-vitro implantation' rates of zona-free blastocysts on a variety of feeder monolayers was 92%, offering some thoughts as to the role of the zona and interaction of the inner cell mass and trophoectoderm with the endometrium in implantation. Based on the in-vitro studies and the high multiple pregnancy rates, it appears that zona-manipulated blastocysts implant relatively well and there would be a need to reduce the number of transferred embryos to one or two, thus reducing multiple pregnancies and having spare blastocysts available for cryopreservation. The results also suggest that using the embryo culture protocol and method of transfer in the present study offers encouraging improvements to assisted reproduction technology, and enzymatic treatment of the zona may allow better anchorage and dialogue of the embryo with the endometrium, helping us to improve and understand implantation.
Human Reproduction 11/1998; 13(1O):2926-32. · 4.47 Impact Factor
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ABSTRACT: A 35 year old women with premature ovarian failure and another 30 year old women with gonadal dysgenesis were the recipients of donated supernumerous frozen embryos after successfully prepared with cyclic steroid replacement therapy as described previously. One patient received 4 and the second 2 frozen embryos, transferred transcervically on the 3rd day of progesterone administration. Both patients had viable twin pregnancies. The plasma beta hCG levels for both patients at 2 weeks post replacement (4 weeks gestation) were lower than the median values in our normal, uncomplicated singleton pregnancy for the same gestation. The level after 4 weeks post-replacement (6 weeks gestation) became comparable. Plasma progesterone profiles suggested a level of above 70 ng/ml would be enough to support the twin pregnancies. The first patient developed antepartum haemorrhage of unknown origin at 34 weeks of gestation preceding preterm premature rupture of membranes and subsequently had preterm labour. The second patient developed proteinuric hypertension at 33 weeks of gestation. Both ended in a lower segment cesarean section. Both sets of twins and their mothers were discharged well.
Journal of Obstetrics and Gynaecology Research 07/1998; 24(3):203-9. · 0.94 Impact Factor
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ABSTRACT: Recent reports suggest that transfer of day 5 blastocysts improves implantation rates in in-vitro fertilization programmes. This paper reports a successful ongoing pregnancy after the transfer of zona-free day 6 expanded and hatching blastocysts. The patient was 37 years old and had undergone six stimulated and two thaw cycles previously, all of which had failed. Stimulation was by down-regulation and oocytes recovered transvaginally by ultrasound guidance. Two pronuclear embryos were co-cultured on Vero cells to day 6. The zonae of two hatching and two fully expanded blastocysts were removed using 0.5% pronase, and the zona-free blastocysts were then transferred. Pregnancy was confirmed on day 18 with a positive human chorionic gonadotrophin (HCG) and ultrasound at 6 weeks showed a single healthy fetal heart inside a clear sac. At 14 weeks a triple test (oestriol, J-HCG and alpha-fetoprotein) was normal and at 22 weeks a detailed ultrasound scan showed no congenital anomalies. This is the first report in the human of a normal ongoing pregnancy after the transfer of zona-free day 6 embryos.
Human Reproduction 04/1997; 12(3):557-60. · 4.47 Impact Factor
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Cell Biology International 01/1995; 18(12):1181-9. · 1.48 Impact Factor
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ABSTRACT: Totipotent non-committed inner cell mass (ICM) cells from human blastocyts, if demonstrated to be capable of proliferating in vitro without differentiation, will have several beneficial uses, not only in the treatment of neurodegenerative and genetic disorders, but also as a model in studying the events involved in embryogenesis and genomic manipulation. Nine patients admitted to an in-vitro fertilization programme donated 21 spare embryos for this study. All 21 embryos were grown from the 2-pronuclear until blastocyst stages on a human tubal epithelial monolayer in commercial Earle's medium (Medicult, Denmark) supplemented with 10% human serum. The medium was changed after blastocyst formation to Chang's medium supplemented with 1000 units/ml of human leukaemia inhibitory factor (HLIF) and the embryos left undisturbed for 72 h to allow the hatched ICM and trophoblast to attach to the feeder monolayer. Nineteen of the 21 embryos from nine patients produced healthy ICM lumps which could be separated and grown in vitro. Two of the lumps differentiated into fibroblasts while the remaining 17 (eight patients) produced cells with typical stem cell-like morphology, were alkaline phosphatase positive and could be maintained for two passages. It was possible to retain the stem cell-like morphology, alkaline phosphatase positiveness and normal karyotype through the two passages in all of them using repeated doses of HLIF every 48 to 72 h. This is the first report on the successful isolation of human ICM cells and their continued culture for at least two passages in vitro.
Human Reproduction 12/1994; 9(11):2110-7. · 4.47 Impact Factor
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ABSTRACT: Embryonic behavior to blastocyst, hatching, and hatched stages were evaluated in 77, four-cell human embryos that were first grown in oviductal cell coculture and then equally allotted at the eight-cell stage to two coculture systems in a serum-free medium (34 continued on oviductal monolayers, 32 on endometrium monolayers). Sixty-three percent and 40% of embryos expanded and hatched in the sequential oviductal-endometrial coculture system when compared with 41% and 9% in the oviductal system alone, respectively. The sequential coculture system appears to be an improved system over the single human oviductal coculture system.
Fertility and Sterility 06/1994; 61(5):976-8. · 3.56 Impact Factor
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ABSTRACT: It is well over a decade since the birth of the first test-tube baby and still the in-vitro conditions for early embryonic development remain suboptimal. The ideal culture medium to increase longevity and improve viability of human embryos is not available. Since the metabolic requirements of the human embryo changes from one cleavage stage to another, the development of a single culture medium for all stages could not be expected. The use of helper cells (coculture) in vitro offers much promise as there are numerous documentations in both man and animals describing their ability to increase blastulation rates and improve embryo viability. This paper reviews the effect of coculture on human zygote development. The selection and establishment of cell-lines, biologic actions of coculture of gametes and zygotes, the outcome, and future prospects are discussed.
