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Biomedical subjects

A Trounson

Publications and source records attributed to A Trounson.

At least 73 records · Page 4Linked to original sources
PubMed1989

Problems in the cryopreservation of unfertilized eggs by slow cooling in dimethyl sulfoxide.

The survival, fertilization, development, and viability in vitro and in vivo of unfertilized mouse eggs frozen by slow cooling to -36 degrees C or -80 degrees C in 1.5 M dimethyl sulphoxide (DMSO) was examined in a series of experiments which explored some of the problems in freezing the egg. DMSO was added to the eggs at either room temperature or at 0 degrees C. Maximum success rate (42% of frozen eggs developing to two cells) was obtained when DMSO was added at 0 degrees C and the eggs slow cooled to -80 degrees C. Removal of cumulus failed to improve freezing success rates. Addition of DMSO at temperatures above 0 degrees C significantly reduced the fertilizing capacity of eggs. Excessive exposure of eggs to temperatures around 15 degrees C also caused a significant reduction in fertilization rates. The effects of DMSO and cooling on fertilization are likely to be due to zona hardening by cortical granule release and to disorganization of the egg cytoskeleton and plasma membrane. These problems will be difficult to overcome if cryopreservation of the unfertilized human egg is preferred to the fertilized egg or early cleavage stage embryo in clinical in vitro fertilization.

Animals↗
PubMed1989

Human micro-insemination by injection of single or multiple sperm: ultrastructure.

The process of micro-insemination by single or multiple sperm transfer into the perivitelline space (PVS) or by direct sperm injection into oocytes was examined by transmission electron microscopy. Spermatozoa from normal and oligozoospermic men were injected into oocytes, obtained from consenting IVF patients, mostly by zona-puncture using micromanipulators. Spermatozoa were washed by the Percoll or Ficoll methods and capacitated using Whittingham's T6 or modified Tyrode's medium or incubated in strontium medium before injection. The women were stimulated by three IVF methods and oocytes were recovered by laparoscopy or ultrasonography. Sixty-one oocytes were cultured in T6 or Ham's F-10 media (3-24 h) and were subjected to micromanipulation. Four oocytes were also studied after zona-drilling. Normal 2-pronuclear ova were developed after single-sperm transfer satisfying all morphological criteria of fertilization. Both monospermic and polyspermic fertilization resulted after multiple sperm transfer, indicating that a vitelline block to polyspermy may exist in humans. The majority of oocytes examined were unfertilized. Spermatozoa with intact or reacted acrosomes and those undergoing the acrosome reaction were found in the PVS and in the ooplasm. Abnormal spermatozoa were also seen in these locations. Quantitation of acrosomal status in 16 oocytes after multiple-sperm transfer, revealed that 24% of spermatozoa were acrosome-reacted or reacting in the PVS following Ficoll entrapment, while 76% of spermatozoa were intact (33% of these abnormal). Sperm transfer seemed to be the least invasive, while direct sperm injection was comparatively destructive to oocytes. Drilling with acid made larger breaches in the zona when compared with mechanical perforation and spermatozoa occasionally escaped through breaches. Three 2-pronuclear ova obtained after multiple sperm transfer have resulted in two pregnancies, in cases of severe oligozoospermia, during the course of this study.

Acrosome↗
PubMed1988

Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses.

Vitrification of mouse oocytes adversely affected the subsequent developmental potential of embryos and fetuses derived from the fertilization of such oocytes after thawing. Only 5% of oocytes vitrified formed viable fetuses on the 15th day of gestation as compared to 47% in the controls. The incidence of chromosomally aneuploid zygotes, derived from cryopreserved oocytes, was approximately threefold higher than the controls irrespective of whether the oocytes were cryopreserved by vitrification or DMSO slow-freezing. Malformed fetuses were obtained from oocytes that had been vitrified as well as those that had been exposed to vitrification solutions only, whereas no malformed fetuses were obtained in oocytes slow-frozen by DMSO or fresh controls--thus demonstrating that the exposure of oocytes to the vitrification chemicals was responsible for the fetal malformations. The data in this study suggest that the vitrification technique should be cautiously applied to human oocyte cryopreservation. Furthermore, the data also demonstrate that the exposure of female gametes to carcinogenic and/or teratogenic chemicals may result in malformed embryos when such oocytes are subsequently fertilized.

Aneuploidy↗
PubMed1988

Ultrarapid freezing of early cleavage stage human embryos and eight-cell mouse embryos.

