Search

Mitochondria play a critical role in cell function and embryo development. Recently, increasing studies have investigated whether mitochondrial DNA (mtDNA) can be used as a predictive biomarker of embryo implantation. However, the results of its effect on implantation are still controversial. To further understand the clinical application value of mtDNA content for reproductive potential, we analyzed the influence of relative mtDNA quantity on embryo quality and transfer outcomes based on the results of second-generation sequencing of preimplantation genetic testing patients in our center. Biopsied trophectoderm (TE) from aneuploid blastocysts contained much larger amounts of mtDNA than those from euploid blastocysts ( p  < 0.000). In an analysis of only euploid blastocysts ( n  = 769), female age had no effect on mtDNA content ( p  = 0.216). TE cells biopsied on day 5 ( n  = 355) contained significantly higher amounts of mtDNA compared to those biopsied on day 6 ( n  = 388) or day 7 ( n  = 26) ( p  < 0.000). Higher quality trophoblast was associated with lower mtDNA content ( p  = 0.026), but quality of inner cell mass was not correlated with quantity of mtDNA ( p  = 0.112). For transferred embryos, the biopsied date and mtDNA content were significantly associated with embryo implantation and live birth outcomes. Day-5 euploid blastocysts with lower quantities of mtDNA exhibited higher implantation rate and live birth rate. However, our data indicated that mtDNA content may not be considered an independent predictive marker, it may be a useful reference for the selection of day-5 transferred euploid blastocysts.

Aim: The objective of this study was to assess whether PGT conducted with previously untested vitrified embryos affect the clinical outcomes. Methods: A total of 49 patients who underwent biopsy on vitrification‐warming embryos for PGT were enrolled from January 2016 to January 2019. The cleavage‐stage embryos were thawed and cultured into the blastocyst stage for biopsy. During this period, 195 patients underwent routine PGT and FET, whose embryos were biopsied before frozen were used as the control group. The clinical outcomes were further compared between the two groups after a 1:2 PSM. Results: There were 47 transferable blastocysts in 30 patients, while 19 patients without transferable embryos, who performed biopsy on vitrification‐warming embryos for PGT. During this study period, 27 patients have already underwent FET with the clinical pregnancy rate was 66.7% (18/27). After 1:2 PSM, 24 patients in the biopsy on vitrification‐warming embryo group and 48 patients in the control group were compared, the clinical pregnancy rate (68.8% vs. 70.8%, p = 0.86), miscarriage rate (18.2% vs. 11.8%, p = 0.86), or live birth rate (52.1% vs. 62.5%, p = 0.40) had no significant difference. And the transferable blastocyst rate or the clinical pregnancy rate in the vitrification‐warming cleavage‐stage embryo group was not significantly different from those in the vitrification‐warming blastocyst group. In addition, the PGT clinical outcomes of biopsy on vitrification‐warming embryos had no significant difference between IVF‐fertilized embryos and ICSI‐fertilized embryos. Conclusion: Biopsy on the vitrification‐warming embryos with a dual vitrified cryopreservation does not affect the embryo quality or the PGT clinical outcomes. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the clinical factors that could be used predict the number of transferable blastocysts in preimplantation genetic testing (PGT) cycles based on next-generation sequencing (NGS) and formed form a mathematical model to predict the chance likelihood of obtaining one transferable blastocyst, which is helpful for genetic counseling. Methods: This retrospective study enrolled couples undergoing PGT cycles for chromosomal structural rearrangement (PGT-SR, n = 363, 202 with reciprocal translocation carriers, 131 with Robertsonian translocation carriers, 30 with inversion carriers), monogenic diseases (PGT-M, n = 47), and for Aneuploidies (PGT-A, n = 132) from January 2015 to October 2018. Stepwise multiple linear regression analysis was used to identify the factors relevant for obtaining at least one transferable blastocyst. The factors that predict the number of biopsied blastocysts were further analyzed. Results: The transferable blastocyst rates were 29.94, 41.99, 49.09, 41.42, and 44.37% in the reciprocal translocation carrier, Robertsonian translocation carrier, inversion carrier, PGT-M, and PGT-A cycles, respectively. The number of transferable blastocysts in these cycles were 0.3004 × the number of biopsied blastocysts (NBB) − 0.0031, 0.4063 × NBB + 0.0460, 0.5762 × NBB − 0.3128, 0.3611 × NBB + 0.1910, and 0.4831 × NBB − 0.0970, respectively. Furthermore, the number of MII oocytes and female age were clinical predictors of NBB in reciprocal translocation and PGT-A couples, while the number of MII oocytes was the only clinical predictor in Robertsonian translocation carriers, inversion carriers, and PGT-M couples. Conclusions: The number of biopsied blastocysts was the only clinical predictor of the ability to obtain a transferable blastocyst in PGT cycles; therefore, for clinical practice, theoretically the minimum numbers of biopsied blastocysts is 4 in reciprocal translocation carrier and 3 in couples undergoing PGT for other reasons. The number of MII oocytes and female age were clinical predictors of the number of biopsied blastocysts. With the mathematical models in our study as a reference, in clinical practice, clinicians will be able to conduct a more targeted genetic consultation for different kinds of PGT patients. [ABSTRACT FROM AUTHOR]

