Keli Luo

Single-nucleotide polymorphism microarray-based preimplantation genetic diagnosis is likely to improve the clinical outcome for translocation carriers

STUDY QUESTION Is preimplantation genetic diagnosis (PGD) for translocation carriers more effective when done with a single-nucleotide polymorphism (SNP) array using trophectoderm (TE) biopsy and frozen embryo transfer (FET) compared with traditional PGD based on fluorescence in situ hybridization (FISH-PGD) using blastomere biopsy and fresh embryo transfer? SUMMARY ANSWER The procedure using the SNP array combined with TE biopsy and FET significantly improves the clinical pregnancy rate for translocation carriers. The miscarriage rate also slightly decreases. WHAT IS KNOWN ALREADY FISH-PGD has been widely used in translocation carriers but the clinical outcomes have not been ideal. SNP arrays can detect both chromosome segmental imbalances and aneuploidy, and may overcome the limitations of FISH in PGD for translocation carriers. STUDY DESIGN, SIZE AND DURATION This was a retrospective study of 575 couples with chromosomal translocations, including 169 couples treated by SNP-PGD between October 2011 and August 2012, and 406 couples treated by FISH-PGD between January 2005 and October 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS The study was set in an IVF center at the Reproductive and Genetic Hospital of CITIC-Xiangya, China. In total, 169 couples underwent SNP analysis, including 52 Robertsonian translocation carriers and 117 carriers of reciprocal translocations. Blastocysts (n = 773) were biopsied and FET was carried out on the balanced embryos. Four hundred and six couples underwent FISH-PGD, including 149 Robertsonian translocation carriers and 257 reciprocal translocation carriers. In total, 3968 embryos were biopsied and balanced embryos were transferred fresh. The SNP-PGD results and clinical outcomes were compared with those of FISH-PGD. MAIN RESULTS AND THE ROLE OF CHANCE Reliable SNP-PGD results were obtained for 717 out of 773 (92.8%) biopsied blastocysts. The proportions of normal/balanced embryos, embryos with translocation-related and translocation-unrelated abnormalities, the median number of embryos per patient, the ongoing pregnancy rate per embryo transfer and the miscarriage rate were 58, 23, 19, 2, 69 and 12%, respectively, for Robertsonian translocation carriers and 36, 52, 12, 1, 74 and 11%, respectively, in reciprocal translocation carriers. Reliable FISH-PGD results were obtained for 3452 out of 3968 (87.0%) biopsied embryos. The proportions of normal/balanced embryos, unbalanced embryos, the median number of embryos per patient, the ongoing pregnancy rate per transfer and the miscarriage were 36, 64, 3, 38 and 17%, respectively, for Robertsonian translocation carriers and 20, 80, 1, 39 and 16%, respectively, for reciprocal translocation carriers. Thus, SNP-PGD achieved a higher pregnancy rate but a lower miscarriage rate than FISH-PGD. There were no significant differences in maternal age, basal endocrine level and the average number of retrieved oocytes and good-quality D3 embryos in the SNP-PGD group compared with the FISH-PGD group. LIMITATIONS, REASONS FOR CAUTION This was a retrospective study with the two groups treated in different periods; therefore, there is a chance of sample bias and a possibility that the results were influenced by other factors that changed over time. Furthermore, the two treatment protocols differ in several respects and we cannot say which makes the greatest contribution to the difference in success. Complete pregnancy outcomes of SNP-PGD have not been obtained as some embryos have not been transferred yet. We cannot exclude differences between the final data and the data in the present manuscript. WIDER IMPLICATIONS OF THE FINDINGS The adoption of SNP-PGD combined with TE biopsy and FET may significantly improve the clinical pregnancy rate, and decrease the miscarriage rate after PGD for translocation carriers.

