Regulation of human and mouse oocyte maturation in vitro with 6-dimethylaminopurine

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.     

Intracytoplasmic injection of human sperm into the hamster oocyte (hamster ICSI assay) as a test for fertilizing capacity of the severe male-factor sperm

Objective: Our objective was to investigate the fertilizing ability of human sperm from severe male-factor patients, by microinjection of single sperm into the hamster oocyte. Design: Semen samples of severe male factor either with a 0% penetration rate in the zona-free hamster test or with a very low number of motile sperm for which performing the standard penetration test was impossible were used. For the control study, oligozoospermic semen samples with at least 10% penetration rate in zona free hamster test were used. Setting: All materials were collected from the National University Hospital, Singapore. Methods: There were 10 patients in both the experimental and the control groups. Intracytoplasmic sperm injection (ICSI) was carried out. The main outcome measures were sperm head decondensation and pronuclear formation. Results: Twenty-one percent of the injected sperm could decondense and undergo male pronuclear formation. This rate was not significantly different from that in the control study group (28%; P=0.13). A small proportion of the oocytes was damaged during the procedure (9.2 and 8.75% in experimental and control groups, respectively). Conclusions: Hamster-ICSI assay may be of benefit in predicting the sperm's ability for further development before allowing the patient to undergo the clinical program.     

ART practice in Singapore

Conclusions ART practice in Singapore is monitored by the MOH, with guidance from an Advisory Committee on HRE. All centers and practitioners must be licensed. Data collection is by means of three forms. Based on the submissions, the averages for all centers in Singapore for the 5 years 1990 to 1995 were as follows: clinical pregnancies averaged 16.4% per cycle and 22.6% per replacement, and take-home baby rates averaged 11.5% per cycle and 15.9% per replacement.The opinions presented in this column are those of its authors and do not necessarily reflect those of the journal and its editors, publisher, and advertisers.     

Effect of sperm motility on human in vitro fertilization

Several sperm motility parameters in semen prepared by the swim-up technique were compared with IVF rates in 84 patients. The patients were either on clomiphene + human menopausal gonadotrophin or follicle stimulating hormone + human menopausal gonadotrophin stimulation regimens. Motility ratings were assessed both manually according to World Health Organization guidelines as well as computer-automated semen analysis (Cellsoft, Cryoresources, USA). Motility ratings of greater than or equal to 2 yielded significantly higher fertilization rates (78-82%) than ratings below 2 (20-23%) (p less than 0.001) for patients on both regimens. Velocity (41, 55, 78 microns/sec) and mean amplitude of lateral head displacement (1.96, 3.29, 4.91 microns) correlated significantly with and between manual ratings of 1, 2, and 3, respectively (r = 0.83; p less than 0.01). No significant differences were observed in linearity and beat/cross frequency between the manual ratings, although beat/cross frequencies tended to reduce linearly with increases in intensity of motility. The velocity of sperm motility has a significant effect on fertilization rates, and cut-off points of greater than or equal to 2 or greater than or equal to 50 microns/sec predict the actual potential and likely success of in vitro fertilization. These criteria on the swim-up semen should be used in the selection of patients admitted to IVF programs, and they justify the necessity of research investigations to improve motility in those patients with sluggish motility.     

Nine-day-old human embryo cultured in vitro: a clue to the origins of embryonic stem cells

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.     

History and perspective of stem cell research

Several types of stem cell have been discovered from germ cells, the embryo, fetus and adult. Each of these has promised to revolutionize the future of regenerative medicine through the provision of cell-replacement therapies to treat a variety of debilitating diseases. Stem cell research is politically charged, receives considerable media coverage, raises many ethical and religious debates and generates a great deal of public interest. The tremendous versatility of embryonic stem cells versus the unprecedented reports describing adult stem cell plasticity have ignited debates as to the choice of one cell type over another for future application. However, the biology of these mysterious cells have yet to be understood and a lot more basic research is needed before new therapies using stem-cell-differentiated derivatives can be applied. Stem cell research opens-up the new field of 'cell-based therapies' and, as such, several safety measures have also to be evaluated.     

Human blastocyst culture and derivation of embryonic stem cell lines

Human embryonic stem cell (hESC) biology is expected to revolutionize the future of medicine by the provision of cell-based therapies for the treatment of a variety of deliberatig diseases. The tremendous versatility of hESCs has reinforced this hope. To understand the biology of these my sterious cells and attempt to differentiate them into desirable tissues, bona fide hESCs that maintain their stability with time are required for research and clinical application. This review discusses the various protocols to derive and propagate hESCs from high quality embryos. The nature and properties of hESCs are also described together with unanswered questions that need to be addressed if this science is to be taken to the bedside.     

Embryonic behavior of two-cell mouse embryos frozen by the one- and two-step ultrarapid techniques

Purpose A modified two-step ultrarapid freezing technique was compared to the one-step ultrarapid freezing technique. Two-cell mouse embryos were frozen-thawed using the two freezing protocols, and postthaw cryoprotectant removal was carried out in either a single- or a multiple-step procedure.Results Statistically similar cryosurvival (96.95–100%) and blastocyst formation rates (87.95–91.47%) were obtained with both freezing groups. In addition, the method of cryoprotectant removal did not have any significant effect on the survival and development of the frozenthawed embryos in both groups. Blastocysts formed following single-step cryoprotectant removal had significantly lower inner cell mass counts in the one-step than in the two-step group (26.14 and 27.59, respectively; P <0.05). Embryo transfer studies showed that the implantation and fetal formation rates of embryos frozen by the two-step technique (61.67 and 60.0%, respectively) were similar to those of embryos frozen by the one-step technique (74.12 and 71.76%, respectively). Conclusion These results demonstrate that the ultrarapid two-step technique is as effective in cryopreserving two-cell mouse embryos as the ultrarapid one-step technique.     

Amniocentesis and Its Complications

This study was conducted in order to evaluate whether the performance of an experienced operator had any significant influence in reducing the incidence of complications in amniocentesis; 1,459 women had amniocentesis performed under ultrasound guidance; 1,324 were performed by experienced operators and 135 cases by less experienced operators. Complications like fetal loss, blood-stained amniotic fluid, culture failure, multiple needle puncture, leaking liquor, fetal trauma and error in results were compared in the 2 groups. This study demonstrated that amniocentesis performed by an experienced operator decreased the various complications associated with amniocentesis.