This study investigated the usefulness of next-generation sequencing (NGS)-based single nucleotide polymorphism (SNP) haplotyping for preimplantation genetic diagnosis (PGD) of hemoglobin H (Hb H) disease. Multiple displacement amplification (MDA) was used for whole genome amplification (WGA) of biopsied trophectoderm (TE) cells. Gap-PCR and NGS-based SNP haplotyping was used to distinguish the two genotypes of -α3.7/αα and –SEA/αα for PGD of Hb H disease. One out of the ten blastocysts (B11) was successfully diagnosed as genotype -α3.7/αα by Gap-PCR, whereas the others revealed allele dropout (ADO) (B1, B2, B4, B5, B7, B8, B12, and B15) or amplification failure (B10). However, NGS-based SNP haplotyping successfully diagnosed the -α3.7/αα and –SEA/αα genotypes from the MDA products of the biopsied TE cells. The haplotyping result showed that B4, B7, B8, B10, B11, B12, and B15 were carriers of the -α3.7deletion (-α3.7/αα), whereas B1, B2, and B5 were carriers of the –SEAdeletion (–SEA/αα). A blastocyst (B11) was transferred into the uterus in a subsequent frozen embryo transfer (FET) cycle after PGD. A healthy infant with a -α3.7/αα genotype weighing 2,800 g was born by cesarean section at the 38thweek of gestation. This result indicates that NGS-based SNP haplotyping is a valid screening tool for the PGD of Hb H disease. [ABSTRACT FROM PUBLISHER]

Purpose: To investigate the usefulness of preimplantation genetic diagnosis (PGD) for the patient affected by congenital contractural arachnodactyly (CCA) and spinal and bulbar muscular atrophy (SBMA). Methods: Multiple displacement amplification (MDA) was performed for whole genome amplification (WGA) of biopsied trophectoderm (TE) cells. Direct mutation detection by sequencing and next-generation sequencing (NGS)-based single nucleotide polymorphism (SNP) haplotyping were used for CCA diagnosis. Direct sequencing of the PCR products and sex determination by amplification of sex-determining region Y (SRY) gene were used for SBMA diagnosis. After PGD, the unaffected blastocyst (B4) was transferred in the following frozen embryo transfer (FET). Results: In this PGD cycle, sixteen MII oocytes were inseminated by ICSI with testicular spermatozoa. Four blastocysts (B4, B5, B10, B13) were utilized for TE cell biopsy on day 5 after ICSI. After PGD, B4 was unaffected by CCA and SBMA. B5 was affected by CCA and carried SBMA. B10 was unaffected by CCA and carried SBMA. B13 was affected by CCA and unaffected by SBMA. B4 was the only unaffected blastocyst and transferred into the uterus for the subsequent FET cycle. The accuracy of PGD was confirmed by amniocentesis at 21 weeks of gestation. A healthy boy weighing 2850 g was born by cesarean section at the 38th week of gestation. Conclusions: PGD is a valid screening tool for patienst affected of CCA and SBMA to prevent transmission of these genetic diseases from parents to children. [ABSTRACT FROM AUTHOR]

