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Biomedical subjects

A Trounson

Publications and source records attributed to A Trounson.

At least 37 records · Page 2Linked to original sources
PubMed1999

Birth following vitrification of a small number of human oocytes: case report.

We report the birth of a healthy baby girl at 37 weeks gestation to a 47 year old recipient, after vitrification of mature oocytes from four in-vitro fertilization (IVF) patients. A total of 17 oocytes was vitrified in 1-2 microl of ethylene glycol (40%) and 0.6 mol/l sucrose (20.54%) in open pulled straws. Eleven oocytes survived after vitrification and five pronuclear zygotes were obtained after intracytoplasmic sperm injection (ICSI). Three embryos were transferred to three patients, two of whom were the original oocyte donors and pregnancy was not established. The third embryo was donated to a 47 year old infertile woman after preimplantation diagnosis had confirmed euploidy for chromosomes X, 13, 14, 15, 16, 18, 21 and 22. The successfully completed pregnancy is encouraging for further research to explore the potential benefits of vitrification for the cryopreservation of human oocytes, given the relatively low success of conventional freezing of human oocytes by slow cooling methods.

Adult↗
PubMed1998

Potential benefits of cell cloning for human medicine.

The successful cloning of a mammal from an adult somatic cell nucleus opens new avenues for major advances in reproductive medicine, biotechnology and cellular-based transplantation therapies for degenerative diseases. At the same time, this breakthrough has generated much heated discussion concerning the ethics of cloning. Twinning is a form of cloning, and there are instances in clinical assisted reproduction in which the deliberate formation of twins by embryo dissection would seem ethically acceptable. Nuclear transfer technology might facilitate the derivation of human embryonic stem cells, capable of differentiation into a wide variety of somatic cell lineages. Directed differentiation of human embryonic stem cells into specific cell types in vitro could provide a universal source of cells for transplantation therapy. The potential benefits of therapeutics based on cloning technologies are considerable, and hasty legislation to ban all such procedures could block progress in critical arenas of biomedical research.

Animals↗
PubMed1998

Reprogramming cattle somatic cells by isolated nuclear injection.

The development of a somatic cell nuclear transfer procedure for the production of blastocyst stage cattle embryos is described. Bovine fetal fibroblasts were used for fusion experiments with surgically enucleated oocytes (cytoplasts) following the establishment of optimal parameters for electrofusion from isofusion contours. Fusion rates were increased by decreasing size of the cytoplasts used but cleavage was decreased by decreasing size of the cytoplast used (quarter, half and whole cytoplasts). The use of double cytoplasts did not improve cleavage, and development to blastocysts could not be achieved. In a comparison of electrofusion of fibroblasts with cytoplasts in the subzonal perivitelline space with intracytoplasmic injection of nuclei and parthenogenetically activated oocytes, 2%, 14% and 24% developed to blastocysts respectively. In the group injected with isolated nuclei, the passage number (4 to 9) had no apparent influence on developmental competence to blastocysts. The embryos produced by nuclear injection of somatic cell nuclei showed the normal pattern of cell surface appearance of TEC-3 and TEC-4 stage-specific epitopes during development, as seen in fertilized oocytes. We conclude that the nuclear injection of somatic cell nuclei is a relatively efficient way to clone bovine embryos.

Animals↗
PubMed1998

Novel method for demonstrating nuclear contribution in mouse nuclear transfer.

Confirmation of nuclear contribution is essential to all nuclear transfer experiments. Contribution is easily demonstrated in nuclear transfer progeny but more difficult to confirm in nuclear transfer embryos. The use of donor nuclei isolated from lacZ transgenic mice offers a clear and simple method to demonstrate contribution in nuclear transfer embryos and offspring. The unique line of transgenic mice (Zin40) used in this study displays nuclear localised lacZ expression in all cells, including embryonic blastomeres, and demonstrates distinctive blue nuclei when treated with X-gal substrate. This characteristic staining pattern provided an ideal marker for demonstrating nuclear contribution. Nuclear transfer embryos were generated following serial nuclear transfer of metaphase-arrested nuclei from transgenic and non-transgenic 4-cell embryos. Totipotency of nuclear transfer blastocysts was confirmed by the generation of live born offspring. Transgenic blastocysts and all tissue samples from fetuses and pups generated by nuclear transfer displayed distinctive blue nuclei when stained with X-gal. This staining pattern was characteristic of the transgenic mice from which the donor nuclei were isolated and clearly confirmed nuclear origin. The use of this marker will also allow the opportunity to investigate the developmental potential of nuclear transfer embryos by examining the contribution of nuclear transfer embryonic cells in chimaeric embryos.

