Blastomere Biopsy Research Articles

The Technological Advances in Embryo Selection and Genetic Testing: A Look Back at the Evolution of Aneuploidy Screening and the Prospects of Non-Invasive PGT

Since the birth of the first IVF baby, Louise Brown, in 1978, researchers and clinicians have sought ways to improve pregnancy outcomes through embryo selection. In the 1990s, blastomere biopsy and fluorescence in situ hybridization (FISH) were developed in human embryos for the assessment of aneuploidy and translocations. Limitations in the number of chromosomes that could be assayed with FISH lead to the development of comparative genomic hybridization (CGH); however, pregnancy rates overall were not improved. The later development of trophectoderm biopsy with comprehensive chromosome screening (CCS) technologies, as well as the subsequent development of next-generation sequencing (NGS), have shown much greater promise in improving pregnancy and live birth rates. Recently, many studies are focusing on the utilization of non-invasive preimplantation genetic testing (niPGT) in an effort to assess embryo ploidy without exposing embryos to additional interventions.

Impact of trophectoderm biopsy on obstetric and perinatal outcomes following frozen-thawed embryo transfer cycles.

Does trophectoderm biopsy for preimplantation genetic testing (PGT) increase the risk of obstetric or perinatal complications in frozen-thawed embryo transfer (FET) cycles? Trophectoderm biopsy may increase the risk of hypertensive disorders of pregnancy (HDP) in pregnancies following FET cycles. Trophectoderm biopsy has replaced blastomere biopsy as the standard of care to procure cells for PGT analysis. Recently, there has been concern that trophectoderm biopsy may adversely impact obstetric and perinatal outcomes. Previous studies examining this question are limited by use of inappropriate control groups, small sample size or reporting on data that no longer reflects current IVF practice. This was a retrospective cohort study conducted at a single university-affiliated fertility center. A total of 756 patients who underwent FET with transfer of previously vitrified blastocysts that had either trophectoderm biopsy or were unbiopsied and resulted in a singleton live birth between 2013 and 2019 were included. Obstetric and perinatal outcomes for patients aged 20-44 years who underwent FET with transfer of previously vitrified blastocysts that were either biopsied (n = 241) or unbiopsied (n = 515) were analyzed. Primary outcome was odds of placentation disorders including HDP and rate of fetal growth restriction (FGR). Binary logistic regression was performed to control for potential covariates. The biopsy group was significantly older, had fewer anovulatory patients, was more often nulliparous and had fewer embryos transferred compared to the unbiopsied group. After controlling for potential covariates, the probability of developing HDP was significantly higher in the biopsy group compared with unbiopsied group (adjusted odds ratio (aOR) 1.943, 95% CI 1.072-3.521; P = 0.029).There was no significant difference between groups in the probability of placenta previa or placenta accreta. There was also no significant difference in the rate of FGR (aOR 1.397; 95% CI, 0.815-2.395; P = 0.224) or the proportion of low (aOR 0.603; 95% CI, 0.336-1.084; P = 0.091) or very low (aOR 2.948; 95% CI, 0.613-14.177; P = 0.177) birthweight infants comparing biopsied to unbiopsied groups. This was a retrospective study performed at a single fertility center, which may limit the generalizability of our findings. Trophectoderm biopsy may increase the risk of HDP in FET cycles, however, a prospective multicenter randomized trial should be performed to confirm these findings. No specific funding was obtained for this study. The authors declare no conflict of interest. NA.

Modification of late human embryo development after blastomere removal on day 3 for preimplantation genetic testing

ABSTRACT The purpose of our study was to use a time-lapse monitoring (TLM) system to determine if day 3 blastomere biopsy for preimplantation genetic testing (PGT) had an impact on subsequent morphokinetic parameters at the morula and blastocyst stages. In this retrospective monocentric study conducted between May 2013 and August 2017, we compared late morphokinetic parameters in embryos undergoing day 3 blastomere biopsy for PGT and in control non-biopsied embryos obtained in intracytoplasmic sperm injection (ICSI) cycles for male infertility. All embryos in both groups were cultured in a TLM system. The biopsy group was composed of 1691 embryos (386 PGT cycles). The control group was composed of 2578 embryos (786 ICSI cycles). Early morphokinetic parameters up to day 3 were similar in both groups. Concerning late morphokinetic parameters, the onset of compaction (tSC), fully-compacted morula stage (tM), onset of cavitation/early blastulation (tSB), and blastocyst stages (tB and tEB) appeared significantly earlier in the biopsy group than in the control group. We found that late morphokinetic events at the morula and the blastocyst stages occurred significantly earlier in biopsied embryos than in control non-biopsied-embryos. The mechanisms underlying these modifications of embryo development after biopsy should be investigated in order to determine precisely, and this phenomenon could be associated with embryo, fetal, and offspring development. Abbreviations: TLM: time-lapse monitoring; PGT: preimplantation genetic testing; ICSI: intracytoplasmic sperm injection; tSC: the onset of compaction; tM: fully-compacted morula stage; tSB: onset of cavitation/early blastulation; tB and tEB: blastocyst stages; OHSS: ovarian hyperstimulation syndrome