Purpose: To analyse the meiotic segregation modes of chromosomal structural rearrangements (PGT-SR) of reciprocal translocation in biopsied blastocysts from preimplantation genetic testing and to investigate whether any features of reciprocal translocation, such as carrier gender or the presence of acrocentric chromosomes or terminal breakpoints, affect meiotic segregation modes. Methods: Comprehensive chromosomal screening was performed by next generation sequencing (NGS) on 378 biopsied blastocysts from 102 PGD cycles of 89 reciprocal translocation carriers. The segregation modes of a quadrivalent in 378 blastocysts were analysed according to the carrier's gender, chromosome type and the location of chromosome breakpoints. Results: The results showed that 122 out of 378 blastocysts (32.3%) were normal or balanced, 209 (55.3%) were translocated chromosomal abnormalities, and 47 (12.4%) were abnormalities of non-translocated chromosomes. The proportion of translocated chromosomal abnormalities in translocations without acrocentric chromosomes was significantly higher than that in blastocysts from carriers with acrocentric chromosomes (14.8% versus 5.9%, P = 0.032). Translocation with acrocentric chromosomes exhibited a significantly higher proportion of 3:1 segregation (24.8% versus 5.1%, P < 0.0001) and a lower rate of 2:2 segregation (70.3% versus 87.0%, P = 0.00028) compared with the proportions in blastocysts from carriers without acrocentric chromosomes. The frequency of adjacent-2 segregation was significantly different in translocations with terminal breakpoints compared to the frequency in blastocysts from carriers without terminal breakpoints (6.7% versus 15.5%, P = 0.013). Conclusions: This study indicates that the segregation modes in blastocysts were affected by the presence of acrocentric chromosomes and terminal breakpoints, but not by the carrier's sex. Our data may be useful for predicting the segregation pattern of a reciprocal translocation and could support genetic counselling for balanced translocation carriers for PGT cycles using blastocyst biopsy. [ABSTRACT FROM AUTHOR]

Abstract Research question Can next-generation sequencing (NGS) based on copy number variation sequencing (CNV-Seq) identify normal/balanced embryos in balanced reciprocal translocation carriers and what are their reproductive outcomes? Design One hundred couples with balanced reciprocal translocation who underwent a total of 134 preimplantation genetic testing (PGT) cycles between January 2015 and October 2017 were evaluated. Trophectoderm cells of blastocysts were biopsied for CNV-Seq-based NGS. All the balanced/normal blastocysts were vitrified and cryopreserved. Single balanced/normal blastocysts were warmed and transferred in the subsequent frozen embryo transfer (FET) cycle. Results During the study period, 400 blastocysts were analysed by NGS-PGT, of which 109 (27.25%) were balanced and euploid. A total of 52 blastocysts were transferred in the FET cycle. Clinical pregnancy was confirmed in 34 women (65.38%), with a miscarriage rate of 2.94%; 26 healthy term babies were born, including 24 singletons and one set of twins, while eight couples had ongoing pregnancies. Amniocentesis revealed a fetal chromosome status that was consistent with the NGS-PGT results. Female carriers had a significantly higher blastocyst rate than did the male carriers (37.01% versus 31.27%, P = 0.04). The transferable blastocyst rate was higher in couples treated with gonadotrophin-releasing hormone (GnRH) antagonist than in those treated with GnRH agonist (38.20% versus 24.37%, P = 0.01). However, neither carrier sex nor ovarian stimulation protocol influenced the clinical pregnancy rate. Conclusions CNV-Seq-based NGS is an efficient and reliable PGT method for balanced reciprocal translocation. [ABSTRACT FROM AUTHOR]