Animals↗
PubMed1998

Blastocyst transfer after enzymatic treatment of the zona pellucida: improving in-vitro fertilization and understanding implantation.

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.

Adult↗
PubMed1998

Oocyte maturation.

Primary oocytes recovered from small and growing follicles of > or = 3 mm in the ovaries of untreated women, can be matured in vitro, will fertilize and develop in vitro, and when transferred to the patient, develop to term. However, the implantation rate of cleaved embryos has been disappointingly low and when embryos are allowed to develop beyond the 4-cell in vitro, retardation of development and blockage is frequently observed, with relatively few embryos developing to blastocysts. We have devised new culture systems for human embryos to enable high rates of development of in-vivo matured oocytes to blastocysts within 5-6 days of culture, and high implantation rates of these blastocysts when they are transferred to the patients' uterus. These culture systems are now being used for in-vitro matured oocytes. In order to determine whether embryo developmental competence could be improved, a number of factors were examined. Treatment of patients with pure follicle stimulating hormone (FSH) early in the follicular phase, or treatment with oestrogen prior to oocyte recovery, had no apparent effect on any parameters of oocyte developmental competence. There was no indication that a medium made specifically for human oocyte maturation improved oocyte developmental competence. Nuclear and cytoplasmic changes in oocytes matured in vitro appear to be similar to that in vivo, although some lack of synchronization in completing maturation is evident. It is possible that follicles of < 10 mm diameter in the human contain developmentally-incompetent oocytes. However, the development to term and birth of normal babies from germinal vesicle stage oocytes recovered from small follicles and matured in vitro, suggests that further research will identify the factors necessary to improve embryo developmental competence. The application of immature oocyte collection (IOC) and in vitro maturation (IVM) as an alternative to ovulation stimulation with high doses of gonadotrophins for in-vitro fertilization (IVF), remains a priority for research in human medicine.

Animals↗
PubMed1998

Azoospermia associated with a mutation in the ligand-binding domain of an androgen receptor displaying normal ligand binding, but defective trans-activation.

Although male infertility affects a significant proportion of couples trying to conceive, the cause of defective spermatogenesis is not known in a large number of cases. Ligand binding studies indicate that a number of these subjects may have defects of the androgen receptor (AR). Genetic screening in subjects with defective spermatogenesis and in 110 fertile controls identified an azoospermic (no sperm in any ejaculates) patient with an amino acid substitution (Gln-->Glu) in residue 798 of the AR gene. This germline mutation was pathogenic because it was not observed in fertile controls, was associated with features of minimal androgen insensitivity in our patient, has been related to more severe grades of androgen insensitivity, and caused a subtle, but significant, decrease in receptor trans-activation function in vitro that is consistent with the phenotype. Despite being located in the middle of the ligand-binding domain of the receptor, the Q798E mutation did not cause any ligand binding defect, indicating that this highly conserved residue has a trans-activation function but does not directly form part of the ligand binding pocket of the receptor. The trans-activation defect of the mutant receptor can be rectified in vitro with the androgenic drug, fluoxymesterone, but not with mesterolone or nortestosterone. Further studies are required to determine the therapeutic relevance of this finding.

Adult↗
PubMed1997

Is the acrosome reaction a prerequisite for sperm incorporation after intra-cytoplasmic sperm injection (ICSI)?