Current Opinion in Obstetrics and Gynecology 11/1993; 5(5):585-93. · 2.38 Impact Factor
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Human Reproduction 09/1993; 8(8):1155-60; discussion 1160-2. · 4.47 Impact Factor
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ABSTRACT: To evaluate the embryonic behavior in vitro and the pregnancy and implantation rates of embryos grown in a human ampullary cell coculture system.
In a prospective study, two pronuclei embryos were cultured on human ampullary feeder layers up to the two to six-cell and blastocyst stages and replaced either as tubal, uterine, or sequential transfers.
Assisted reproductive technology program in a university-based hospital.
Fifty women with a mean age of 35.6 years who went through a single coculture cycle. Thirty of the patients were admitted for in vitro fertilization (IVF) and 20 for tubal embryo transfer (TET).
The overall clinical pregnancy rate (PR) for all 50 patients was 44% per cycle (IVF, 37%; TET, 55%) and the implantation rate was 31.8% (IVF, 31.0%; TET, 32.6%). Sixty-eight percent of pregnant patients were over 35 years, and 68% had two previously failed assisted reproduction cycles. Five of 9 patients who received sequential transfers became pregnant. Three of the 22 pregnancies aborted (2 after sequential transfer), and there was one ectopic. Overall, 88% of two to six-cell stage embryos were of good quality.
The human ampullary coculture system produces better quality embryos, increased numbers of blastocysts with improved PRs and implantation rates. The beneficial effects of the feeder layer may be through the release of embryotrophic factors and detoxification of the medium by the cells. Coculture is a new concept in assisted reproduction and has tremendous potential in boosting conception rates by mimicking the in vivo environment.
Fertility and Sterility 10/1992; 58(3):569-74. · 3.56 Impact Factor
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ABSTRACT: Although the assisted reproductive techniques (ART) have contributed significantly over the last decade in alleviating subfertility in the childless couple, the implantation and take-home baby rates have been stubbornly low. A major cause for such low success rates has been the reduced viability of replaced embryos perhaps induced by the suboptimal in vitro conditions used in ART laboratories. One approach to improving embryo viability is to provide the growing embryo with a simulated in vivo environment by replicating the conditions existing in the human fallopian tube in vitro. This requires either the maintenance of an intact fallopian tube in vitro or establishment and maintenance of tubal epithelial cell-lines which could be used as feeder layers for early embryonic growth. The concomitant growth of cells with embryos in vitro has been referred to as co-culture. This paper discusses the in vitro behaviour of human tubal epithelial cells, the fertilisation and growth of embryos in ampullary co-culture, the specificity of co-cultures, the mechanism of action of co-cultures and the methods of screening the human ampullary co-culture system for microbes. The pregnancy and implantation results on 50 patients enrolled for a co-culture clinical trial are presented and the future use of this system discussed.
Annals of the Academy of Medicine, Singapore 08/1992; 21(4):571-5. · 1.25 Impact Factor
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ABSTRACT: To examine the fertilization rates of 48-hour unfertilized oocytes inseminated with fertile donor sperm and to evaluate the cleavage and cytogenetics of ensuing embryos.
Prospective.
Assisted reproductive technology (ART) program.
Four hundred ninety-seven unfertilized oocytes from 97 ART patients were categorized into four groups. A (zona-intact) and B (zona-free) were from patients with partial fertilization failure, whereas C (zona-intact) and D (zona-free) were total fertilization failures.
Fertilization rates in groups A and B were significantly higher than C and D (33.2% to 60.9% versus 20.0% to 48.1%; P less than 0.01). Zona-free oocytes had higher fertilization rates than zona-intact oocytes (48.1% to 60.9% versus 20.0% to 32.2%). Multiple pronuclei were high in zona-free oocytes (33.1% to 41.3%). Forty-eight to 54% of embryos generated after donor insemination had chromosome anomalies (mosaicism, aneuploidy, pulverization).
One cause of total fertilization failure appears to lie in intrinsic oocyte problems confined to the zona and oolemma. The fertilization of 48-hour unfertilized oocytes may be of some value in diagnosing fertilization failure in ART patients.
Fertility and Sterility 02/1992; 57(1):129-33. · 3.56 Impact Factor
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ABSTRACT: Passaged human tubal ampullary epithelial cells from cell lines were used to evaluate fertilization rates and the support of early human embryonic cleavage in vitro. A total of 225 mature oocytes from 32 patients was used in this study. Oocytes from each patient were equally allotted at random to two groups. One hundred thirty-two oocytes were inseminated in the presence of passaged human ampullary cells, while 123 oocytes were inseminated in culture medium alone. Fertilized oocytes were transferred to tubal ampullary cell monolayers and monitored for up to 48 hr. Eighty-five percent of oocytes fertilized in coculture, as compared to 67% in medium alone (P less than 0.01). A higher percentage of good-quality embryos was observed in cocultures than in controls (79 vs 67%; P less than 0.05). Binding of sperm to ampullary monolayer cells was observed. Human ampullary-cell cocultures may be useful to improve fertilization rates and embryonic viability and thus increase take-home baby rates for in vitro fertilization programs.
Journal of In Vitro Fertilization and Embryo Transfer 09/1991; 8(4):216-21.
