Biomedical subjects
M Dhont
Publications and source records attributed to M Dhont.
Cytogenetic analysis of human oocytes parthenogenetically activated by puromycin.
PURPOSE: Treatment of aged human oocytes by puromycin allows a high rate of parthenogenetic activation and development until the first cleavage division. This technique was used for the study of the chromosome complement of oocytes which remained unfertilized after in vitro fertilization. Three hundred four unfertilized oocytes were treated with 10 micrograms/ml puromycin for 6-8 hr and further cultured for 12-15 hr. RESULTS: Activation occurred in 90.5% of the oocytes. Heterozygous diploids with two pronuclei predominated (61%), which is in contrast to the mouse, where the majority of oocytes activated by puromycin are uniform haploids (89%). CONCLUSIONS: Therefore we conclude that puromycin treatment induces retention of the second polar body in human oocytes, unlike in mouse oocytes treated in the same way. Chromosome analysis performed on 182 oocytes suggested a nondisjunction (ND) rate for the second meiotic division of 12.7%. This is a low figure considering the fact that puromycin itself has been reported to induce nondisjunction. For the first meiotic division a ND rate of only 5.6% was found. This rate is lower than the one found in metaphase II arrested oocytes and we believe that this difference is due to the technical differences between the study of meiotic and that of mitotic chromosomes.
Behaviour of spermatozoa in human oocytes displaying no or one pronucleus after intracytoplasmic sperm injection.
The behaviour of sperm cells after intracytoplasmic sperm injection (ICSI) was investigated by analysing 192 unfertilized and 37 one-pronuclear (1PN) oocytes following ICSI. Eighty-two unfertilized oocytes were directly fixed whereas 110 were first parthenogenetically activated by puromycin. In contrast to the findings in unfertilized oocytes after in-vitro fertilization, most unfertilized oocytes after ICSI (n = 76) contained evidence of the presence of spermatozoa in the cytoplasm. Few oocytes (n = 6) contained prematurely condensed sperm chromosomes (PCC), whereas the majority contained either intact sperm heads (n = 31) or swollen sperm nuclei (n = 39) along with metaphase II chromosomes of the oocyte. Following activation by puromycin, swollen sperm nuclei and PCC were no longer observed, whereas unchanged sperm heads persisted in 12 oocytes displaying a single pronucleus. A non-decondensed sperm nucleus along with decondensed maternal chromatin were also discovered in 32 out of 37 oocytes displaying a single pronucleus after ICSI. The findings in unfertilized and 1PN oocytes after ICSI indicate that successful sperm injection, even followed by oocyte activation, is not sufficient to guarantee normal fertilization. It seems that partial sperm membrane damage prior to injection is also required to ensure normal sperm decondensation.
Aminoterminal propeptide of type III procollagen in cord blood and amniotic fluid of appropriate-for-gestational-age infants: a predictor of age-related fetal growth rate.
Procollagen propeptide serum levels reflect the rate of collagen production. Because the prenatal period is unrivaled in terms of relative amounts of collagen synthesized per unit of time, this life episode must be the most sensitive period for the study of these biochemical markers of growth variability. N-terminal propeptide of type III procollagen (PIIINP) concentration was measured by two different methods (Fab'-fragment and whole antibody-based RIA assay) on paired samples of cord serum and amniotic fluid from a study cohort of 602 perinates with gestational age ranging from 20 to 41 wk. The aim of this study is to assess the clinical usefulness of the PIIINP assay for the evaluation of fetal somatic growth variation during the second half of normal pregnancies. It was demonstrated that the PIIINP level in cord serum, as well as in amniotic fluid, reflects age-related growth activity in "healthy" fetuses (n = 504) with normal intrauterine growth. This reflection was independent of the cumulative body mass or length already attained at the time of investigation. The PIIINP concentration closely mirrors the shape of the fetal somatic growth velocity curve, expressed as weight-specific gain (g/kg/d) during the second half of pregnancy. It can be concluded that PIIINP level in cord blood or amniotic fluid of fetuses with normal intrauterine growth is an interesting parameter for the assessment of maturity-related fetal growth potential.