There is debate as to whether the acrosome reaction is necessary for sperm incorporation after intra-cytoplasmic sperm injection (ICSI). Ultrastructural evidence is presented to show that the acrosome reaction could occur in the ooplasm before sperm incorporation in mature human oocytes or the acrosome could be discarded intact before sperm incorporation in immature oocytes, matured in vitro. Both germinal vesicle and growing follicular oocytes showed sperm chromatin decondensation, with discarded acrosomes close to the sites of incorporation, and were able to form male pronuclei. This is probably the first report of microfertilization of a growing oocyte with a reticulate nucleolus by ICSI. The acrosome reaction, when it occurs, is preceded by acrosome swelling and is followed by vesiculation of surface membranes exposing the inner acrosome membrane, as observed on the surface of the zona during IVF or in the perivitelline space after subzonal sperm injection. These sperm were probably capacitated at the time of ICSI. There was subtle evidence of leaching of the acrosomal matrix from intact discarded acrosomes and from partially depleted acrosomes attached to decondensing spermheads. These sperm were probably not fully capacitated at the time of ICSI. It is concluded that both the acrosome reaction and acrosome deletion are possible prerequisites to sperm incorporation after ICSI.

Acrosome↗
PubMed1997

Ongoing normal pregnancy after transfer of zona-free blastocysts: implications for embryo transfer in the human.

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.

Adult↗
PubMed1996

Production of embryos from in vitro-matured primary human oocytes.

OBJECTIVE: To determine the factors that influence the number and quality of embryos produced from primary oocytes collected from untreated regularly ovulating and irregular or anovulatory polycystic women. DESIGN: A direct comparison between two patient groups whose oocytes were matured in vitro and a comparison of the embryo development of in vitro-matured oocytes from untreated patients with in vivo-matured oocytes of superovulated IVF-ET patients obtained during the same period. SETTING: The Monash IVF Clinic, involving patients who expressed the desire to avoid super-ovulation with fertility drugs. MAIN OUTCOME MEASURES: The completion of nuclear maturation of oocytes after 36 or 48 hours culture, fertilization in vitro, and embryo development ratio. RESULTS: Oocytes from regular cycling patients matured and fertilized at significantly higher rates than irregular cycling and anovulatory women and their embryos had significantly higher mean embryo development ratio. The mean embryo development ratio of embryos of regular cycling patients was similar to superovulated IVF patients but irregular cycling and anovulatory patients had a significantly lower embryo development ratio. Culture of oocytes for 48 hours increased maturation of oocytes from 57% to 82% but did not affect fertilization or cleavage rates. Embryo development was not affected significantly by the grade of follicular cell cover of oocytes. CONCLUSIONS: The developmental capability of primary oocytes is higher in regular cycling women than in irregular cycling and anovulatory women with polycystic ovary disease.

Anovulation↗
PubMed1996

The sperm centriole: its inheritance, replication and perpetuation in early human embryos.

The inheritance, replication and perpetuation of the sperm centriole in the early human embryo are reported. Both normal monospermic and abnormal dispermic embryos (n = 127) were examined by transmission electron microscopy. Centrioles were traced from fertilization to the hatching blastocyst stage. The sperm proximal centriole is introduced into the oocyte at fertilization and remains attached to the expanding spermhead during sperm nuclear decondensation, as it forms the male pronucleus. A sperm aster is initially formed after the centriole duplicates at the pronuclear stage. At syngamy, centrioles occupy a pivotal position on opposite spindle poles, when the first mitotic figure is formed. Bipolar spindles were found in the majority of embryos, while tripolar spindles were seen in four dispermic embryos at syngamy. Two single centrioles were detected at two poles of two tripolar spindles, while two additional centrioles were located on the sides of a bipolar spindle of a dispermic embryo. Sperm tails were detected near spindle poles at syngamy and in later embryos. Typical centrioles showing the characteristic pin-wheel organization of nine triplets of microtubules were evident. During centriolar replication, the daughter centriole grows laterally from the parent and gradually acquires pericentriolar material (PCM). The two centrioles are surrounded by a halo of electron-dense PCM, which nucleates microtubules, thus making it a typical centrosome. The usual alignment of diplosomes at right angles to each other was maintained. Centrioles were detected at all stages of embryonic cleavage from the 1-cell through 8-cell stages, right up to the hatching blastocyst stage. They were closely associated with nuclei at interphase, when they were often replicating, and were prominently located at spindle poles during the first four cell cycles. In blastocysts, they were detected in trophoblast, embryoblast and endoderm cells respectively. It is evident that the sperm centrosome is the functional active centrosome in human, while the female is inactive but may contribute some centrosomal material to the zygote centrosome. It is very likely that the paternal centriole is the ancestor of the centrioles in fetal and adult somatic cells.