Blastomere removal affects homeostatic control leading to obesity in male mice.

Preimplantation embryos are particularly vulnerable to environmental perturbations, including those related to assisted reproductive technologies. Invasive embryo manipulations, such as blastomere biopsy, are applied worldwide in clinical settings for preimplantation genetic testing. Mouse models have previously shown that blastomere biopsy may be associated with altered phenotypes in adult offspring. The aim of the present study was to investigate the specific contribution of blastomere removal to the physiological, behavioral, and molecular regulators of energy homeostasis, as compared to sham manipulation (re-introducing the blastomere into the embryo after its removal) and in vitro culture. Mice derived from 8-cell embryos subjected to blastomere removal displayed: (i) higher body weight and adiposity, (ii) increased food intake and sucrose preference, (iii) decreased time of immobility in the tail suspension test, and (iv) resistance to weight loss after social isolation or following 3 days of physical exercise - compared to mice derived from sham biopsy or from in vitro-cultured embryos. Mice generated after blastomere removal also had increased circulating leptin and leptin gene expression in adipose tissue, as well as increased ghrelin receptor gene expression in the hypothalamus, compared to control mice. The effects of blastomere biopsy on offspring phenotype were sexually dimorphic, with females not being affected. These results indicate that blastomere deprivation, rather than other perturbations of the blastomere biopsy procedure, programs male embryos to develop physiological, behavioral, and molecular dysregulation of energy homeostasis, leading to postnatal obesity.

Is Day-4 morula biopsy a feasible alternative for preimplantation genetic testing?

ObjectiveTo assess the efficacy and clinical outcome of PGT-M undertaken on Day-3, Day-4 and Day-4 “delayed” embryos that were unsuitable for biopsy on Day-3.Design and settingCohort-historical study of all consecutive patients admitted to the IVF-PGT-M program in a large tertiary center.Main outcome measure(s)The pregnancy rates and the percentages of complete, incomplete diagnosis, PCR failure, abnormal embryos in PGT of Day-3 cleavage-stage, Day-4 and Day-4 “delayed” embryos.Patients and methodsWe reviewed the medical files of all consecutive patients admitted to our IVF for a fresh IVF-PGT-M cycle. Patients were divided into 3 groups according to the day of blastomere biopsy: Day 3 cleavage-stage, Day-4 morula and Day-4 “delayed” embryos. The laboratory data, genetic diagnostic and clinical results were collected and compared between the different study groups.ResultsNine hundred and six patients underwent PGT-M cycles in our PGT program: 747, 127 and 32 in the Day-3, Day- 4 and Day-4 “delayed” groups, respectively. Ongoing pregnancy rates per transfer and per patient (15.8% and 9.4%, respectively) were non-significantly lower in the Day-4 “delayed”, compared to Day-3 (21.4% and 17.5%, respectively) and Day-4 (24.3% and 19.7%, respectively). When comparing ALL morulas (Day-4 and Day-4 “delayed”) to ALL cleavage-stage embryos (Day-3, Day-4 and Day-4 “delayed”), a significantly higher ongoing pregnancy rate was demonstrated following the transfer of embryos derived from morula biopsy, as compared to biopsy at the cleavage-stage (33.3% vs 20.5%, p<0.03, respectively).ConclusionDay-4 embryo biopsy is feasible and yields comparable and even higher ongoing pregnancy rate if undertaken at the morula stage. Further studies evaluating the cumulative live-birth rate per started cycles in Day-3 vs Day-4 embryo biopsy for PGT-M are warranted.

