Impact of blastomere biopsy and cryopreservation techniques on human embryo viability.
(33/2509)
The purpose of the present study was to evaluate the effect of cryopreservation on 55 embryos which had one blastomere biopsied for preimplantation genetic diagnosis of aneuploidy before freezing. The thawing outcome was compared to that obtained in 94 embryos which derived from our conventional freezing programme in patients with comparable characteristics who were treated in the same period. Their embryos were morphologically similar but the incidence of aneuploidy was 100% in the biopsy group and unknown in the controls. The percentage of embryos which survived intact after thawing was significantly lower in the biopsied group compared to the controls (9 versus 25% respectively; P < 0.025), whereas the rate of lysis was superior among biopsied embryos (34 versus 13% in the controls; P < 0.001). Similarly, the survival index was higher in the frozen-intact embryos than in the embryos which were frozen after biopsy (61 versus 38%; P < 0.001). No empty zonae resulted in the control group, while six were found after thawing biopsied embryos. In the second part of the study, blastomere biopsy was implemented on 102 thawed embryos generated by 16 patients. The chromosomal analyses revealed that 49 were normal, leading to the transfer of 2.5 +/- 0.8 embryos per patient. Only three clinical pregnancies were obtained, and are presently ongoing. In conclusion, the present findings discourage the use of conventional cryopreservation protocols in strategies involving preimplantation genetic diagnosis in human reproductive medicine. Adequate protocols are required for freezing and thawing embryos which have been subjected to biopsy procedures. (+info)
Comparison of human blastulation rates and total cell number in sequential culture media with and without co-culture.
(34/2509)
Recent interest in delayed embryo transfers necessitated the evaluation of two improved in-vitro systems that could generate viable blastocysts. A total of 178 two-pronucleated embryos (entire cohorts) from 19 patients was cultured in IVF50 medium (100 microl) under oil for 24 h until day 2. Each patient's day 2 embryos were then equally allotted to two in-vitro systems. Embryos in system A were grown until the morning of day 3 on Vero cells covered with IVF50 medium (100 microl) under oil. The medium was then replaced on day 3 with a 1:1 mixture (100 microl) of IVF50:S2 medium and on day 4 with S2 medium only. The same culture protocol was used for system B without Vero cells. Throughout the 5 days all dishes were housed in sealed humidified modular chambers containing a triple gas atmosphere. Separately, 175 spare embryos from 80 patients were grown in system A and B up to days 6 and 7 for total cell number (TCN) analysis. Blastulation rates were not significantly different between system A and B (67.4 versus 68.5%; P > 0.01) although co-cultured embryos cleaved slightly faster by day 4. The overall pregnancy and implantation rates were 52.0% and 32.1% for the 19 patients each of whom received a mixed cohort of three day 5 embryos from both systems. TCN values for the day 6 and 7 blastocysts from both systems were high and increased steadily from days 6-7 and from expanded to hatching stages. There were no significant differences in TCN for day 6 expanded blastocysts between the two systems although day 6 hatching and hatched co-cultured blastocysts had greater values than non-co-cultured blastocysts (246.0 +/- 18.5 and 236.7 +/- 17.8 versus 173.0 +/- 13.5 and 166.5 +/- 16.0; P < 0.01). The results demonstrated that the culture protocol using the sequential IVF50-S2 media combination was a good substitute for Vero cell co-culture for the transfer of viable day 3-6 embryos. (+info)
Blastomere development after embryo biopsy: a new model to predict embryo development and to select for transfer.
(35/2509)
One of the most important and unsolved problems in in-vitro fertilization is to decide which embryos are more suitable to implant and therefore should be transferred. We analysed the in-vitro development of isolated biopsied blastomeres and compared it to the development of the original embryo, in order to find a relationship that could show the embryo's potential future development and so increase implantation rates. A total of 66 normally fertilized human embryos were biopsied at the 6- to 10-cell stages. At day 6, blastomeres were counted by nuclear labelling. A total of 33 embryos (50%) reached the blastocyst stage. Of the isolated blastomeres, 63% divided and 53% cavitated over 3 days in culture. Of the blastomeres taken from embryos that developed to the blastocyst stage, 88% divided, 79% cavitated, 76% divided and cavitated and 9% neither divided nor cavitated. In those from arrested embryos, 39% divided (P < 0.001), 21% cavitated (P < 0.001), 15% divided and cavitated (P < 0.001) and 55% neither divided nor cavitated (P < 0.001). Blastomeres biopsied from embryos that reached the blastocyst stage showed a significantly higher proportion of division and cavitation than those originated from arrested embryos. Culture of the isolated blastomeres can demonstrate those embryos more likely to develop to the blastocyst stage and that are probably more suitable to implant. Cryopreserving biopsed embryos and culturing blastomeres would increase implantation rates. Embryos can then be selected according to the blastomere development and thawed for transfer in a future cycle. (+info)
Broad ligament twin pregnancy following in-vitro fertilization.
