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Biomedical subjects

A Trounson

Publications and source records attributed to A Trounson.

At least 55 records · Page 3Linked to original sources
PubMed1995

Intracytoplasmic sperm injection in mice: increased fertilization and development to term after induction of the acrosome reaction.

A technique has been developed for intracytoplasmic sperm injection (ICSI) in the mouse with a relatively low rate of lysis of oocytes (range 5-25% across experiments) and pronuclear formation in around one-third of the intact oocytes (range 30-38% across experiments) for untreated spermatozoa. The treatment of spermatozoa with calcium ionophore, to induce the acrosome reaction (increases acrosome-free spermatozoa from 28 to 58%) before ICSI, increased pronuclear formation to approximately 60% (range 59-62% across experiments) in intact oocytes. The pronuclear oocytes developed to blastocysts in vitro and to term when transferred to recipient foster mothers at rates equivalent to zygotes formed after insemination in vitro. There was no benefit for fertilization rates of activating oocytes with 8% ethanol before or after ICSI, nor was there any evidence of parthenogenetic activation by the sperm solution used for ICSI. This technique adds to other in-vitro fertilization techniques which can be used to explore gamete interactions and to recover breeding in infertile strains and reproductively unfit mice.

Acrosome↗
PubMed1994

In vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients.

OBJECTIVE: To determine the maturational and developmental competence of immature oocytes recovered in situ from anovulatory and ovulatory patients with polycystic ovaries (PCO). DESIGN: A newly designed method for recovery of immature oocytes from 2 to 10 mm follicles by transvaginal ultrasound or laparoscopy was used to compare the recovery and maturation of oocytes from 9 anovulatory polycystic ovarian syndrome (PCOS) patients and 10 ovulatory patients without polycystic ovaries (PCO) (experiment 1). In a second study (experiment 2), we compared the maturation, fertilization, and development of oocytes recovered from another 10 anovulatory PCOS and 13 ovulatory PCO patients. Two types of culture methods and time intervals for maturation were also examined. RESULTS: In experiment 1, a significantly higher number of immature oocytes were recovered from PCOS patients (15.3) compared with non-PCO patients (2.8). Sixty-five percent of oocytes cultured in medium with gonadotropins, estrogen, and fetal calf serum matured to metaphase II by 43 to 47 hours, and 81% were mature at 48 to 54 hours of culture. Thirty-four percent of the inseminated oocytes fertilized and 56% of the cultured pronuclear oocytes cleaved to eight cells or more. In experiment 2, there was no significant difference between anovulatory PCOS and ovulatory PCO patients in the number of oocytes recovered or their maturation, fertilization, and development. There was no difference between oocytes matured in medium or in coculture with mature granulosa cells, with or without added hCG. However, significantly fewer oocytes were immature and more fertilized when oocytes were inseminated after 34.5 to 35.5 hours of maturation than 29.5 to 32.5 hours of maturation. A pregnancy and the birth of a normal baby occurred in one of the anovulatory PCOS patient receiving an abbreviated steroid replacement protocol after ET. CONCLUSION: Immature oocyte recovery could be developed as a new method for the treatment of women with infertility due to PCO because the oocytes of these patients retain their maturational and developmental competence.

Anovulation↗
PubMed1994

Microinjection: choice of embryo transfer technique.

Subzonal sperm microinjection (SUZI) is indicated in severe oligoasthenozoospermia, in which the total count of motile sperm is inadequate for in vitro fertilization (IVF), and in cases with repeated failure of fertilization. Sperm for microinjection are selected following centrifugation on a Percoll gradient and stimulation with pentoxifylline and 2-deoxyadenosine. Motile sperm (2-10 per egg) are injected into the perivitelline space and fertilized oocytes are then cultured for two days prior to transfer into the Fallopian tube (tubal embryo stage transfer, TEST) or uterus. During 1992, SUZI results showed a total fertilization rate of 30% (19% were 2 pronuclear, 11% were polyspermic), a transfer rate of 55% and pregnancy rates of 15.2% per transfer and 8.3% per cycle. Recent pregnancy data in mild-moderate male factor infertility showed that gamete intrafallopian transfer (GIFT) results were consistently superior to TEST or IVF, suggesting a beneficial effect of the tubal environment on fertilization and early embryonic development. Accordingly, the combination of SUZI followed by the immediate transfer of injected oocytes into the Fallopian tube, the MIFT procedure, was explored. An initial study of 21 consecutive microinjection candidates showed a clinical pregnancy rate of 24% per cycle. Information regarding fertilization and polyspermy rates was available from supernumerary oocytes in 90% of patients. A randomized, controlled trial comparing MIFT with SUZI or TEST in severe male factor infertility is required to confirm the improved pregnancy rate in MIFT cycles.

