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Biomedical subjects

A Bongso

Publications and source records attributed to A Bongso.

72 records · Page 4Linked to original sources
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↗
PubMed1989

Improved quality of human embryos when co-cultured with human ampullary cells.

Cultured human, ampullary, epithelial cells obtained from fertile women undergoing hysterectomy were evaluated for the support of human embryonic cleavage and growth in vitro. Twelve patients provided 23 embryos for co-culture with subcultured ampullary cells grown in T6 + 15% patient's serum and 18 embryos for growth in T6 + 15% patient's serum alone (controls). Of embryos co-cultured with ampullary cells, 78% cleaved to the compacted embryo stage and 69% cavitated as compared with 50 and 33% respectively for controls (P less than 0.01). Only 30% of co-cultured embryos reached the expanded blastocyst and 26% underwent hatching as compared with 28% for both stages in controls. At the 2 - 4- and 6 - 8-cell stages, 91 and 87% of co-cultured embryos showed an absence or slight fragmentation as compared with 72 and 61% respectively for embryos grown in medium alone (P less than 0.01). None of the co-cultured embryos showed unequal-sized blastomeres while 22% of controls showed unequal cleavage. Embryos grown with ampullary cells cleaved slightly faster than controls. Scanning electron micrographs showed that ampullary cells collected from co-cultures were all of the secretory type with several microvilli and apical protrusions. It is clear that subcultured human ampullary cells support human embryonic cleavage and yield a reasonable number of good quality embryos up to the cavitation stage. Development past the expanded blastocyst and hatching stages seems to involve another critical phase with its own specific requirements.

Cells, Cultured↗
PubMed1988

The effects of ultrarapid freezing on meiotic and mitotic spindles of mouse oocytes and embryos.

Preovulatory mouse oocytes and 2-cell embryos were frozen with dimethyl sulfoxide and propanediol by an ultrarapid method. The survival of frozen oocytes was low (33-34%) compared to that of 2-cell embryos (78-79%) with either cryoprotectant. Development to blastocysts after postthaw culture was about 7-15% for oocytes and 79-80% for the embryos. Ultrarapid freezing preserves cell structure quite well as revealed by electron microscopy, but meiotic oocytes and late 2-cell embryos undergoing mitosis showed evidence of spindle disorganization involving loss or clumping of microtubules resulting in some scattering of chromosomes. Embryos developed from frozen eggs showed clear evidence of micronuclear formation and incomplete incorporation of chromosomal material into main nuclei. These experiments confirm our observations on freezing of human oocytes and show that spindle microtubules are sensitive to freeze-thawing and that cryopreservation could cause chromosomal aberrations during early development. A cautious approach to the introduction of oocyte freezing in human in vitro fertilization (IVF) programs is advocated.

Animals↗
PubMed1988

Chromosome analysis of two-cell mouse embryos frozen by slow and ultrarapid methods using two different cryoprotectants.

Three freezing methods (slow-1,2 propanediol; ultrarapid-dimethyl sulphoxide; ultrarapid-1,2 propanediol) were used to assess the effects of low temperature storage on morphologic features and chromosome make-up of 2-cell mouse embryos. Weekly batches (15 weeks) of 2-cell mouse embryos were frozen for a period of 7 days using these three freezing methods, then thawed and subjected to chromosome analysis. After thawing, all three freezing regimens yielded 70.2% to 75.8% of healthy intact 2-cell embryos, and 5.5% to 8.1% of embryos with one healthy blastomere intact, respectively, out of 817 embryos examined. Chromosome analysis was possible in all 1- and 2-cell embryos. The incidence of aneuploidy and polyploidy in frozen-thawed embryos of all three methods (2.8% to 3.4%; 4.0% to 6.5%) was not significantly different from that of control unfrozen embryos (3.0%; 6.0%) (P greater than 0.01). Mitotic crossing over was observed in 3.5% of embryos frozen-thawed by the ultrarapid-dimethyl sulphoxide method only. It is not clear how the mitotic crossing over was induced by ultrarapid freezing in the presence of dimethyl sulphoxide.

Animals↗
PubMed1988

Chromosome anomalies in human oocytes failing to fertilize after insemination in vitro.