Timing day-3 vitrification for PGT-M embryos: pre- or post-blastomere biopsy?

To assess the efficacy and clinical outcomes of preimplantation genetic testing for monogenic diseases (PGT-M), following blastomere biopsy prior or following vitrification. A cohort-historical study of all consecutive patients admitted to IVF in a large tertiary center for PGT-M and PCR cycle from September 2016 to March 2020. Patients were divided into 4 groups: Group A1 consisted of patients undergoing day-3 embryos biopsy followed by a fresh transfer of unaffected embryos. Group A2 consisted of Group A1 patients that their surplus unaffected embryos were vitrified, thawed, and transferred in a subsequent FET cycle. Group B1 consisted of patients that their day-3 embryos were vitrified intact (without biopsy) for a subsequent FET cycle. Later embryos were thawed and underwent blastomere biopsies, and the unaffected embryos were transferred, while the surplus unaffected embryos were re-vitrified for a subsequent FET cycle. Group B2 consisted of Group B1 patients that their surplus unaffected embryos were re-vitrified, thawed, and transferred in a subsequent FET cycle. The laboratory data and clinical results were collected and compared between groups. A total of 368 patients underwent 529 PGT-M cycles in our center: 347 with day-3 embryos biopsied before undergoing vitrification (Group A1) and 182 following vitrification and thawing (Group B1). There were no between group differences in embryo survival rate post-thawing, nor the ongoing implantation and pregnancy rates. In PGT-M cycles, the timing of embryos vitrification, whether prior or following blastomere biopsy, has no detrimental effect on post-thawing embryo survival rate, nor their potential ongoing implantation and pregnancy rates.

Author

Y chromosome microdeletions (YCM) represent a genetic disorder, frequently responsible for the disruption of the spermatogenesis process. Its prevalence is about 20% among patients diagnosed with oligozoospermia. Here we report the successful live birth of a female baby after morphologically selected sperm injection (IMSI) and PGD for a couple with severe oligospermia related to Y chromosome microdeletions. Blood samples from the couple were obtained and the cytogenetic analysis was carried out on in vitro cultured peripheral lymphocytes. DNA extraction and molecular analysis performed on blood samples from the male patient using multiplex-PCR method enabled the identification of 24 microdeletions in AZFa, AZFb or AZFc loci, the absence of a series of markers being observed. The couple underwent an IVF cycle associated with PGD. The resulting embryos were cultured until day 3 and subsequently, the blastomere biopsy was carried out on 7 of the viable embryos. The entire genome was scanned using the Microarray-CGH technique with the Agilent GenetiSure Pre-Screen test.

A blastocyst biopsy approach for preimplantation genetic diagnosis technique that affects the expression of SNAP-α in mice

Preimplantation genetic diagnosis (PGD) is a technique that is commonly used during assisted reproduction in the clinics to eliminate genetically abnormal embryos before implantation. The blastomere biopsy technique has risks related to the embryo, but blastocyst biopsy has not been systematically evaluated in relation to effects after birth, and the resulting offspring have not been followed up on. We designed a series of experiments to evaluate the risk of blastocyst biopsy on the resulting progeny. Mice were divided into a PGD group and a control group. The former was the progeny of mice that underwent blastocyst biopsy and the latter was delivered through a normal pregnancy without blastocyst biopsy. Each group consisted of 15 animals. We found no effects of blastocyst biopsy on reproductive capacities and weight gain. As for neurobehavioral evaluation between both groups, there were no significant differences in tail suspension test, sucrose preference test, the open field test and the elevated plus maze. Western blotting, immunohistochemistry and quantitative RT-PCR results showed that the expression levels of MBP, PRDX5 and UCHL1 in the PGD group were not significantly different compared to the control group, but SNAP-α expression in the PGD group was lower than that in control group. In summary, we concluded that blastocyst biopsy had no adverse effect on the general growth and behavior in mice. However, blastocyst biopsy effected the expression of SNAP-α. Therefore, the safety of blastocyst biopsy requires further evaluation.

Preimplantation Genetic Screening and The Success Rate of In Vitro Fertilization: A Three-Years Study on Iranian Population.

