Biomedical subjects
Publications and source records attributed to M Plachot.
We report a case of monozygotic triplet pregnancy after the transfer of three frozen-thawed embryos. The outcome of the pregnancy was the birth of three healthy normal boys. The pregnancy was a monochorionic triamniotic triplet pregnancy. This is the first such case of triplets, although it seems that zygote splitting is significantly more common than normal in patients undergoing in-vitro fertilization.
We report a case of monozygotic triplet pregnancy after transfer of 3 frozen thawed embryos. The pregnancy ended by the birth of 3 healthy normal boys and all the analysis showed that it was really a monochorionic triamniotic triplet pregnancy, that as far as we know is the first reported one induced by frozen thawed technics. We shall discuss: the etiology of monozygotic pregnancies after IVF; the sonographic diagnosis of placentation; the obstetrical care which allowed the birth of 3 healthy normal boys.
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A total of 17 couples with repetitive implantation failure after transfer of fresh or frozen-thawed embryos had half of their zygotes cultured in standard conditions and frozen at day 2 after insemination, and the other half cocultured with autologous granulosa cells and transferred at the morula or blastocyst stage at day 5 or 6 after oocyte retrieval. At the end of the culture period, supernatants of cocultures were recovered for steroid assays. Monolayers were stained for granulosa cell growth and morphological assessment. We observed that granulosa cells improve embryo development in vitro since 32 out of 60 (53%) reached the morula stage and 18 (30%) the blastocyst stage, leading to a total of 83% embryos available for transfer (compared with 3% without coculture). The ongoing pregnancy rate of these patients who were selected because they had at least three previous implantation failures, is only 5.9%, however, which is similar to the control group without coculture (6.3%). To conclude, granulosa cells improve embryo development but not the pregnancy rate after transfer of cocultured embryos in patients with multiple previous implantation failures.
Ten couples included in our in-vitro fertilization programme, selected because of one of more previous total fertilization failures or a low fertilization rate (< 20%), were entered in an experimental protocol of egg insemination on autologous granulosa cells. Half the oocytes from each patient were randomly assigned to either a control or a co-culture group. We observed no difference in the fertilization rate between the control (16.2%) and co-culture groups (12.1%). Only two couples benefited from this technique since fertilization was obtained only in co-culture. The poor efficiency of this protocol led us to propose the use of sperm micro-injection if all classical attempts to improve fertilization were unsuccessful.
Twenty six normo-ovulatory patients < 38 years with tubal or idiopathic infertility and normal sperm were treated for IVF by FSH +/- hMG and randomized into four groups: with and without LHRH agonist, "step-up" or "step-down" stimulation. In the step down protocol, after 4 days with 4 amps plasmatic FSH reached its top level, then decreased. Despite reduction to only two amps, estradiol continued to increase in all the 5 women without agonist, whereas 3/7 with the long protocol were considered as low responders and stopped. The fertilization/cleavage rate was 69.2% in step-down and 45.7% in step-up (p < 0.04) with the short protocol, and respectively 65.6% and 41.9% (p < 0.03) with the long one. The pregnancy rate was equivalent in all groups.
Two hundred and thirty three-isolated blastomeres originating from 82 abnormal human 2 to 11-cell embryos were fixed separately for cytologic and cytogenetic analyses. These embryos were rejected from our in vitro fertilization program because of either the presence of 1 or 3 pronuclei when observed 17 hours after insemination, or a wide fragmentation of normally fertilized eggs, or delayed fertilization. Twelve per cent of the blastomeres had no nuclei, whereas 7% were binucleated. When comparing the different types of embryos, parthenogenetic ones were those displaying the closest to normal development, since only 3% were cytologically abnormal. Seventy-six percent of the embryos had at least one analysable mitosis. Only 33% had 2 analysable mitoses, and 11% at least 3. Embryos with more than 1 analysable mitosis were the most often mosaicisms. When compared with classical techniques involving the fixation of whole embryos, cytogenetic analysis of isolated blastomeres permits ensuring that the number of chromosomes per cell is not overestimated as a consequence of mixed mitoses and demonstrates the very high incidence of mosaicism in abnormal human embryos.
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OBJECTIVE: To determine whether levels of human chorionic gonadotropin (hCG), 17 beta-estradiol (E2), and progesterone (P) are different in the peri-implantation phase of fresh versus frozen embryos. DESIGN: Hormonal secretions were measured on days 9 and 11 after implantation and at 4, 5, and 6 weeks gestation. PATIENTS: Thirty-one pregnancies were achieved in 65 patients with ovarian failure. Seventeen singleton pregnancies developed after implantation of 4 frozen and 13 fresh embryos. RESULTS: Human chorionic gonadotropin and E2, contrary to P, were higher in cases of fresh embryos from the 9th day after transfer to the 5th week at which time they become statistically significant (respectively, for hCG and E2, 5,800.3 +/- 332.3 versus 2,027.3 +/- 916.3 [mean +/- SD] mIU/mL for hCG and 562.3 +/- 215 versus 291 +/- 152 pg/mL for E2). CONCLUSIONS: This difference might be explained by either the higher number of fresh embryo replaced or by the fact that the number of blastomeres and also their metabolic activity could be reduced after freezing and thawing.