Embryo Transfer↗
PubMed1993

The effects of the sperm motility activators 2-deoxyadenosine and pentoxifylline used for sperm micro-injection on mouse and human embryo development.

The sperm motility stimulants 2-deoxyadenosine (DOA) and pentoxifylline (PTF), used to improve the success of insemination and sperm micro-injection for low motility sperm samples, were studied for their effects on the developmental capacity of mouse and human oocytes. When human oocytes were micro-injected with spermatozoa in 3 mM DOA 80% of them became blocked at the 1-cell pronuclear stage. However, when spermatozoa in 3 mM PTF were used for micro-injection or when spermatozoa were washed to remove DOA before micro-injection only a few oocytes (9-10%) were blocked. Pregnancies occurred in five of 14 patients into whom cleaving embryos from all three treatments had been transferred, indicating that once cleavage was initiated, development of embryos occurred at expected rates. Exposure of mouse oocytes to DOA for a short period during insemination (4-6 h) or a longer period during the pronuclear cell cycle (18-20 h) significantly reduced cleavage beyond the 2-cell stage, resulting in a dramatic reduction in blastocyst formation. PTF did not significantly reduce mouse embryo development. Similar results were obtained for oocytes inseminated in vitro or micro-injected with a spermatozoon into the perivitelline space. Neither DOA nor PTF increased fertilization of mouse oocytes. PTF reduced fertilization, particularly in cumulus-enclosed oocytes and oocytes micro-injected with spermatozoa in PTF. We conclude that DOA is a potent inhibitor of embryo development and oocytes should not be exposed to DOA. Exposure of oocytes to PTF had little effect on their subsequent development but treatment of cauda epididymal mouse spermatozoa can reduce their fertilizing capacity.

Animals↗
PubMed1992

The effects of cooling mouse oocytes.

The effects of cooling and warming on meiotic spindles of mouse oocytes have been assessed by transmission electron microscopy. Intact cumulus-oocyte complexes were immediately cooled from 37 to 15, 4, 0, and -7 degrees C (seeding temperature) for 15 min in a programmed biological freezer and fixed at these temperatures. Other complexes, cooled to these temperatures, were rapidly warmed to 37 degrees C and incubated for 2 hr before fixation at 37 degrees C. Of 334 oocytes assessed at various temperatures, at least 100 were examined for metaphase II spindles. Spindle microtubules completely disappear at 0 and -7 degrees C, while complete or partial depolymerization of microtubules was observed at 4 degrees C. Cooling to 15 degrees C did not cause major disruptions of spindle structure in most oocytes. Chromosomes tended to rotate or clump at lower temperatures but chromosome scatter outside the spindle zone was rarely observed. Centrosomal material was fragmented at 4 degrees C and occasionally at 15 degrees C and was not evident at the spindle poles at 0 and -7 degrees C. Kinetochores were seen at all temperatures. Spindle structure was evidently restored in the majority of oocytes on rewarming at 37 degrees C. Changes in the ooplasm induced by cooling were elongation and disruption of vesicular smooth endoplasmic reticulum, especially between lipid globules and disappearance of fibrillar inclusions. Cortical granule exocytosis was not observed on cooling, while microfilaments were intact. Swelling of membranous organelles was also observed in cumulus cells. Most of the cytoplasmic changes were also reversed on rewarming. The response of mouse oocytes to cooling is compared to that of human oocytes, reported previously.

Animals↗
PubMed1992

Subzonal sperm microinjection in cases of severe male factor infertility and repeated in vitro fertilization failure.