In vitro fertilization (IVF) is one of the most efficient approaches within the context of assisted reproductive technology (ART) to treat infertility. High pregnancy rates have become the major index of successful IVF in clinical studies. It is not clear yet which factors are certainly responsible for IVF success, as various outcomes were obtained in different IVF centers with different settings. In this study, we aimed to address controversies in the interpretation of promising results of IVF with respect to preimplantation genetic screening (PGS). In this retrospective case series study, we built a dataset containing data from 213 IVF patient candidates for PGS (654 embryos) with blastomere biopsy at day 3 and trophectoderm biopsy in day 5, referred to Royan Institute, Tehran, Iran from 2015 to 2018. Next, the data were analyzed to find influential factors affecting success rate of ART cycles. Data analyses showed that regardless of PGS indications (ART failures, recurrent miscarriage, chromosomal abnormalities, etc.), the pregnancy rate is influenced by maternal and embryonic factors such as the age of mother as well as quantity and quality of transferred embryos. Furthermore, genotyping of embryos using array comparative genomic hybridization (aCGH) depicted the highest rate of chromosomal aberrations for chromosomes 1, 16 and 19 while the lowest frequency for chromosomes 11 and 17. Similarly, we detected 463 genetically abnormal embryos by aCGH, among which only 41.9% could be detected by classical fluorescent in situ hybridization (FISH) method. This study not only highlighted the advantages of aCGH over the FISH method in detection of chromosomal abnormalities, but also emphasized the importance of genetic abnormality as an indication for determination of IVF success rate.

Adjusted mitochondrial DNA quantification in human embryos may not be applicable as a biomarker of implantation potential.

To evaluate the feasibility of adjusted mitochondrial DNA quantification in human embryos as a biomarker for implantation potential. Double-blind, observational, prospective analysis of an Asian population in a single university-affiliated in vitro fertilization center. A total of 1617 embryos derived from 380 infertile couples were collected. The DNA from blastomere biopsy (n = 99) or trophectoderm biopsy (n = 1518) were analyzed with next-generation sequencing. The adjusted mtDNA quantification followed a non-normal distribution in both types of the embryos. When stratified by ploidy status, the adjusted mtDNA quantification was significantly higher in aneuploid trophectoderm than in euploid cells, but not in blastomeres. The adjusted mtDNA quantification of embryos showed significant but very weak positive correlation in total trophectoderm cells with maternal age (Spearman's correlation, r = 0.095, p = 0.0028) but neither in blastomeres nor stratified by ploidy status. The median adjusted mtDNA quantification was also significantly higher in aneuploid blastocysts than in euploid ones while corrected with embryo morphology. Viable embryos did not contain significantly different quantities of adjusted mtDNA compared with nonviable embryos (implanted n = 103, non-implanted n = 164; median 0.00097 vs. 0.00088, p = 0.21) in 267 transferred blastocysts. Quantification of adjusted mitochondria DNA in human embryos was significantly lower in euploid blastocysts than in aneuploid blastocysts. However, no statistically significant differences regarding implantation outcome were evident. To our best knowledge, this study provides the largest scale and the first correlation data between mitochondria copy number and human embryo implantation potential in Asians.

22. A NOVEL, FAST, SINGLE TUBE AMPLIFICATION AND INDEXING APPROACH FOR PGT-A FOR ILLUMINA® SEQUENCING INSTRUMENTS

Introduction Most methods to prepare embryo biopsy samples for Preimplantation Genetic Testing for Aneuploidy (PGT-A) for Illumina® sequencing instruments use a two-step approach of Whole Genome Amplification (WGA) followed by library preparation. A single tube, PCR indexing approach offers several advantages for laboratories performing PGT-A including protocol time efficiencies and reduced hands on time. A novel protocol has been developed based on the PerkinElmer DOPlify® WGA kit. The aim of this study was to determine the accuracy and performance characteristics of this protocol on 5 cell samples from euploid, single and double trisomy cell lines. Material and methods Five-cell samples representative of trophectoderm biopsy were manually sorted from aneuploid cell lines (Coriell Institute) and euploid lymphocytes. Cell lysis and WGA were performed using a modified DOPlify® kit protocol (PerkinElmer) followed by incorporation of Illumina specific adapter sequences and unique indexes in a novel single tube approach. Amplified, indexed cell samples were purified, quantified then pooled before 50 sample multiplex and 1 × 75bp read length sequencing on the Illumina MiSeq® Instrument using v3 chemistry. Sequencing data was analysed for correct aneuploidy calling using the PG-Find™ software (PerkinElmer). Results A total of 104 multicell samples were processed with the following karyotypes: 47,XY,+15 (n=22), 47,XX,+18 (n=20), 48,XXY,+21 (n=20), 46,XY (n=22) and 46,XX (n=20). Three samples were excluded from final analysis due to weak amplification (3%). All other samples were processed through to sequencing and displayed the expected karyotype when analysed with the PG-Find™ software, with no false positives or false negatives observed. The 50-sample multiplex generated an average of 510,000 mapped reads per sample with a 98.9% mapping rate to hg19. The novel protocol took on average 4.5 hours (2.5 hours hands on) to process 50 samples from sample receipt to MiSeq® Instrument loading. Conclusions The novel one tube amplification and indexing method was shown to be a fast, accurate protocol for PGT-A utilising Illumina® sequencing instruments. Sequencing ready, 50 sample multiplex pools were prepared in 4.5 hours, readily enabling a less than 24 hour turn-around time from sample receipt to reporting. Studies processing single cells (representative of blastomere biopsy) and cell samples with smaller segmental aberrations to determine the resolution of the protocol are underway, which will be followed by clinical validation using embryo biopsy samples.