Blastocyst↗
PubMed1996

Evaluation of motility, freezing ability and embryonic development of murine epididymal sperm after coculture with epididymal epithelium.

Murine sperm from the caput, corpus and cauda epididymis were cocultured with epididymal epithelial cells of their own region or more distal regions, in the presence and absence of androgens (testosterone and dihydrotestosterone). Epithelial cell cultures were used 3 or 10 days after preparation in a complex tissue culture medium (Chang's) as plated tubules. The coculture studies involving spermatozoa and oocytes with epithelial cells were carried out in T6 medium. Motility of caput spermatozoa was maintained for 24 h in the presence of day 3 corpus and cauda epithelial cells and hormones but not under other conditions. Likewise, the motility of corpus spermatozoa was maintained for 24 h in the presence of day 3 cauda epithelial cells and hormones but not other conditions. Fertilization of zona-intact oocytes by epididymal spermatozoa was not affected by their coculture for 24 h with epithelial cells but fertilization rates for zone-free oocytes were increased for caput spermatozoa cocultured with more distal epithelial cells. Fertilization rates for both zona-intact and zone-free oocytes were increased for corpus spermatozoa cocultured with more distal cauda epithelial cells. The developmental capacity of embryos derived from caput spermatozoa was not significantly increased by coculture with epithelial cells but those derived from corpus spermatozoa cocultured with cauda epithelial cells were significantly increased. We conclude that the presence of more distal epithelial cells of the mouse epididymis maintains motility in culture, increases the ability of caput and corpus spermatozoa to fertilize zona-free oocytes and increases the developmental capacity of embryos formed from corpus spermatozoa. These observations demonstrate the function of epididymal regions in the maturation of murine spermatozoa for fertilization and embryo development.

Animals↗
PubMed1996

Update on micromanipulation techniques for assisted conception.

This article reviews the progress in the field of micromanipulation in the past year. The major event has been the wide application of intracytoplasmic sperm injection. Other advances include haploid spermatid injection and the development of successful mouse models for intracytoplasmic sperm injection. There is now increasing awareness of the genetic basis of male infertility and its possible impact on how we offer treatment.

Animals↗
PubMed1995

Mouse sperm fertilising capacity following subzonal microinjection is dependent on sperm washing and response to solubilised zonae pellucidae.

Spermatozoa obtained from hybrid male mice were treated with solubilised zonae pellucidae after a period of capacitation in vitro to induce the acrosome reaction. Single spermatozoa were selected and microinjected into the perivitelline space of mature oocytes. A high proportion of the spermatozoa acrosome-reacted after treatment with solubilised zonae (63%). However, the fertilisation rate (37%) after subzonal microinjection of oocytes by the treated spermatozoa was not different to the fertilisation rate (40%) of oocytes microinjected with untreated spermatozoa which had a lower rate of acrosome reaction (39%). When spermatozoa were washed by high-speed centrifugation before treatment with solubilised zonae pellucidae and subzonal microinjection, the fertilisation rate (68%) was significantly higher (p < 0.001) than that (29%) for oocytes microinjected with untreated spermatozoa and was found to be correlated with a high acrosome reaction rate (74%) (r = 0.8). The washing of spermatozoa by centrifugation itself did not increase the acrosome reaction rate or fertilisation rate of oocytes after microinjection. The results of this study suggests that some modifications other than the acrosome reaction are needed to enable capacitated mouse spermatozoa to fuse with the oocyte plasma membrane. These modifications were achieved by washing spermatozoa by high-speed centrifugation and the replacement of the supernatant with fresh culture medium used for capacitation. Induction of the acrosome reaction by solubilised zonae pellucidae following this treatment leads to a high fertilisation rate of oocytes by subzonal sperm microinjection.

Acrosome↗