OBJECTIVE: To examine (1) fertilization rates obtained with subzonal sperm microinjection when different numbers of sperm are injected into the perivitelline space; (2) when subzonal sperm microinjection is combined with dilute insemination; and (3) the association of semen quality characteristics with fertilization. DESIGN: Subzonal sperm microinjection and subzonal sperm microinjection combined with dilute insemination was performed in 109 and 41 cycles on patients in two clinical trials in Melbourne, Australia, and Bologna, Italy, respectively. PATIENT PARTICIPANTS: Couples who have experienced repeated in vitro fertilization failure or in whom the husband has severe male factor infertility. PRIMARY OUTCOME MEASURES: The number of oocytes fertilized after injection of different numbers of sperm into the perivitelline space, the number of patients transferred, and pregnancy outcome. RESULTS: The injection of multiple numbers of sperm into the perivitelline space failed to improve monospermic fertilization rates but caused an increase in polyspermic fertilization. In patients with initial semen parameters exhibiting greater than 50% motility or greater than 50% normal morphology fertilization rates were improved when subzonal sperm microinjection-treated eggs were incubated in a dilute insemination medium. Six pregnancies were obtained, two of which have progressed to term. CONCLUSIONS: When applied to male factor patients, the subzonal sperm microinjection technique results in a 14% to 15% fertilization rate. However, of the 102 embryos transferred only three (2.9%) fetal heart beats were obtained.

Female↗
PubMed1992

The incidence of chromosomal aneuploidy in stimulated and unstimulated (natural) uninseminated human oocytes.

The incidence of chromosomal aneuploidy in human oocytes is higher than for various animal species. Since this estimate for aneuploidies is based on data obtained from in-vitro fertilization (IVF) patients, it is possible that superovulation could be contributing to this phenomenon. In this study we determine the incidence of chromosomal aneuploidy in nonstimulated uninseminated human oocytes donated by IVF patients. Furthermore, we compare this incidence of aneuploidy to that obtained after superovulation using two different protocols for induction of multiple follicular growth. The rate of aneuploidy in non-stimulated oocytes was 20% (4/20). This is not significantly different from the rate of aneuploidy in oocytes obtained after superovulation with clomiphene/human menopausal gonadotrophin (HMG)/(HCG) (15/43 = 35%, chi 2 = 1.11; P > 0.20), buserelin-flare (8/25 = 32%; chi 2 = 0.32; P > 0.05), and the rate of aneuploidy in the total number of superovulated oocytes (23/68 = 34%; chi 2 = 82; P < 0.30). Furthermore, the incidence of chromosome aneuploidy in non-stimulated uninseminated oocytes (20%) was well within the range and not significantly different from that reported in the literature for both superovulated uninseminated oocytes (range, 21-57%; total aneuploidy rate, 67/216 = 31%; P < 0.30) and superovulated inseminated oocytes (range, 3-56%; total aneuploidy, 339/1480 = 23%; P < 0.95). Consequently, the data provide evidence that superovulation protocols used in IVF may not be responsible for the higher rate of aneuploidy in human oocytes. These results are discussed in relation to hypotheses on the occurrence of meiotic non-disjunction.

Adult↗
PubMed1992

Dopamine treatment for severe ovarian hyperstimulation syndrome.

Seven oliguric patients with severe ovarian hyperstimulation syndrome following gonadotrophin treatment for in-vitro fertilization or gamete intra-Fallopian transfer, were treated with low doses of dopamine by peripheral infusion. Five patients were pregnant. The rationale for this therapeutic approach was to increase renal blood flow and glomerular filtration. In addition to dopamine, fluid intake was restricted to 500 ml/day and a protein and salt-rich diet was provided in order to increase serum osmolarity. Within 24-48 h from the beginning of the dopamine treatment, the syndrome started to regress in all cases. No adverse maternal or fetal effects occurred. We conclude that dopamine therapy may constitute a major advance towards the management of severe ovarian hyperstimulation syndrome.

Dopamine↗
PubMed1991

Centrioles in the beginning of human development.

We demonstrate the presence of centrioles in fertilized human oocytes at syngamy. Single or double centrioles within centrosomes were detected by transmission electron microscopy at one pole of the first cleavage spindle in normal and dispermic embryos (25-26 hr after insemination). Sperm centrioles were also closely associated with the male pronucleus (16-20 hr after insemination) in pronuclear stage embryos. A tripolar spindle derived from a tripronuclear embryo is also demonstrated with two centrioles at one pole. The data provide evidence that human centrioles, as those in most other animals, and unlike the mouse, are paternally derived, thus supporting Boveri's classical theory. Furthermore, this study provides insights to the proposed mechanisms of aberrant cleavage patterns of dispermic human embryos.

Blastocyst↗
PubMed1991

Fertilizing capacity of epididymal and testicular spermatozoa microinjected under the zona pellucida of the mouse oocyte.