44. CAN PGT-A USING ARRAY CGH IMPROVE IVF CLINICAL RESULTS? (THE EGYPTIAN IVF-ET CENTER EXPERIENCE)

Introduction Chromosomal aneuploidies are considered the main cause of failed development of embryos, implantation failures, and early reproductive losses. Preimplantation genetic testing (PGT) using array comparative genomic hybridization (array-CGH) allows selection of embryos with normal karyotype, before embryo transfer. This should increase the implantation rate and reduce the frequency of early pregnancy loss. The aim of this study is to assess if PGT using array-CGH improve IVF clinical results. Material & methods During 2016 - 2018, a total of 97 cycles for 94 patients underwent blastomere biopsy by laser on day 3, and 90 fresh transfer cycles were performed. The indications were repeated IVF failure (30), repeated pregnancy loss (26), previous history of aneuploid offspring (10) and for aneuploidy screening and sex selection (28). The maternal ages ranged from 25 to 42yrs. 732 embryos from 97 cycles were subjected to day-3 biopsies, the DNA was amplified using modified whole genome amplification (WGA) protocol. The amplified DNA from 710 embryos was labeled and analyzed using array-CGH software (IBSA Genetics., Switzerland). Results 710 embryos was tested, 69.44% (493/710) of the embryos were aneuploid267, and 30.46% (217/710) were euploid.70 euploid embryos were cryopreserved, and 147 euploid embryos were transferred to 90 patients resulting in 50% (45/90) pregnancies, 22 deliveries with healthy babies and 20 ongoing pregnancies and 3 miscarriages. Conclusion: Our results show that array CGH has the potential to provide high pregnancy and delivery rates after transfer of euploid embryos.

Cleavage stage mitochondrial DNA is correlated with preimplantation human embryo development and ploidy status.

To evaluate whether the mitoscore of cleavage stage embryos might correlate with developmental kinetics and the ploidy status. This retrospective single-center study involved all cycles between April 2016 and April 2018 in which preimplantation genetic testing for aneuploidy (PGT-A) on day 3 was performed. The mitochondrial DNA (mtDNA) content and embryo ploidy were determined on 375 single blastomere biopsies by next generation sequencing (NGS). After intracytoplasmic sperm injection, a time-lapse imaging system (embryoscope) was used to follow the development. The median mtDNA content of cleavage stage embryos (49.4) was used to stratify the embryos into two groups to compare embryo development and ploidy status: low mitoscore group (≤ 49.4) and high mitoscore group (> 49.4). The total number of euploid embryos was equal between both mitoscore groups (32.1% versus 33.5%; p = 0.854). However, embryos in the low mitoscore group had a significantly higher cell number on day 3 (8.13 ± 1.59 versus 7.62 ± 1.5; p = 0.0013) and showed a significantly faster development up until the 8-cell stage. Mitoscore was not different between euploid and aneuploid embryos, with the same blastomere number at the time of biopsy. Furthermore, absence of cavitation within 118h after insemination was correlated with higher mitoscore values (60.22 ± 42.23 versus 50.97 ± 13.37; p = 0.006) and a lower chance of being euploid (17.1% versus 47.4%; p = 0.001). mtDNA content of cleavage stage embryos correlates with time-lapse parameters. Early blastulation is correlated with a lower mtDNA content and a higher chance of euploidy.