A 45,X/46,XX/47,XXX female mosaic detected by cytogenetic analysis of unfertilized IVF oocytes.
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Tubal hydatidiform mole.
We report a tubal hydatidiform mole, most likely resulting from dispermic fertilization. Early hatching of the embryo, because of a defective zona pellucida, may have favored tubal implantation.
The ovarian hyperstimulation syndrome in in-vitro fertilization: a Belgian multicentric study. I. Clinical and biological features.
The multicentric study regroups 128 cases of the ovarian hyperstimulation syndrome (OHSS) in in-vitro fertilization (IVF) and 256 selected controls. Values of serum oestradiol obtained from different laboratories were found to be normally distributed after logarithmic transformation. Comparative study of clinical and biological characteristics indicates that among OHSS patients (i) mean age was lower; (ii) tubal indications for IVF were less frequent; (iii) polycystic ovary-like conditions (i.e. hyperandrogenism, anovulation, luteinizing hormone/follicle stimulating hormone ratio > 2) were more frequent. OHSS patients displayed ovarian hypersensitivity reflected by higher oestradiol peak concentrations in response to lower dosage of human menopausal gonadotrophin and by a steeper slope of oestradiol increment during stimulation. In these patients, the collection of greater numbers of fertilizable oocytes allowed replacement of more embryos with a good vitality score. Ongoing pregnancy rate was found to be higher among the OHSS patients. The following complications were recorded among OHSS cases: abdominal fluid at echographic examination or clinical ascites (86.7 and 71.1%, respectively); pleural and pericardial effusion (21 and 3%, respectively); haemoconcentration (71.1%); electrolytic disorders (6.2%). Although significantly different between groups, clinical and biological parameters under study showed considerable overlap of their distributions in control and OHSS cases. Therefore, these data must be submitted to discriminant analysis in order to derive a formula predictive of the risk of OHSS.
Hormonal stimulation for in vitro fertilization: a comparison of fertilization rates and cytogenetic findings in unfertilized oocytes.
OBJECTIVE: The purpose of this study was to compare fertilization and aneuploidy rates after two stimulation protocols in an IVF program. DESIGN: This was a retrospective study. SETTING: The study took place in the IVF laboratory of an Infertility Department. METHODS: In 349 treatment cycles, clomiphene citrate (CC) and human menopausal gonadotropin (hMG) were used in one group (N = 233) and hMG after treatment with a gonadotropin-releasing hormone agonist (GnRHa) in two other groups (long protocol): goserelin (N = 73) and buserelin (N = 43). Cytogenetic analysis was performed on all unfertilized oocytes in both groups. RESULTS: Fertilization rates were significantly higher in the GnRHa/hMG group than in the CC/hMG group, but cleavage rates and embryo quality were not different. Of 736 oocytes prepared for cytogenetic analysis, 256 were karyotyped: 172 were found to be euploid and 84 aneuploid. More oocytes were aneuploid in the GnRHa/hMG group than in the CC/hMG group and this difference was statistically different after analysis of the data using a specially designed mathematical model. CONCLUSION: If no selection against chromosomally abnormal oocytes takes place at the time of fertilization, more abnormal oocytes are harvested with GnRHa/hMG protocols than with CC/hMG. If, on the other hand, there is a selection against oocytes with some chromosomal imbalance, there is no intrinsic effect of GnRH agonists on the chromosomal complement of the oocyte, and the real aneuploidy frequency in all oocytes, fertilized and unfertilized, is the same in the GnRHa/hMG and in the CC/hMG group.
Contralateral tubal pregnancy after gamete intrafallopian transfer.
