Assessment of aneuploidy formation in human blastocysts resulting from donated eggs and the necessity of the embryos for aneuploidy screening.

PURPOSE: To examine the prevalence of aneuploidy in human blastocysts resulting from donated eggs and embryo implantation after transfer of normal euploid embryos. Also, to assess the necessity of preimplantation genetic screening (PGS) for embryos produced with donor eggs. METHODS: Blastocysts from donor-recipient cycles were biopsied for PGS (PGS group) and the samples were analyzed with DNA microarray. Euploid blastocysts were transferred to the recipients, and both clinical pregnancy and embryo implantation were examined and compared with embryos without PGS (control group). RESULTS: After PGS, 39.1 % of blastocysts were abnormal, including aneuploidy and euploid with partial chromosome deletion and/or duplication. Transfer of normal euploid blastocysts brought about 72.4 % of clinical pregnancy, 65.5 % of ongoing/delivery and 54.9 % of embryo implantation rates; these rates were slightly higher than those in the control group (66.7, 54.0 and 47.8 %, respectively), but there was no statistical difference between the two groups. By contrast, the miscarriage rate was higher in the control group (19.2 %) than in the PGS group (9.5 %), but no statistical difference was observed. Transfer of two or more embryos did not significantly increase the ongoing/delivery rates in both groups, but significantly increased the twin pregnancy rates (50.0 % in the PGS group and 43.8 % in the control group). CONCLUSION(S): High proportions of human blastocysts derived from donor eggs are aneuploid. Although pregnancy and embryo implantation rates were increased, and miscarriage rates were reduced by transfer of embryos selected by PGS, the efficiency was not significantly different as compared to the control, suggesting that PGS may be necessary only in some specific situations, such as single embryo transfer.

Mosaic pregnancy after transfer of a "euploid" blastocyst screened by DNA microarray.

BACKGROUND: High proportions of human embryos produced by in vitro fertilization are aneuploidy and mosaic. DNA microarray is one of the most practical screening methods to select euploid embryos for transfer. However, mosaic pregnancy is still possible due to embryonic mosacism. Here we report a successful pregnancy after transfer of a mosaic blastocyst with euploid inner cell mass. METHODS: A woman with a previous trisomy 13 pregnancy pursued infertility treatment with preimplantation genetic screening by a trophectoderm biopsy and DNA microarray. NimbleGen oligonucleotide DNA microarray was applied to biopsied samples from 13 blastocysts. A euploid blastocyst was transferred to the patient and subsequent prenatal cytogenetic tests were performed by FISH and/or G banding. RESULTS: Following DNA microarray, it was found that 5 blastocysts were euploid and 8 were aneuploidy. Transfer of one euploid blastocyst resulted in a clinical pregnancy. Prenatal cytogenetic tests of samples biopsied from chorionic villi sample showed both trisomy 21 (47 XX, +21) and euploid (46, XX) cells. Further prenatal cytogenetic test with a sample from amniotic fluid indicated that all cells were euploid (46, XX). The pregnancy was continued and a healthy girl was delivered after 41 weeks of gestation. CONCLUSIONS: This is the first report to indicate a mosaic pregnancy after transfer of a "euploid" blastocyst that was screened by DNA microarray, and the case further confirms that mosaicism is present in human blastocysts produced by in vitro fertilization.

Identification of chromosomal errors in human preimplantation embryos with oligonucleotide DNA microarray.

A previous study comparing the performance of different platforms for DNA microarray found that the oligonucleotide (oligo) microarray platform containing 385K isothermal probes had the best performance when evaluating dosage sensitivity, precision, specificity, sensitivity and copy number variations border definition. Although oligo microarray platform has been used in some research fields and clinics, it has not been used for aneuploidy screening in human embryos. The present study was designed to use this new microarray platform for preimplantation genetic screening in the human. A total of 383 blastocysts from 72 infertility patients with either advanced maternal age or with previous miscarriage were analyzed after biopsy and microarray. Euploid blastocysts were transferred to patients and clinical pregnancy and implantation rates were measured. Chromosomes in some aneuploid blastocysts were further analyzed by fluorescence in-situ hybridization (FISH) to evaluate accuracy of the results. We found that most (58.1%) of the blastocysts had chromosomal abnormalities that included single or multiple gains and/or losses of chromosome(s), partial chromosome deletions and/or duplications in both euploid and aneuploid embryos. Transfer of normal euploid blastocysts in 34 cycles resulted in 58.8% clinical pregnancy and 54.4% implantation rates. Examination of abnormal blastocysts by FISH showed that all embryos had matching results comparing microarray and FISH analysis. The present study indicates that oligo microarray conducted with a higher resolution and a greater number of probes is able to detect not only aneuploidy, but also minor chromosomal abnormalities, such as partial chromosome deletion and/or duplication in human embryos. Preimplantation genetic screening of the aneuploidy by DNA microarray is an advanced technology used to select embryos for transfer and improved embryo implantation can be obtained after transfer of the screened normal embryos.

Optimized protocol for cryopreservation of human eggs improves developmental competence and implantation of resulting embryos.