Mouse spermatozoa were obtained from the testis and caput, corpus, and cauda epididymis incubated for 2 h in capacitation medium and a single spermatozoon from the capacitated samples microinjected into the pervitelline space of mature mouse oocytes. Spermatozoa from the testis were unable to fertilize oocytes and few spermatozoa from the caput were capable of fertilization (0-7% depending on the method of preparation). Similar fertilization rates (30-45%) were obtained with spermatozoa with forward progressive motility from the proximal and distal corpus and the cauda epididymis, but those that were vibratory or with local circular motility had significantly reduced fertilizing capacity (6-10%). The capacity of spermatozoa from the different regions of the testis and epididymis to bind to zona-free oocytes followed the same pattern as fertilization rate after microinjection. There was a progressive increase in acrosome reactions after 2 h incubation in capacitation medium in samples obtained from the testis to the cauda epididymis. Maturation of the capacity to bind to the perivitelline membrane and to develop forward progressive motility, rather than the capacity to acrosome react, appears to govern the fertilization of oocytes in which the zona has been bypassed by microinjection. These characteristics are obtained in the proximal segment of the corpus. However, there was evidence that the embryo developmental capacity of oocytes fertilized by spermatozoa from the higher segments of the epididymis is reduced, particularly in oocytes fertilized by caput spermatozoa. These observations suggest that sperm microinjection may have only a limited benefit for improving fertilization rates for men with high epididymal obstructive azoospermia.

Acrosome↗
PubMed1991

Rapid freezing of the mouse blastocyst: effects of cryoprotectants and of time and temperature of exposure to cryoprotectant before direct plunging into liquid nitrogen.

This study investigates the effects of time and temperature of exposure to a high concentration (4.5 M) of dimethyl sulfoxide (DMSO), glycerol, 1,2-propanediol (PROH), or a mixture of DMSO and glycerol (DG) in a solution containing 0.25 M sucrose, on the survival and development of rapidly frozen mouse blastocysts. Embryos had significantly (P less than 0.01) higher rates of survival and development when exposed to cryoprotectant at 0 degree C compared with room temperature. The time of exposure to cryoprotectant at either 0 degree C or room temperature before being plunged into liquid nitrogen significantly (P less than 0.01) affected the survival and development of frozen-thawed embryos. Survival and development of blastocysts in vitro and in vivo was significantly (P less than 0.05) higher when exposed at 0 degree C for 10 min to DG, DMSO and glycerol than to PROH. It is concluded that, unlike early-cleavage stage embryos, blastocysts need to be equilibrated at a low temperature (0 degree C) with high concentrations of cryoprotectant before rapid freezing. Exposure of blastocysts to 4.5 M cryoprotectant and 0.25 M sucrose at room temperature either was toxic or else markedly reduced their viability after freezing and thawing, depending on the duration of the initial exposure.

Animals↗
PubMed1990

Developmental capacity of mechanically bisected mouse morulae and blastocysts.

Mouse embryos were mechanically bisected at the morula, early blastocyst or expanded blastocyst stages of development and cultured in vitro to the expanded blastocyst stage. Their capacity for postimplantation development was assessed after transfer to pseudopregnant foster mice. Embryos bisected at blastocyst stages had a higher survival rate in vitro than those bisected at the morula stage. Half-embryos had approximately half the number of cells at the blastocyst stage as control embryos, but the proportion of cells in the inner cell mass (ICM) was unaltered. The implantation rate of blastocysts derived from bisected embryos was only slightly lower than that of control embryos, but bisected embryos had a significantly reduced capacity to form fetuses. Histological analyses showed that failure to form a fetus is due to the absence of egg cylinder development, which correlates with the reduced number of cells in the ICM of bisected embryos. Postimplantation viability of half-embryos was significantly higher when blastocysts were transferred to Day-3 rather than Day-4 pseudopregnant recipients, presumably because of an increase in cell number in vivo prior to implantation.

Animals↗
PubMed1990

Chromosomal analysis of human oocytes fertilized by microinjection of spermatozoa into the perivitelline space.