Three-hundred-and-two unfertilized oocytes left over from successful in-vitro fertilization (IVF) attempts in 143 women (27-42 years) on a follicular stimulating hormone-human menopausal gonadotrophin (FSH-HMG) stimulation regime were subjected to chromosome analysis. Ten oocytes were degenerated with no visible chromosomes and 41 metaphases had chromosomes that were clumped together which could not be interpreted either numerically or structurally. Of the remaining oocytes, 76.6% (192/251) had a normal haploid complement (n = 23), 13% (33/251) were hypohaploid (n = 19-22), 8% (20/251) were hyperhaploid (n = 24-26), 2% (5/251) were diploid (2n = 46) and 0.4% (1/251) had structural rearrangements. The 21% aneuploidy was from 24 different patients and hypohaploid sets had chromosomes missing mainly from the A, B, C, D and G groups while the hyperhaploid sets had extra chromosomes from A, B, D, G and E groups of the human karyotype. The mean age of patients showing aneuploid oocytes was 36.7 years which was above the mean for the entire group. The aneuploidy may have been brought about by errors in oogenesis (anaphase lagging or non-disjunction) and may offer one explanation for fertilization failure and overall low pregnancy rates after IVF.

Adult↗
PubMed1988

Establishment of human endometrial cell cultures.

Epithelial and stromal endometrial cells from 19 patients at different phases of the menstrual cycle were enzymatically separated, isolated by successive centrifugation and primary cultures established for in-vitro studies on implantation. The behaviour of cells in vitro was evaluated using Nomarski's inverted optics, May-Grunwald-Giemsa stained coverslips and scanning electron microscopy. Epithelial and stromal cells from all patients grew successfully in Chang's medium and formed a mixed confluent monolayer of epithelioid and fibroblastic cells in 3-7 days and such monolayers could be maintained alive up to 3-4 weeks. Epithelioid cells were polyhedral and grew as islands in a whorl-like wavy pattern around glandular fragments. Fibroblasts were spindle-shaped, more long-lived and grew rapidly to form parallel bundles of cells. Significant differences were observed in the number of multinucleated cells and cells with intracytoplasmic vacuoles between endometrium from proliferative, postovulatory and secretory phases (P less than 0.01). Scanning electron micrographs showed cells with cilia with varying densities of microvilli and apical protrusions. Endometrial cells in culture showed structural features remarkably similar to those described for cells in situ. The method described allows the propagation in vitro of separate endometrium cell types which can be used to study implantation mechanisms in unstimulated and stimulated cycles.

Cells, Cultured↗
PubMed1988

Gamete intrafallopian transfer (GIFT): an alternate method of conception in couples with unexplained infertility, endometriosis and oligo-asthenospermia.

138 treatment cycles of GIFT were carried out in couples with mainly unexplained infertility, endometriosis and male factor. The overall pregnancy rate was 24.6%, abortion 29.2%, multiple pregnancy 20.6% and ectopic pregnancy 0%. Four oocytes seem to be the optimal number of oocytes to be transferred. Patients with male factor infertility fared the worse in comparison with the other groups. Successful in-vitro fertilization of excess eggs did not correlate with a positive outcome of the GIFT procedure.

Endometriosis↗
PubMed1988

Contributions to reproductive medicine through in-vitro fertilization: a review.

In vitro fertilization (IVF) is now an accepted treatment procedure for subfertility. Its use has also contributed greatly to the understanding of our knowledge in reproductive medicine. This review looks at these contributions. In the female, IVF has made it possible to examine more closely ovarian physiology through the follicular micro-environment, follicular dominance and cohorts. In the male, the new sperm preparation technology has assisted the investigation of sperm parameters in-depth. A better understanding of human egg and sperm physiology, the fertilization process and pre-implantation phenomena has also contributed to better management of the infertile couple. Perhaps in the future, the pre-implantation embryo may be considered as a "patient".

Fertilization in Vitro↗
PubMed1988

The use of amniotic fluid and serum with propanediol in freezing of murine 2-cell embryos.

Human and mouse embryos have been cultured in amniotic fluid (AF). Human AF and human serum (HS) are used in the freeze-thaw of 2-cell mouse embryos. Two hundred seventy-five 2-cell embryos were collected into phosphate-buffered saline with 20% HS and 20% AF and into 100% HS and AF. The embryos were cooled with propanediol as cryoprotectant at a controlled rate. After thaw, they were cultured in T6 with 3 mg/ml bovine serum albumin. Blastocyst formation post-thaw was 56/79, 44/70, 51/61, and 56/79 of intact embryos from 20% HS, 20% AF, pure HS, and pure AF (NS). But blastocyst hatching was better from embryos frozen in pure HS (22/61, compared with 16/79 for 20% HS; P less than 0.05). Hence there is no advantage in using AF in freeze-thaw, but pure HS may be of use.