Non-invasive preimplantationsexingusingRT-PCRon the spent embryo culture media

Introduction Preimplantation sexing is an alternative method for Preimplantation GeneticTesting (PGT) to prevent the birth of children suffering from x-linked genetic disorders.Social sexing for non-medical reasons is also legal for some cases. PGT routinely needsto biopsy of embryonic cells or oocyte polar bodies which are invasive and could lead to implantation failure. SRY gene expression begins in male embryos following genome activation in preimplantation embryos. The aim of this study was sexing of preimplantation embryos without biopsy based on the presence of SRY RNA and DNA in the spent culture medium as a biomarker of sexing of human preimplantation embryos. Materials and Methods In this double-blind study, two groups were evaluated. In the first group, on the third day after the biopsy, the human embryos were cultured individually; we received the spent media of the non-degenerated 5-day embryos those have been underwent sexing by Fluorescent In Situ Hybridization(FISH) method via blastomere biopsy on the third day. In the second group, embryos of ART candidateswere cultivated on the third day individuallyuntil development to blastocyst stage; this group of embryos was not biopsied but the blastocysts were fixed for sexing by FISH. Each group was divided to 2 subgroups; in the first subgroup we extracted the RNA from the spent culture medium of each embryo. Following RNA extraction, the total RNA was immediately reverse transcribed to cDNA. In the second subgroup, PCR was performed directly on the culture medium. PCR was performed for SRY and GAPDH genes. The SRY positive embryos were considered as male embryos and those were GAPDH positive and SRY negative considered as females. The results of sexing based on spent culture media was compared with the results of sexing based on FISH. Results For the first group, 12 samples were evaluated, after comparing the results of PCR and FISH, the results for all the samples were concordant.In the second group, we were able to correctly diagnose 12 of the 14 samples. Two embryos with false diagnosis were females that diagnosed as males by direct PCR. Conclusion Preimplantation sexing without embryo biopsy by RT-PCR on the spent embryo culture media seems to be a reliable tool for non-invasive preimplantation sexing. However, direct PCR-based diagnosis might be lead to false results; this is probably due to DNA contamination.

Distinguishing between carrier and noncarrier embryos with the use of long-read sequencing in preimplantation genetic testing for reciprocal translocations

Balanced reciprocal translocation carriers are usually phenotypically normal but are at an increased risk of infertility, recurrent miscarriage or having affected children. Preimplantation genetic testing on chromosomal structural rearrangement (PGT-SR) offers a way to screen against unbalanced embryos. Here, we demonstrated a new method to distinguish carrier from noncarrier embryos. Translocation breakpoints were first delineated by nanopore sequencing followed by polymerase chain reaction (PCR) across breakpoints. High-resolution breakpoint mapping was successful in all (9/9) balanced reciprocal translocation carriers. Retrospective analysis of their embryo biopsies with breakpoint PCR showed 100% concordant results with PGT-SR on trophectoderm biopsies (40/40) and 53% concordance on blastomere biopsies (8/15). The low concordant rate in blastomeres was due to failure in the amplification of derivative chromosomes involving large deletions. Breakpoint PCR also showed 100% concordant results with prenatal/postnatal outcomes on 5 pregnancies, indicating that our new method can accurately distinguish carrier from noncarrier embryos.

Non-invasive chromosome testing (NUCT) improved in vitro fertilization (IVF) outcomes compared to morphological assessment in single blastocyst transfer (SBT)