Only about 10% of embryos obtained after in vitro fertilization (IVF) are able to implant after transfer in utero. Detecting the viable embryos is therefore the prerequisite condition to increase the overall efficiency of the technique. Zygote and embryo morphology is partly related to their viability. Indeed, fertilized oocytes with a timely fertilization and pronuclear growth, leading to embryos with equally sized blastomeres reaching at least the 4-cell stage about 42 h after insemination, have the best chance of implanting. The metabolic approach, although requiring sensitive microassays, seems promising. Indeed, in a small series 100% of the pregnancies were predicted when the level of interleukin 1-alpha was > 60 pg/ml and the suppressive level (antiproliferative activity using tumour cell lines) was > 20% in the supernatant of transferred embryos. Lastly, invasive assessment of the chromosome status of embryos should allow us in future to discard severely abnormal embryos, thus increasing the success rate of IVF.
The selective reduction of embryos in multiple pregnancies poses numerous medical, technical, ethical and psycho-social problems. In a retrospective study, we analysed nine hundred and twenty-two pregnancies obtained using medically assisted procreation between May 1982 and May 1990. Among 922 successful pregnancies, 372 were singleton, 102 were twin and 13 were triplet. Data from this analysis and from a French multicentre study of 262 embryo reduction procedures demonstrated the value of an embryo quality score for minimizing the risk of multiple pregnancy and the existence of extremely infrequent, ethically acceptable indications for embryo reduction. These indications included ultrasound-proven malformations of one fetus, multiple pregnancies in patients with extensive uterine scarring, and multiple pregnancy despite the appropriate use of preventative measures which can be expected to make this technique unnecessary in the future.
Fertilization abnormalities (premature chromosome condensation of spermatozoa (PCC), triploidy, haploidy) were analysed in order to determine their origin. PCC occurs in 9% of unfertilized oocytes and seems to be the consequence of a failure of oocyte activation, leading to the continuing presence of cytoplasmic chromosome-condensing factors, causing the sperm nucleus to undergo chromosome condensation prematurely. This anomaly appears to be related to incomplete nuclear and/or cytoplasmic maturation. Triploid zygotes (6.5% of fertilized oocytes) display an original type of division: half of them divide into 3 and 6 cells, whereas at the same time diploid zygotes divide into 2 and then 4 cells. A cytological study, using both antitubulin antibodies and Hoechst dye, allowed us to demonstrate that they divide into 3 cells by means of a tripolar spindle. Triploidy seems to be correlated with four of 16 clinical or biological parameters examined: semen origin (fresh or frozen), type of stimulation treatment, number of oocytes recovered and embryo morphology. Haploid eggs (1.6% of inseminated oocytes) result from parthenogenetic activation. A correlation was found between a high number of recovered oocytes and triploid zygotes, and the occurrence of oocyte activation. These data show that increasing follicular recruitment decreases the overall oocyte quality and maturity leading to an overall 9% with impaired fertilization.
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Of 44 pregnancies obtained at the Tenon Maternity Hospital by in vitro fertilization (IVF) using donated oocytes, 25 have already been completed by spontaneous delivery or cesarean section: 22 (88%) single; 2 (8%) twins, and 1 (4%) triple. The most frequently seen complications of pregnancy were hypertension (16%) and preterm labor in the last 3 months (12%). However, as compared to a control group of 275 pregnancies resulting from classical IVF, the only difference was a very high percentage of cesarean sections (72%). No fetal deaths or neonatal malformations were reported.
Abnormal embryo development represents the major cause of implantation failures and accounts for the low rate of human infertility in vivo or in vitro. Chromosome abnormalities are widely involved in this process. Indeed, 28.4% of oocytes carry a chromosome aberration, i.e. 25.6% aneuploidy and 2.8% structural anomalies. Fertilisation abnormalities (possibly increased by in vitro procedures) were recorded: 7 to 28% of oocytes from fertilisation failure showed a sperm premature chromosome condensation probably resulting from ooplasmic immaturity. Moreover, 1.6% and 3.8% of inseminated oocytes had either a single or 3 pronuclei demonstrating parthenogenesis or triploidy, respectively. In vitro developmental capacities of embryos depends on the degree of ploidy. Parthenogenetic embryos display a fairly normal development until implantation. Triploid zygotes show an original way of division: half of them divide first into 3 cells and then into 6 cells (via a tripolar spindle) whereas diploid zygotes divide into 2 and then 4 cells. As a consequence of either meiotic or mitotic non disjunctions or fertilisation anomalies, 25 to 71% preimplantation embryos carry a chromosome disorder. As an outgrowth of in vitro fertilisation and embryo transfer, detection of genetic and metabolic defects prior to implantation might be possible in the future. So far, 6 girls have been born in couples at risk of transmitting X-linked disease. This technic will increase the efficiency of IVF and avoid the trauma of repeated abortions.
We retrospectively analyzed the prevalence and prognostic significance of plasma progesterone rises during in vitro fertilization (IVF) protocols including pituitary down-regulation by LHRH agonists in a long protocol and stimulation by hMG (n = 1,116: Group A) or pure FSH (n = 178: Group B). On the day of hCG injection, plasma progesterone level was in the 0.60-0.99 ng/ml range in 7.1% of group A patients and 6.2% of group B patients (NS) and above 1 ng/ml in respectively 4.0% and 3.4% of the cases (NS). On the same day, plasma progesterone was strongly correlated with plasma estradiol (r = 0.26 - p < 0.001 in group A; r = 0.21 - p < 0.01 in group B). However, increased progesterone levels were not associated with changes in plasma LH levels, falls in plasma estradiol levels after hCG injection, or significant modifications in biological and clinical results of IVF. The cause of this pattern of progesterone rise remains to be elucidated.