Amniotic Fluid↗
PubMed1977

Bovine fetal fluid cells in vitro: fate and fetal sex prediction accuracy.

The in vitro fate of bovine fetal fluid cells and the efficiency of fetal sex predication from cultures of these cells are studied using aspirates from live animals and pregnant uteri collected from the slaughterhouse. Over 70 percent of bovine amniotic fluid samples aspirated from pregnant uteri at the time of slaughter yielded cultures adequate for chromosome analysis whereas only 10 percent of allantoic fluid samples produced growth of cells satisfactory for chromosome analysis. Fetal sexing accuracy was 100 percent in all samples studied. Seven readily recognizable cell types were noted in cultures of fetal fluid cells obtained at various stages of gestation. In a majority of cases, the in vitro morphology of cells from both fetal cavities was similar to that observed in primary human amniotic fluid cell cultures.

Allantois↗
PubMed1976

Chromosome anomalies in Canadian Guernsey bulls.

The performance records of a group of Guernsey bulls used for artificial insemination revealed lower 60 to 90 day non-return rates as compared to that for bulls of the Holstein breed, and nine out of the 19 Guernsey bulls in this group exhibited fertility level below the average for this group. A cytogenetic survey to test whether or not the reduced fertility of these bulls was due to chromosome anomalies revealed a relatively high frequency of chromatid breaks and achromatic gaps in three out of 19 bulls while two carried 59 chromosomes each including a translocation chromosome. The 1/29 translocation heterozygote and the bulls showing achromatic gaps and chromatid breaks were among the five bulls which were consistently below the breed average in fertility as evidenced by the t test (P less than 0.01), whereas the bull with translocation 27/29 (G3) exhibited a non-return rate slightly higher than the breed average (P less than 0.01). It is possible that the chromosomally abnormal bulls contribute to increased returns to service not only because of the defective gametes they themselves might produce but also through their influence on the performance of their daughters.

Animals↗
PubMedDate not supplied

Discrepancy between cytogenetic and FISH results on an amniotic fluid sample of 45,X/46,X,idic(Y)(p11).

The presence of abnormal ultrasound markers showing a thick nuchal fold with short middle phalanx of the fifth finger in an otherwise normal-appearing female fetus led to the sampling of amniotic fluid at 16 weeks gestation. Cytogenetic analysis with routine G-banding showed a 45,X karyotype in all 20 cells analysed from two flasks. However, fluorescent in situ hybridization on uncultured cells showed presence of a Y signal in 9 cells, 11 cells showing a single signal for the X. A cytogenetic analysis of the fetal blood at 23 weeks confirmed the presence of two cell lines, 45,X and 46,X, idic(Y)(p11). The couple opted to have the pregnancy terminated. However, the fetus was not available to carry out confirmatory tests.

Amniotic Fluid↗
PubMedDate not supplied

Human sperm function after coculture with human fallopian tubal epithelial cell monolayers: in vitro model for studying cell interactions in early human conception.

It has been difficult to study the behavior of sperm in the tubal environment in vivo. Human tubal epithelial cells were therefore cultured in vitro to simulate tubal conditions and human sperm function was assessed in the presence of such cells in vitro. Ampullary epithelial cell lines were established from fallopian tubes collected from premenopausal women undergoing hysterectomy. Approximately 1 x 10(5) cells/mL from monolayers of the third passage were seeded with 1 mL of culture medium into each well of 4-well plastic dishes. Sperm from 10 ejaculates of 10 different oligoasthenozoospermic men 30-41 years of age were recovered by the swim-up method and 200,000 sperm from each ejaculate were added into each well at the time of cell seeding. Control wells were treated the same but without cells. All dishes were incubated at 37 degrees C in 5% CO2, and sperm motility, acrosome reaction, and sperm-cell binding assessed at 1, 5, and 24 h. Curvilinear velocity and mean amplitude of lateral head displacement were significantly different in ampullary cultures as compared with controls for all three time periods: 1 h (67 +/- 5.2 vs 58 +/- 4.9 microns/s, p < 0.05; 4.48 +/- 0.4 vs 3.29 +/- 0.3 microns; p < 0.05), 5 h (75 +/- 5.8 vs 64 +/- 5.0 microns/s, p < 0.05; 4.92 +/- 0.5 vs 3.68 +/- 0.3 microns, p < 0.05), and 24 h (70 +/- 4.8 vs 59 +/- 4.2 microns/s, p < 0.05; 4.36 +/- 0.4 vs 3.11 +/- 0.3 microns, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMedDate not supplied

Comparative evaluation of three sperm-washing methods to improve sperm concentration and motility in frozen-thawed oligozoospermic and normozoospermic samples.