Introduction Pre-implantation screening (PGS) has been widely used in assisted reproductive technology (ART) and helped improve in vitro fertilization (IVF) clinical outcomes. However, the invasive blastomere or trophectoderm biopsy process involved in PGS not only could generate potential effects on embryo development but requires skills and experience. Non-invasive chromosome screening (NICS) method, which using MALBAC technique to amplify the genomic DNA secreted into the culture medium and following next generation sequencing (NGS) to analysis ploidy condition of the embryos, had been validated through in vitro study(J. Xu et al. 2016 ). Here we further investigate NICS potentials on IVF. Material & Methods A retrospective observational study from September 2015 to September 2016. A total of 353 frozen-thawed single blastocyst transfer (SBT) cycles were divided into three groups with different blastocyst selection criteria: the first was the No-choice group which had only one blastocyst for transfer for each patient (n=285). The second was the Morphology group. The patients of this group had at least two blastocysts available and blastocysts were selected for single blastocyst transfer on the basis of traditional morphological assessments (n=41); the third was the NICS group that had at least two blastocysts for each patient and the euploid blastocysts were selected using NICS for single blastocyst transfer (n=27). Results The biochemical pregnancy rate in the No-choice group, the Morphology group and the NICS group was 65.3%, 65.9% and 85.2%, respectively (P=0.232). Among three groups, the NICS group had the highest implantation rate (63.0%) (P=0.193), compared with 56.1% and 45.3% in the Morphology group and the No-choice group, respectively. There was no incidence of early miscarriage in the NICS group, while the early miscarriage rate in the No-choice group and the Morphology group was 21.1% and 13.0%, respectively (P=0.094). A significant difference was found in the ongoing pregnancy rate among the three groups, which was highest in the NICS group (63.0%) and lowest in the No-choice group (37.9%) (P=0.016). Conclusions NICS is a novel genetic testing that allows an accurate selection of blastocysts with better developmental potential for transfer and might improve the clinical outcomes.

Agilent Technologies OnePGT solution: External verification on both blastomere and trophectoderm biopsies

Objectives OnePGT is a genome-wide next-generation sequencing haplarithmisis-based solution designed to reinforce ranking of IVF embryos. The single workflow solution, consisting of wet-lab reagents and dedicated data analysis software, allows concurrent PGT-M (preimplantation genetic testing of monogenic disorders), PGT-SR (preimplantation genetic testing of structural rearrangements) and PGT-A (preimplantation genetic testing of aneuploidies) hence enabling enhanced genetic profiling. In collaboration with MUMC and KU Leuven, a study was set up to assess the level of concordance between OnePGT and Gold Standard analysis of both single and few cell embryo biopsies for PGT-M, PGT-SR and PGT-A. Methods A total of 227 embryo samples (191 PGT-M; 36 PGT-SR) were included in the study, consisting of 179 blastomere and 48 trophectoderm biopsies, of which 118 embryos were obtained from KU Leuven, and 109 from MUMC. For the KU Leuven site of the study, left-over whole genome amplified (WGAed) DNA from the embryo biopsies was processed, while in the MUMC site, a second biopsy was taken and WGAed. Libraries for NGS were generated using proprietary OnePGT reagents for all WGAed embryo biopsies and for bulk DNA samples from parents and reference family member(s) in case of PGT-M. Libraries were sequenced on NextSeq500 (Illumina) and resulting data were analyzed with the dedicated proprietary OnePGT software suite. Results For PGT-M analysis, a OnePGT conclusion was made for 91 % of embryos determined to be of sufficient quality (n=161). Of these, four were shown to have mitotic aberrations resulting from chromosome instability phenomenon and three did not receive a conclusion from the Gold Standard. The remaining 154 embryos showed 100% concordance with the standard PGT techniques. For PGT-SR analysis (n=36), three embryos were shown to have mitotic aberrations resulted from chromosome instability phenomenon, the remaining 33 embryos 100% concordance with the standard PGT methods. For PGT-A analysis, 100% concordance (> 50 Mb) was shown between OnePGT and standard PGT methods for which a PGT-A result was available (n=118). Conclusion The proprietary OnePGT solution, consisting of all-in-one library preparation reagents and dedicated software suite for concurrent PGT-M, PGT-SR and PGT-A in a single assay, successfully identified the presence of 26 different monogenic disorders, translocations and chromosomal aneuploidies in over 200 embryo biopsies, with a concordance rate of 100%. This indicates that this innovative genome-wide solution is expected to boost the current PGT practice and to facilitate effective ranking of IVF embryos based on enhanced genetic profiling.

Multiplying embryos: experimental monozygotic polyembryony in mammals and its uses.