Clinical outcome of preimplantation genetic diagnosis and screening using next generation sequencing

BackgroundNext generation sequencing (NGS) is now being used for detecting chromosomal abnormalities in blastocyst trophectoderm (TE) cells from in vitro fertilized embryos. However, few data are available regarding the clinical outcome, which provides vital reference for further application of the methodology. Here, we present a clinical evaluation of NGS-based preimplantation genetic diagnosis/screening (PGD/PGS) compared with single nucleotide polymorphism (SNP) array-based PGD/PGS as a control.ResultsA total of 395 couples participated. They were carriers of either translocation or inversion mutations, or were patients with recurrent miscarriage and/or advanced maternal age. A total of 1,512 blastocysts were biopsied on D5 after fertilization, with 1,058 blastocysts set aside for SNP array testing and 454 blastocysts for NGS testing. In the NGS cycles group, the implantation, clinical pregnancy and miscarriage rates were 52.6% (60/114), 61.3% (49/80) and 14.3% (7/49), respectively. In the SNP array cycles group, the implantation, clinical pregnancy and miscarriage rates were 47.6% (139/292), 56.7% (115/203) and 14.8% (17/115), respectively. The outcome measures of both the NGS and SNP array cycles were the same with insignificant differences. There were 150 blastocysts that underwent both NGS and SNP array analysis, of which seven blastocysts were found with inconsistent signals. All other signals obtained from NGS analysis were confirmed to be accurate by validation with qPCR. The relative copy number of mitochondrial DNA (mtDNA) for each blastocyst that underwent NGS testing was evaluated, and a significant difference was found between the copy number of mtDNA for the euploid and the chromosomally abnormal blastocysts. So far, out of 42 ongoing pregnancies, 24 babies were born in NGS cycles; all of these babies are healthy and free of any developmental problems.ConclusionsThis study provides the first evaluation of the clinical outcomes of NGS-based pre-implantation genetic diagnosis/screening, and shows the reliability of this method in a clinical and array-based laboratory setting. NGS provides an accurate approach to detect embryonic imbalanced segmental rearrangements, to avoid the potential risks of false signals from SNP array in this study.

Cyclic regulation of LPA3 in human endometrium

BackgroundLysophosphatidic acid (LPA) belongs to the group of lipid messengers, which plays a pivotal role in the establishment of implantation via its cellular receptor, LPA3. The aims of the present study were to characterize LPA3 mRNA and protein in human endometrium during the normal human menstrual cycle.MethodsForty-three normally cycling volunteers without reproductive disorders were randomized to undergo endometrial sampling on a specific cycle day. Samples were assessed for relative LPA3 mRNA expression using real-time PCR and for LPA3 protein using immunohistochemistry and western blot.ResultsThe expression of LPA3 mRNA significantly increased during the early and late secretory phase compared with other menstrual phases. LPA3 protein was localized to the epithelial and stromal cells and expression levels followed the same pattern as for LPA3 mRNA.ConclusionIn the normal human menstrual cycle, LPA3 mRNA and protein expression also change, indicating that this gene may be related to the function of the endometrium.

Reciprocal Translocation Carrier Diagnosis in Preimplantation Human Embryos.

Preimplantation genetic diagnosis (PGD) is widely applied in reciprocal translocation carriers to increase the chance for a successful live birth. However, reciprocal translocation carrier embryos were seldom discriminated from the normal ones mainly due to the technique restriction. Here we established a clinical applicable approach to identify precise breakpoint of reciprocal translocation and to further distinguish normal embryos in PGD. In the preclinical phase, rearrangement breakpoints and adjacent single nucleotide polymorphisms (SNPs) were characterized by next-generation sequencing following microdissecting junction region (MicroSeq) from 8 reciprocal translocation carriers. Junction-spanning PCR and sequencing further discovered precise breakpoints. The precise breakpoints were identified in 7/8 patients and we revealed that translocations in 6 patients caused 9 gene disruptions. In the clinical phase of embryo analysis, informative SNPs were chosen for linkage analyses combined with PCR analysis of the breakpoints to identify the carrier embryos. From 15 blastocysts diagnosed to be chromosomal balanced, 13 blastocysts were identified to be carriers and 2 to be normal. Late prenatal diagnoses for five carriers and one normal fetus confirmed the carrier diagnosis results. Our results suggest that MicroSeq can accurately evaluate the genetic risk of translocation carriers and carrier screen is possible in later PGD treatment.