A case of EP after GIFT into the contralateral tube is presented. Hydrotubation with Earle's medium immediately before the GIFT cannot be excluded as a possible cause of gamete migration. The complete demise of the CL also demonstrates that trophoblast can implant and develop without steroidal support.
Propofol anaesthesia for ultrasound guided oocyte retrieval: accumulation of the anaesthetic agent in follicular fluid.
Propofol (2,6-diisopropylphenol, Diprivan, ICI-Pharmaceuticals, Manchester, UK) is widely used either as an adjunct in general anaesthesia or as sole anaesthetic agent by the continuous intravenous route and intermittent bolus injections for minor surgical interventions. For several years, we have been using this kind of anaesthesia in transvaginal oocyte retrieval for in-vitro fertilization (IVF), allowing a completely painless puncture on an out-patient basis. From in-vitro studies on mouse oocytes, it appeared that propofol could be deleterious for fertilization in a dose- and time-dependent manner. We therefore investigated the concentrations of propofol in follicular fluid during oocyte retrieval in women. We measured propofol levels in serum and follicular fluid of nine patients at fixed intervals during ultrasound guided oocyte retrieval. Serum levels fluctuated randomly, due to interference from top-off doses of propofol. In follicular fluid, however, we found a steady increase of propofol levels, which was proportional to the total dose of propofol administered. These data indicate that propofol accumulates in follicular fluid. Although it seems unlikely that propofol as used in the present protocol exerts a clinically significant unfavourable effect on IVF, we suggest that the oocyte retrieval procedure should be kept as short as possible, in order to limit the accumulation of the anaesthetic in follicular fluid.
Pilot study with 120 mg Andriol treatment for couples with a low fertilization rate during in-vitro fertilization.
Treatment with 120 mg/day of Andriol (testosterone undecanoate; Organon, The Netherlands) was given to 11 men whose semen had either failed to fertilize, or had resulted in a less than 33% fertilization rate in a first in-vitro fertilization (IVF) trial. Repeat IVF at the end of a 3 month treatment period resulted in a highly significant increase in the number of oocytes fertilized from 4/95 (4.2%) before to 23/87 (26.4%, P less than 0.001) after Andriol treatment. One couple attained spontaneous conception during the second month of Andriol intake and three pregnancies occurred among the remaining 10 cases undergoing repeat IVF, for a total ongoing pregnancy rate of 36.4%. Sperm concentration but not motility increased in the native semen after Andriol intake, but there were no significant changes in sperm characteristics after preparation. The improvement in pregnancy rate contrasts favourably with the results obtained by repeat IVF of untreated historical controls. The result obtained in the present pilot study should encourage the performance of a larger, placebo-controlled investigation protocol.
Delayed implantation. A case report.
In cycles in which conception takes place, serum human chorionic gonadotropin (hCG) becomes detectable between 8 and 12 days after ovulation. A delayed appearance of hCG has been reported in a limited number of cases, most of them ending in spontaneous abortion. We encountered a case of ectopic pregnancy characterized by a delayed appearance of hCG and accompanied by a complete, albeit temporary, halt in the steroidogenic activity of the corpus luteum. Although the patient was at risk of developing an ectopic pregnancy, the findings made an early diagnosis extremely difficult.
Predictive value of early embryonic cardiac activity for pregnancy outcome.
The biologic variation of the embryonic heart rate was investigated by transvaginal ultrasonography during the first trimester of normal (n = 141) and abnormal (n = 29) pregnancies, and the predictive value of a repeatedly diagnosed slow heart rate for pregnancy outcome was determined. The mean heart rate increased from 82 +/- 10.3 beats/min at 5 weeks' gestation to 156 +/- 9.6 beats/min at 9 weeks. A single observation of an abnormally slow heart rate did not necessarily predict subsequent embryonic death. However, a continued decline in embryonic heart activity, observed within a few days' interval, was always associated with a first-trimester abortion.
Steroid determinations in human ovarian follicular fluid using reversed phase liquid chromatography.