Early cleavage stage human embryos and 8-cell mouse embryos were snap-frozen after a brief exposure to high concentrations of dimethyl sulfoxide (DMSO; 2 or 3.5 M) and 0.25 M sucrose and thawed in a warm water bath. Eleven of 12 3- to 8-cell human embryos survived freezing and thawing with more than 50% of their original blastomeres intact. However, pregnancy was not initiated when the 11 embryos were transferred to six patients. It was shown that continuation of embryonic development in vitro and in vivo was significantly better when 8-cell mouse embryos were snap-frozen in 3.5 M DMSO than in 2 M DMSO. When frozen in 3.5 M DMSO, 78% of 8-cell embryos survived on thawing, 84% developed to blastocysts in vitro, 63% implanted, and 42% developed to fetuses. Ultrarapid freezing is a quick and inexpensive method for mouse embryo cryobanking, but further studies are required to confirm the viability of frozen human embryos.

Animals↗
PubMed1988

Ultrarapid embryo freezing: effect of dissolved gas and pH of the freezing solutions and straw irradiation.

While optimizing the ultrarapid embryo freezing procedure we noted that embryo survival was lowest when gas bubbles formed in the straws. Here we report the influences of gassing the freezing solutions with 5% CO2 in air, degassing the solution, the pH of the medium and straw irradiation on the survival and development in vitro of 2-cell mouse embryos. Embryos were ultrarapidly frozen in medium M2 containing 3 M dimethyl sulphoxide and 0.25 M sucrose. Significantly fewer embryos survived freezing and thawing in gassed solutions. The subsequent development of intact embryos to blastocysts was similar in gassed and non-gassed solutions. Survival and development of embryos frozen in solutions of pH 7.0, 7.6 or 8.0 was similar, but fewer embryos developed to blastocysts after freezing in media at pH 6.0. In straws sterilized by gamma irradiation before use, embryo survival after freezing and thawing was dramatically reduced compared with that seen when non-irradiated straws were used. With non-gassed solutions significantly more gas bubbles formed in irradiated straws than in non-irradiated straws. Although bubble formation was significantly reduced in all straws by using degassed freezing solutions, this did not improve embryo survival. We conclude that under conditions normally used for ultrarapid freezing, dissolved gas and pH have less effect on embryo viability than the use of irradiated straws.

Animals↗
PubMed1988

The effects of cooling human oocytes.

Preovulatory human oocytes were cooled to 0 degrees C at 1 degree C/min, with or without the cryoprotectant dimethyl sulphoxide (DMSO), to assess the effects of cooling on the meiotic spindles and on oocyte structure. Batches of oocytes, cultured for 3-9 h, were held at 0 degrees C for 20 or 60 min and then fixed for transmission electron microscopy (TEM) either at 0 or 8 degrees C. Control oocytes were not cooled and were fixed at 22 or 37 degrees C for comparison. TEM revealed that 80% of the oocytes were at metaphase II, while 20% were at metaphase I and most had resumed meiosis recently. Control oocytes had more or less barrel-shaped meiotic spindles composed of microtubules (MT), some associated with chromosomes at kinetochores. Both metaphase I and II spindles were disassembled when cooled and fixed at 0 degrees C, with or without DMSO, due to extensive depolymerization of MT. The few MT that survived were found at the poles or were bundled together or were associated with chromosomes. Kinetochores were not prominent. Some oocytes cooled with DMSO and fixed at 0 or 8 degrees C showed evidence of MT, but the spindles were still disorganized and were abnormal in structure. Chromosomes tended to clump together or were dislocated in the cortical ooplasm in cooled oocytes, but widespread scattering was not observed. This was particularly evident in the absence of DMSO. Elements of the endoplasmic reticulum, Golgi, mitochondria and the cytosol were also adversely affected in some of the cooled oocytes and their surrounding cumulus cells. The results show that meiotic spindles are very sensitive to simple cooling and that DMSO does not provide substantial stabilization of the meiotic spindle even at 0 degrees C. The findings are discussed with reference to recent work on frozen human and mouse oocytes.

Cold Temperature↗
PubMed1987

Morphology and fertilizability of frozen human oocytes.

Human oocytes were frozen and thawed by four methods previously used for cryopreservation of human embryos. Most of these oocytes were inseminated after thawing to assess their capacity to fertilize and form pronuclear ova. Their morphology was assessed by phase-contrast microscopy used in routine IVF. Twenty-three oocytes were examined by electron microscopy to critically evaluate the effects of cooling and cryopreservation and to confirm fertilization. Morphological survival was observed in more than 60% of the oocytes examined after freeze-thawing. The main features of cryoinjury were cracks in the zona pellucida, disruption of the plasma membrane and extensive disorganization of the ooplasm. Subtle changes in the cytosol of cumulus cells was also observed. Cooling to 0 degrees C or -6 degrees C had little effect on cytoplasmic structure. Spindles were damaged in two frozen oocytes. Cumulus cell activity, sperm binding to the zona, sperm penetration of the zona seem to be largely unaffected by freeze-thawing. Fertilization was observed in eight oocytes after postthaw insemination and three embryos (8-cell to morula stages) were developed from pronuclear ova on further culture. Both monospermic and polyspermic fertilization were confirmed by electron microscopy and micronuclei were detected in three pronuclear ova. The genetic implications of these nuclear aberrations are discussed. These preliminary studies indicate that oocyte freezing needs to be integrated cautiously with clinical IVF by further assessment of embryos developed from frozen oocytes.