(36/2509)
We report the first case of an ectopic twin pregnancy in the broad ligament following in-vitro fertilization and embryo transfer in a patient with a previous ipsilateral (left) salpingo-oophorectomy. The previous surgery was for endometriosis. We discuss the possible contribution of the embryo transfer technique, limitations of preventive measures and importance of transvaginal ultrasound in establishing the diagnosis. (+info)
Successful laparoscopic management of adnexal torsion during week 25 of a twin pregnancy.
(37/2509)
Adnexal torsion is a rare occurrence during pregnancy. Here we present a case of adnexal torsion during the 25th week of pregnancy, which was managed laparoscopically. The woman had achieved a successful twin pregnancy after in-vitro fertilization/intracytoplasmic sperm injection. She was admitted to the emergency department with acute abdominal pain. Abdominal ultrasound with colour Doppler mapping of the intra-ovarian blood flow showed adnexal torsion. Laparoscopic management was successfully carried out. (+info)
Advantages of day 4 embryo transfer in patients undergoing preimplantation genetic diagnosis of aneuploidy.
(38/2509)
PURPOSE: Following preimplantation genetic diagnosis of aneuploidy, embryo transfer was executed on day 4, with the aim of providing more time for expanding from six to nine the number of diagnosed chromosomes per single cell (Group 2; 45 cycles). The results obtained were compared to those derived from conventional day 3 transfer (Group 1; 71 cycles). METHODS: For multicolor fluorescence in situ hybridization analysis, two panels of probes were used: the first, specific for chromosomes XY, 13, 16, 18, and 21, was tested in all patients (Groups 1 and 2); the second was implemented only in Group 2 patients for the detection of chromosomes 14, 15, and 22. RESULTS: A total of 406 embryos underwent fluorescence in situ hybridization analysis in Group 1, and 236 in Group 2. Comparable percentages of both chromosomal abnormalities (61% and 62%) and pregnancy and implantation rates (36% and 24.5% in Group 1, 41% and 23.6% in Group 2) resulted, regardless of the higher mean age in Group 2. CONCLUSIONS: The diagnosis of the nine chromosomes which are most frequently associated with aneuploidy in humans could have an immediate impact on the rate of spontaneous abortions. Additional advantages are represented by the more accurate morphological evaluation of euploid embryos; the advanced compaction, which means that embryos are less exposed to damage during the transfer procedure; and the possibility of performing a reanalysis in cases where a fluorescence in situ hybridization diagnosis is not obtained. (+info)
Clinical experience of sex determination by fluorescent in-situ hybridization for preimplantation genetic diagnosis.
(39/2509)
In our centre we started using fluorescent in-situ hybridization (FISH) technique for sexing in couples with sex-linked diseases in May 1995. Probes specific for chromosomes X, Y and 18 were applied, allowing us to detect simultaneously both gender and ploidy status. The efficiency of the FISH procedure is 90.4% per biopsied blastomere or 95.2% per biopsied blastomere with a distinct nucleus visible at spreading. Up to December 1997, we treated 15 couples (20 treatment cycles) at risk for X-linked recessive disease and two couples with Yq deletion (two treatment cycles) with the aim of transferring only female embryos. In one cycle, no embryos suitable for biopsy were obtained and in five cycles no normal female embryos were available at diagnosis. In the remaining 16 cycles, transfer was possible and six pregnancies ensued: one miscarriage has occurred and six children have been born from the other five pregnancies. The implantation rate (fetal sacs) per transferred embryo was 20.8%. In 98 (61%) of the 161 diagnosed embryos, a diploid status was observed in one or in both biopsied blastomeres. In 10 out of the 161 (6.2%) embryos a heterogeneity among the two biopsied blastomeres was found: a diploid nucleus in one blastomere and a non-diploid pattern or binuclear status in the other. In the remaining 53 (32.9%) out of 161 diagnosed embryos, the biopsied blastomeres were abnormal. The embryos that were not transferred or frozen were further analysed. When two sex chromosomes and two autosomes were present in the biopsied blastomere, the sex determination of the biopsied blastomere was never in conflict with the sex determination in the rest of the embryo. Furthermore, if the biopsied cell was diagnosed as abnormal (triploid, aneuploid, chaotic) the embryo was indeed completely abnormal or at least mosaic. A FISH error could not be excluded in two embryos (1.2%); however, a wrong gender determination did not result from this. (+info)
Mammalian transgenesis by intracytoplasmic sperm injection.
(40/2509)
Coinjection of unfertilized mouse oocytes with sperm heads and exogenous DNA encoding either a green fluorescent protein (GFP) or beta-galactosidase reporter produced 64 to 94 percent transgene-expressing embryos, reflecting DNA-sperm head association before coinjection. Nonselective transfer to surrogate mothers of embryos in the GFP series generated about 20 percent offspring expressing the integrated transgene. These data indicate that exogenous DNA can reproducibly be delivered into an oocyte by microinjected spermatozoa and suggest an adaptable method of transgenesis. (+info)