In-vitro fertilization by sperm microinjection techniques have been shown to be effective (the birth of one child has resulted), but concerns about the chromosomal normality of embryos derived from this process have been raised. The British Interim Licensing Authority has set, as a pre-condition to the clinical application of sperm microinjection, the chromosomal evaluation of pre-embryos derived by this technique. In this study, we demonstrate that four of 18 (22%) eggs fertilized by sperm microinjection into the perivitelline space are chromosomally abnormal as compared with nine of 30 (30%) fertilized by conventional IVF procedures. These results are not significantly different. In addition, we demonstrate that nine out of 20 couples who did not previously have fertilization in multiple cycles of IVF had between one and three oocytes fertilized by sperm microinjection. These findings demonstrate that sperm microinjection does not increase the incidence of chromosomally abnormal eggs and provide reassurance and support for the clinical implementation of a technique that appears to be effective for the treatment of certain forms of male infertility.

Aneuploidy↗
PubMed1989

A controlled study of luteinizing hormone-releasing hormone agonist (buserelin) for the induction of folliculogenesis before in vitro fertilization.

Treatment with clomiphene citrate and human menopausal gonadotropin (HMG) is often used to induce folliculogenesis before in vitro fertilization, but not all women have an adequate response. It has been hypothesized that abnormally high levels of luteinizing hormone (LH) may contribute to the reduced folliculogenesis. We therefore performed a controlled, open trial in which treatment with buserelin, an agonist of luteinizing hormone-releasing hormone citrate and HMG in 44 consecutive women in whom no oocytes or only one had been produced by standard treatment with clomiphene and HMG. Twenty-nine women received buserelin with HMG, and 15 received clomiphene citrate with HMG. The median number of oocytes per patient recovered from those who received buserelin with HMG was 4 (range, 0 to 19), as compared with 0 (range, 0 to 5) in those who received clomiphene citrate with HMG. The fertilization rates of oocytes recovered from both groups of patients were similar (75.8 percent and 76.5 percent, respectively). Fifty-four percent of patients given buserelin with HMG underwent triple-embryo transfer, as compared with 13 percent of those given clomiphene citrate with HMG. Pregnancy (n = 3) occurred only among the patients receiving buserelin with HMG. In the buserelin-HMG group, significantly fewer oocytes were recovered from patients with occult ovarian failure (infertility and elevated follicular-phase levels of follicle-stimulating hormone, with regular menses) (median, 1; range, 0 to 4) than from those with other causes of infertility (median, 8; range, 0 to 19). Our data suggest that, except in women with occult ovarian failure, buserelin and HMG improve embryologic and clinical outcomes in patients with previously unsatisfactory stimulation of the ovaries for in vitro fertilization.

Administration, Intranasal↗
PubMed1989

Fertilization and development of mouse eggs injected under the zona pellucida with single spermatozoa treated to induce the acrosome reaction.

Mouse spermatozoa were capacitated and acrosome reacted by incubation for 30 min to 6 h in modified Tyrode's medium (T6) and in cGMP. The fertilization rates of eggs microinjected with a spermatozoon from samples incubated for 30 min, 2 h, and 6 h in T6 and in cGMP were 36%, 34%, 29%, and 43%, respectively. These rates were not correlated to the percentage of acrosome-reacted spermatozoa identified after these treatments. The lack of association could be explained by the selection of an increased proportion of acrosome-reacted spermatozoa actually used for microinjection, particularly in samples incubated for 30 min and 2 h in T6. Both acrosome-intact and acrosome-reacted spermatozoa were identified under the zona in the eggs which failed to fertilize. Fertilized eggs developed at very high rates to the blastocyst stage in vitro.

Acrosome↗
PubMed1989

Human amniotic fluid for fertilization and culture of human embryos: results of clinical trials in human in vitro fertilization (IVF) programs.

We report the outcome of clinical trials carried out in two IVF programs, comparing the use of human amniotic fluid (HAF) as a complete medium to Whittingham's T6 medium containing human serum (T6 + 10% HS) for egg incubation, insemination, embryo culture, and embryo transfer. There were no significant differences in the clinical trials between HAF used alone as a complete medium and T6 + 10% HS in fertilization rates of eggs, cleavage rates of embryos up to 48 hours in culture, pregnancy success rates after embryo replacement or the outcome of pregnancies. There was no advantage in using T6 + 10% HS for fertilization of eggs and HAF as a complete medium for embryo culture and transfer in any of the parameters examined. We conclude that HAF does not meet the complete requirements of human eggs and embryos in vitro and further developments of culture media are required to obtain embryo development equivalent to that in vivo.

Amniotic Fluid↗