BACKGROUND: Successful egg cryopreservation has many potential benefits to a variety of patients. However, a superior standard protocol describing all aspects of oocyte cryopreservation has not yet been identified. Oocyte cryopreservation is still a technical challenge for many infertility clinics. To maintain satisfactory clinical outcomes, there is a need to develop an easy to use, yet efficient laboratory protocol. The present study was designed to examine if human embryos resulting from eggs frozen with an optimized vitrification protocol have similar developmental competence as those from fresh eggs. METHODS: Twenty recipients received donated eggs vitrified with a protocol in which short exposure time to the vitrification solution was used and 23 recipients received donated eggs and 6 patients had their own eggs vitrified with a modified protocol in which long exposure time to the vitrification solution was used. After warming, egg survival, fertilization, cleavage, blastocyst formation, clinical pregnancy and implantation rates were compared. The developmental competence of eggs vitrified with the optimized protocol was further compared with fresh eggs donated from the same donors. RESULTS: There was no difference in the oocyte survival, fertilization, cleavage, clinical pregnancy or implantation rates between the short and long protocol groups. However, blastocyst formation rate was significantly (P < 0.001) higher in the long protocol group (50.8%) than that in short protocol group (26.5%), resulting in more blastocysts being transferred and frozen. When frozen eggs vitrified with long protocol and fresh eggs from the same donors (12) were compared in 39 recipients, no differences were observed in terms of fertilization (86.4 vs 80.1%), blastocyst formation (50.0 vs 59.2%), clinical pregnancy (63.2 vs 60.0%) and implantation (41.7 vs 44.7%) rates. Four out of 6 patients had ongoing pregnancy after transfer of embryos from their own frozen eggs with a 46.2% implantation rate. CONCLUSIONS: These results indicate that blastocyst development is an appropriate measure for egg survival after cryopreservation and frozen eggs have similar developmental potential as fresh eggs if they are frozen with an optimized method.

DNA microarray reveals that high proportions of human blastocysts from women of advanced maternal age are aneuploid and mosaic.

Trophectoderm (TE) biopsy and DNA microarray have become the new technologies for preimplantation genetic diagnosis in humans. In this study, we comprehensively examined aneuploid formation in human blastocysts produced in vitro with microarray and investigated the clinical outcome after transfer of euploid embryos. Biopsied cells from either TE or inner cell mass (ICM) were processed for microarray to examine the errors in 23 pairs of chromosomes and the consistency between TE and ICM. It was found that 56.6% of blastocysts were aneuploid. Further analysis indicated that 62.3% of aneuploid blastocysts had single and 37.7% had multiple chromosomal abnormalities. Chromosome errors could occur in any chromosome, but errors in chromosome 21 accounted for the most (11.3%) among the 23 pairs of chromosomes. Transfer of array-screened blastocysts produced high pregnancy (70.2%) and implantation (63.5%) rates. Microarray of TE and ICM cells in the same blastocysts revealed that high proportions of aneuploid blastocysts (69.2%) were mosaic, including aneuploid TE and euploid ICM, inconsistent anomalies between ICM and TE, or euploid TE cells and aneuploid ICM in the same blastocyst. These results indicate that high proportions of human blastocysts produced in vitro from women of advanced maternal age are aneuploid and mosaic. Errors can occur in any of the 23 pairs of chromosomes in human blastocysts. Biopsy from TE in blastocysts does not exactly predict the chromosomal information in ICM if the embryos are aneuploid. Some mosaic blastocysts have euploid ICM, which may indicate important differentiate mechanism(s) of human preimplantation embryos.

Comparison of development and implantation of human embryos biopsied with two different methods: aspiration and displacement.

OBJECTIVE: To compare the development and implantation of human embryos biopsied with two different methods for preimplantation genetic diagnosis (PGD). DESIGN: Technique and method. SETTING: A regional hospital IVF laboratory and private reproductive medicine clinic. PATIENT(S): Women undergoing IVF and PGD. INTERVENTION(S): Day 3 embryos were biopsied with aspiration and displacement; the embryos were cultured to blastocyst stage and then transferred. MAIN OUTCOME MEASURE(S): Blastocyst rate, pregnancy rate, and implantation rate. RESULT(S): One hundred fifty-one embryos from 14 patients were biopsied with the blastomere displacement method and 51 embryos from 5 patients were biopsied with the aspiration method. Displacement used less time than aspiration; thus, the time of embryos exposed to biopsy solution was shorter when displacement was used. Blastocyst formation (55.6%-56.8%) and ongoing pregnancy rate (50%) were not different between the two biopsy methods. However, the implantation rate was significantly higher in patients with embryos biopsied using the displacement method (64.7%) than with the aspiration method (25%). CONCLUSION(S): Blastomere displacement uses less time and is an easy and simple method for embryo biopsy and could be used as an alternative method for embryo biopsy. Our results indicate that the displacement method minimizes embryo damage during biopsy that was indicated by a higher implantation rate.

A simplified technique for embryo biopsy for preimplantation genetic diagnosis.

OBJECTIVE: To report a simplified embryo biopsy method for preimplantation genetic diagnosis (PGD). DESIGN: Technique and method. SETTING: A regional hospital in vitro fertilization (IVF) laboratory and private reproductive medicine clinic. PATIENT(S): Women undergoing IVF and PGD. INTERVENTION(S): Blastomeres were successfully isolated from day-3 embryos at various stages. MAIN OUTCOME MEASURE(S): Blastomere integrity after biopsy, time of biopsy procedure, and subsequent blastocyst developmental rate. RESULT(S): Twenty embryos derived from abnormally fertilized oocytes (one pronucleus or three pronuclei) were used for biopsy at four-cell to 10-cell stages (day 3) by a laser zona drilling and assisted hatching micropipette delivery of culture medium inside the zona to push one blastomere out. Biopsies of all embryos using this method were successful. In two cases for PGD, fourteen 6-9-cell and four 3-4-cell stage embryos were successfully biopsied by this method. Ten out of 14 embryos from the 6-9-cell stage developed to hatching or hatched blastocysts. When two hatched blastocysts were vitrified, warmed, and cultured, both reexpanded, showing normal morphologic features. CONCLUSION(S): This technique is easy to learn, less damaging to the embryos, and less time consuming. It can be used for all stages of embryos without damage to either embryos or isolated blastomeres. It is an alternative method for embryo biopsy in PGD.