Female↗
PubMed1987

Effect of growth factors in culture medium on the rate of mouse embryo development and viability in vitro.

The development of mouse embryos from two cells to blastocysts in Whittingham's T6 medium containing the growth factor insulin or the serum replacement Nu-Serum was studied and compared with development in vivo. The rate of embryo development to blastocysts was increased by the addition of Nu-Serum to T6 and there was a significant increase in the implantation rate of embryos grown in T6 + Nu-Serum compared with T6 alone or with insulin. However, the rate of embryo development in vitro was retarded compared with that in vivo and the number of normal fetuses following transfer to recipients of blastocysts grown in T6 + Nu-Serum was not different from the number for those grown in T6 alone or with insulin. The addition of neither insulin nor Nu-Serum to T6 optimizes embryo development or viability.

Animals↗
PubMed1987

Transfundal transfer of embryos using ultrasound.

Ultrasonically guided transfer of embryos was performed transabdominally in 10 patients. The uterus was punctured by a 16-gauge needle with a stylet through a full urinary bladder. When the needle tip was in the uterine cavity the stylette was removed and a transfer catheter loaded with the embryos was inserted. No complications or technical difficulties were experienced. None of the patients became pregnant.

Adult↗
PubMed1987

Fertilization of human oocytes by microinjection of a single spermatozoon under the zona pellucida.

The authors report a method of micromanipulation for the insertion of a single spermatozoon under the zona pellucida of human oocytes that results in a high rate of fertilization without damage to the oocyte. Spermatozoa were exposed to calcium-depleted medium containing strontium chloride for 20 to 24 hours before resuspension in medium containing calcium to induce capacitation. Single spermatozoa treated in this way were injected under the zona pellucida of morphologically mature oocytes and fertilization was confirmed by electron-microscopy. Five of seven oocytes manipulated within 9 hours of aspiration from the follicle and 3 of 12 oocytes manipulated 23 to 28 hours after recovery from the follicle fertilized. This technique has profound implications for the possible treatment of severe male infertility.

Cell Nucleus↗
PubMed1987

Ultrarapid freezing: a new low-cost and effective method of embryo cryopreservation.

The authors describe a rapid freezing method (ultrarapid freezing) that has been developed for cryopreservation of early cleavage stage embryos. In the present experiments, 2-cell mouse embryos were frozen under a wide range of conditions in an attempt to optimize their survival and viability in vitro and in vivo. The experiments show that embryos exposed briefly (2 to 2.5 minutes) to relatively high concentrations of dimethyl sulfoxide (3 to 4 M) and 0.25 M sucrose survive and develop when plunged directly into liquid nitrogen and thawed in a 37 degrees C waterbath when sealed in 0.25-ml plastic pailettes. Survival and viability rates of ultrarapidly frozen embryos after thawing were comparable to those obtained with conventional slow-freezing techniques. The authors believe that this freezing technique can be further improved and that the speed, ease, and low cost of the method make it a very attractive alternative to more conventional methods for freezing early cleavage stage embryos.

Animals↗
PubMed1987

Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos.

Between 1 and 4% of human oocytes fertilized in vitro are tripronuclear. It has been reported that these tripronuclear oocytes can develop to grossly normal-appearing morulae and that chromosomally, these embryos could be triploid, diploid, or severely depleted. The etiology and proportion of apparently diploid and aneuploid embryos deriving from tripronuclear human oocytes is unknown. This study provides evidence for the first time that most (18 of 29) tripronuclear human oocytes cleave directly to 3-cells at the first cleavage division. These embryos have a severely abnormal (but not triploid) chromosomal complement. Furthermore, some (4 of 29) tripronuclear human oocytes cleave to 2-cells plus an extrusion, and these embryos are diploids, whereas some (7 of 29) cleave to 2-cells, and these embryos are triploid after the first cleavage division. These findings demonstrate that most tripronuclear human oocytes have an altered cleavage pattern at the first cleavage division, that most tripronuclear human oocytes (76% in this study) do not develop into triploid embryos, and that a correlation exists between the pattern of the first cleavage division and the subsequent karyotype of these embryos. Insight into the mechanisms by which these oocytes fail to develop into triploid embryos is also provided.

Aneuploidy↗