Three sperm-washing methods (direct layering without centrifugation, DL; ficoll entrapment, F; and percoll gradient, P) were evaluated for sperm recovery and motility using computer-automated semen analysis on frozen-thawed samples from normozoospermic and oligozoospermic men. The motile sperm recovery after freeze-thawing was significantly greater for normozoospermic compared with oligozoospermic samples (43 +/- 5.08% vs 15.76 +/- 3.53%; p < 0.001). The P-separated prelayer gave significantly higher recovery of motile sperm than the DL and F methods for both oligozoospermic (0.51 +/- 0.14 vs 0.19 +/- 0.05; p < 0.05) and normozoospermic samples (9.74 +/- 3.11 to 43 +/- 6.01 vs 0.88 +/- 0.23 to 15.30 +/- 3.12; p < 0.001). The P-separated postlayer had higher curvilinear velocities than the DL, F, and prelayered P methods for oligozoospermic samples only (71 +/- 5.13 microns/s vs 53 +/- 3.67 to 59 +/- 2.72 microns/s; p < 0.05). Mean amplitude of lateral head displacement values were significantly different for postlayered P-treated sperm compared with DL, F, and prelayered P-treated sperm for normozoospermic samples only (4.25 +/- 0.16 to 4.67 +/- 0.32 vs 3.01 +/- 0.14 to 3.67 +/- 0.19; p < 0.05). Percoll separation without layering may be the best washing method for both normozoospermic and oligozoospermic frozen-thawed sperm, and the treatment of such recovered sperm with a motility-enhancing agent such as pentoxyfylline may yield optimum fertilization rates.

Adult↗
PubMedDate not supplied

Does sperm morphology play a significant role in increased sex chromosomal disomy? A comparison between patients with teratozoospermia and OAT by FISH.

Eight semen samples from men with teratozoospermia (T), along with samples from 3 men with normal fertility and 5 samples from men with oligoasthenoteratozoospermia (OAT) were analyzed for X and Y chromosomal anomalies with the use of fluorescently labeled centromeric probes. This study was undertaken to determine whether patients with abnormal sperm morphology (teratozoospermia) have increased or decreased incidence of a sex chromosomal anomaly. The sex chromosome disomy for the T samples was 0.36% and for the OAT group it was 0.61%, compared with baseline value for the normozoospermia group (0.09%).

Adult↗
PubMedDate not supplied

Comparative evaluation of fresh and washed human sperm cryopreserved in vapor and liquid phases of liquid nitrogen.

Infectious organisms such as hepatitis B were recently shown to survive in liquid nitrogen. To prevent cross-contamination of semen samples via liquid nitrogen, studies were undertaken to evaluate human sperm survival in the vapor phase of liquid nitrogen at -189 degrees C. The study was conducted in 2 separate experiments. In the first experiment, a total of 30 unwashed, fresh semen samples (15 normozoospermic and 15 oligozoospermic) were evaluated for motility, vitality, and morphology after freeze-thaw survival in vaporous (-189 degrees C) and liquid nitrogen (-196 degrees C; control) phases. Similar evaluations were carried out in a second experiment on 27 samples (15 normozoospermic and 12 oligozoospermic) that were previously washed by the swim-up method. Motile sperm recovery rates were significantly different between liquid and vapor phases (unwashed, normozoospermic: 42.76% +/- 3.23% vs 45.52% +/- 4.44%, P < .05; washed, normozoospermic: 34.44% +/- 4.41% vs 37.58% +/- 3.90%, P < .05; unwashed, oligozoospermic: 16.53% +/- 3.34% vs 18.25% +/- 4.36%, P < .05; washed, oligozoospermic: 10.32% +/- 2.54% vs 12.25% +/- 2.81%, P < .05). Recovery rates for motility were much higher for unwashed samples compared with washed semen samples. In all experiments the recovery of normal and live forms showed no significant differences between the vapor and liquid nitrogen storage phases (P > .05). The results demonstrate that both washed and unwashed human sperm survive satisfactorily with good recovery in the vapor of liquid nitrogen and can be recommended for future storage in medically assisted conception programs.

Adult↗