A method is presented, based on high performance liquid chromatography (HPLC) with u.v. absorbance detection, to simultaneously analyse all major unconjugated steroids in ovarian follicular fluids. The total analysis time is only 30 min. The use of a 3 mm i.d. column allows us to obtain detection limits for 3-oxo-4-ene steroids of 2 ng/ml. Calibration curves are linear in the 10-20,000 ng range per injection. Excellent agreement is obtained with the results using a previously published gaschromatography method.
Follicular fluid steroid levels in relation to oocyte maturity and in vitro fertilization.
Steroid levels in follicular fluid (FF) obtained from stimulated ovaries in patients undergoing in vitro fertilization (IVF) were measured by capillary gas chromatography. The correlation between these levels and the maturity of the oocyte, judged from the morphology of the oocyte corona cumulus complex (OCCC) and the fertilizability of the oocytes was analysed. Oocyte maturity was associated with higher FF levels of progesterone, 17-hydroxyprogesterone, 16 alpha-hydroxyprogesterone and 20 alpha-dihydroprogesterone. Follicular fluids containing oocytes that became fertilized had significantly higher levels of 20 alpha-dihydroprogesterone and progesterone and lower levels of androstenedione. Of all the steroids determined, 20 alpha-dihydroprogesterone provides the most significant group differences. Enhanced 20 alpha-dihydrogenation in the presence of decreased 16 alpha- and 17-hydroxylation appears to be an important characteristic of the ultimate ripening stages and early luteinization, at least in stimulated cycles.
Correlations between follicular fluid steroid analysis and maturity and cytogenetic analysis of human oocytes that remained unfertilized after in vitro fertilization.
OBJECTIVE: Is there any correlation between follicular fluid (FF) steroid levels and the occurrence of cytogenetic abnormalities in unfertilized human oocytes? DESIGN: Cytogenetic analysis was carried out on 397 oocytes, and the steroid content of 104 corresponding FF was analyzed using high-pressure liquid chromatography. Ovarian stimulation was performed by clomiphene citrate and human menopausal gonadotropin (hMG) or by hMG combined with a gonadotropin-releasing hormone agonist (GnRH-a) pretreatment. RESULTS: Oocyte maturity was correlated with an increasing FF progestin content and a significant decrease of androstenedione (A) levels. Chromosomal analysis revealed 84 of all oocytes to be abnormal (polyploid or aneuploid and/or prematurely condensed chromosomes present). In this group, A levels and A to estradiol ratios were significantly higher. Although progestin levels were higher in GnRH-a/hMG cycles, the incidence of oocyte normality was not different between the two stimulation schemes. More abnormal oocytes were found in patients with good sperm morphology. CONCLUSIONS: Oocyte abnormality correlates with higher A levels in the corresponding FF. Oocyte fertilization is also determined by intrinsic oocytic factors other than maturity.
Value of ultrasound-guided embryo transfer.
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Chromosome analysis in human oocytes unfertilized in vitro: a mathematical model for the estimation of the first meiotic non-disjunction frequency.
The results of cytogenetic analysis of unfertilized human oocytes in an in-vitro fertilization programme are to a large extent influenced by the methodology used. Exact conclusions cannot be drawn due to technical and interpretational errors. A mathematical model was constructed, therefore, to estimate the weight of these errors and to calculate the exact frequency of first meiotic non-disjunction in IVF oocytes. Of 246 prepared oocytes only 105 could be karyotyped exactly. Using the classical method of calculation, an aneuploidy frequency of 39% was obtained. By applying the maximum likelihood method a correlation between the frequency of first meiotic non-disjunction and the interpretational error level was found. Loss of chromosomes by anaphase lagging seemed to be undetectable, but the probability of chromosome loss due to fixation and unhomogeneous spreading was found to be 20%. Accepting an error level of 10-20%, the true non-disjunction frequency expected should range between 20 and 40%. This model yields a standard curve which can offer a basis for comparison of results obtained in different studies.