Blastocysts can be rebiopsied for preimplantation genetic diagnosis and screening

OBJECTIVE To evaluate the clinical value of re-examining the test-failure blastocysts in preimplantation genetic diagnosis/screening cycles. DESIGN Retrospective study. SETTING University-affiliated center. PATIENT(S) Women with test-failure blastocysts cryopreserved in preimplantation genetic diagnosis/screening cycles. INTERVENTION(S) Cryopreserved test-failure blastocysts were warmed and underwent a second round of biopsy, single nucleotide polymorphism microarray analysis, and vitrification, and the normal blastocysts were warmed again for ET. MAIN OUTCOME MEASURE(S) The percentage of test-failure blastocysts for transfer, the implantation rate per transferred blastocyst, and the live birth rate. RESULT(S) A total of 106 test-failure blastocysts from 77 cycles were warmed for re-examination. A total of 73 blastocysts that completely expanded were considered to have survived the warming process and were successfully rebiopsied. After single nucleotide polymorphism array analysis, 70 blastocysts yielded whole genome amplification product, and 31 had normal chromosomes (44.3%). A total of 19 normal blastocysts were warmed for ET, of which 18 survived and were transferred. The clinical pregnancy rate (implantation rate) was 50.0% in 10 single blastocyst transfer cycles, and all the implanted blastocysts resulted in healthy live births. CONCLUSION(S) Test-failure blastocysts that survived from the first warming procedure can tolerate a second round of biopsy, vitrification, and warming, have a high chance of having normal chromosomes, and are worth being re-examined.

Using SNP array to identify aneuploidy and segmental imbalance in translocation carriers

Translocation is one of the more common structural rearrangements of chromosomes, with a prevalence of 0.2%. The two most common types of chromosomal translocations, Robertsonian and reciprocal, usually result in no obvious phenotypic abnormalities when balanced. However, these are still associated with reproductive risks, such as infertility, spontaneous abortion and the delivery of babies with mental retardation or developmental delay. In recent years, array-based whole-genome amplification (WGA) technologies, including microarray comparative genomic hybridization (array CGH; aCGH) and single-nucleotide polymorphism (SNP) micro-arrays, have enabled the screening of every chromosome for whole-chromosome aneuploidy and segmental imbalance. These techniques have been shown to have clinical application for translocation carriers. Promising studies have indicated that array-based PGD of translocation carriers can lead to transfer pregnancy rates of 45–70% [2]. In addition to genetic testing techniques, the embryo biopsy stage (polar body, cleavage embryo or blastocyst) and the mode of embryo transfer (fresh or frozen embryos) can affect the outcome of PGD. It is now generally recommended that blastomere biopsy should be replaced by blastocyst biopsy to avoid a high mosaic rate and biopsy-related damage to cleavage-stage embryos, which might affect embryo development. However, more clinical data are required to confirm that the technique of SNP array-based PGD (SNP-PGD) combined with trophectoderm (TE) biopsy and frozen embryo transfer (FET) is superior to traditional FISH-PGD combined with Day 3 (D3) blastomere biopsy and fresh embryo transfer.

Clinical outcomes in carriers of complex chromosomal rearrangements: a retrospective analysis of comprehensive chromosome screening results in seven cases

OBJECTIVE To evaluate the clinical outcomes in carriers of complex chromosomal rearrangements (CCRs). DESIGN Case series. SETTING An institute for reproductive and stem cell engineering. PATIENT(S) Seven couples with CCRs. INTERVENTION(S) Assisted reproduction with preimplantation genetic diagnosis (PGD). MAIN OUTCOME MEASURE(S) PGD results, embryo rating, pregnancy outcomes. RESULT(S) In cases 1, 2, 3, 4, 5, and 6, each woman underwent one cycle of PGD. Case 7 underwent two PGD cycles. We obtained 51 blastocysts from seven couples with CCR, of which 47 were eligible for biopsy; only 3 (5.9%) were normal/balanced, and 2 (3.9%) conceptions resulted. One healthy baby girl was born (the other was not yet born at the time of publication). Karyotyping revealed that the healthy baby girl was 46,XX. Although the patient with both a balanced translocation and a CCR (case 7) had 12 embryos available for biopsy, all were chromosomally unbalanced. It is interesting that 22 (57.9%) of the total 38 blastocysts were of high quality for type A CCRs, and 2 (15.4%) of the total 13 blastocysts were of high quality for type B CCR at day 6 after fertilization. CONCLUSION(S) The chances of identifying normal/balanced blastocysts in patients with CCR are <6%; the chances of a pregnancy are <4%. Greater complexity CCRs result in fewer transplantable embryos. Moreover, CCRs of greater complexity have a lower rate of high quality blastocysts than CCRs of less complexity.