Monozygotic (MZ) polyembryony is a strategy to increase the output of a single zygote, thereby producing more offspring from a limited number of oocytes. However, MZ twins and multiples (multiplets) of mammals occur rarely in nature, while their generation has been more successful experimentally. In this work, we review some of the methodological, biological and field aspects of experimental MZ polyembryony in mammals. First attempts of mechanical bisection of 2-cell stage rodent embryos provided a proof-of-principle for the survival and independent development of both blastomeres. Subsequently, experiments in other species, particularly sheep and bovine, allowed 2 methods of embryo multiplication to become routine: the separation or biopsy of blastomeres from cleavage-stage embryos and the bisection of morulae and blastocysts. We discuss how the preferable stage of bisection and the success rate can be species-specific. The scope that profited most from experimental MZ polyembryony is the production of additional copies of elite livestock individuals, the reduction of interindividual variation in test groups and the possibility of investigating discordant phenotypic traits in the same genomic background, for instance, comparing an affected twin with its healthy co-twin. By contrast, the original motivation for experimental polyembryony - efficiently generating more offspring out of the same zygote - has not been fulfilled yet. Although embryo splitting leads to an increase in quantity, there is a loss of embryo quality, thus, there is no real gain from artificially generated embryos (yet) in the field of medically assisted reproduction. In conclusion, mammalian zygotes have the regulative capacity to be polyembryonic, but this is not obligate.

Does blastomere removal alter the expression level of miR-Let7a and its target genes following mouse embryo biopsy?

Embryo manipulations may cause the misexpression of various genes, most of which play critical roles in the regulation of implantation. This study aimed to evaluate the effects of embryo biopsy on the expression of miR-Let-7a and its gene targets including ErbB4, Tgf-α, Itg-αv, Itg β3 on the implantation of mouse embryo. Embryos were produced by in vitro fertilization followed by blastomere biopsy at the eight-cell stage. The effects of blastomere removal on the expression of genes ErbB4, Tgf-α, Itg αv, Itg β3, and miR-Let-7a as well as the alteration of the blastocyst cell number were compared in both biopsied and non-biopsied groups. Finally, blastocyst attachment was assessed on culture dishes precoated with Fibronectin. The results revealed that there were no significant differences between the biopsied and non-biopsied embryos with reference to the blastocyst formation rates, the average inner cell mass, trophectoderm cell number, and percentage of attachment of blastocysts (P > 0.05). The expression of ErbB4, Itg-β3, Itg-αv, TGF-α transcripts, and miR-Let-7a in blastocysts biopsied embryos did not differ from the non-biopsied blastocysts (P > 0.05). The results demonstrated that the preimplantation embryo development and attachment of biopsied embryos in vitro is not adversely affected by one blastomere biopsy at the eight-cell stage embryo.

Increased Elasticity of the Zona Pellucida Affects Micromanipulation Procedures for Preimplantation Genetic Screening at the Cleavage Stage: Possible Role of Blastomere Alterations on Inconclusive or “No Results” Interpretations

Objective Occasionally, submission of blastomere(s) or trophectoderm cells for preimplantation genetic screening (PGS) may be interpreted as inconclusive (“no results”). The main reasons for those results include failure of DNA amplification, degraded DNA, or failure to load cell(s) into the biopsy tube. In a particular case at our clinic, blastomere biopsies were submitted for PGS and all embryos were labeled as “no results”. Interestingly, the zona pellucida (ZP) of all embryos was extremely elastic and blastomere displacement was difficult. We theorize that the high elasticity and extended micromanipulation procedures may have affected the DNA in such a way as to interfere with the accurate assessment/interpretation of results. Design Case Report. Materials and Methods A total of 43 oocytes were harvested at retrieval. Oocytes were inseminated via standard IVF or ICSI. Ten cleavage stage embryos with a minimum of 6 blastomeres were selected for biopsy/PGS screening. Assisted hatching (AH) via partial zona dissection (PZD; tridimensional) was performed prior to blastomere biopsy. Embryo biopsy was performed via blastomere displacement. Results The ZP of all biopsied embryos was highly elastic. In addition, the inner layer of the ZP appeared to be detached in certain areas for some embryos. Those embryos acquired an oval-elliptical shape. The lack of rigidity prevented generation of internal pressure inside the embryo, which is necessary to displace the blastomere(s). The ZP of each embryo had to be probed at different angles in order to displace the selected blastomere(s). The time required to harvest blastomeres was extended to approximately ≥5 min per embryo. Selected blastomeres were eventually isolated without evidence of damage to the remaining blastomeres. All embryos were labeled as “no results” after assessment by the genetic laboratory. The report and additional options were discussed with the patients, and it was decided to proceed with embryo transfer (D5) based on blastocyst morphology. Two high quality blastocysts were transferred, resulting in a singleton term pregnancy. The remaining viable embryos were considered for vitrification. Conclusions Zona pellucida elasticity and extended manipulation of embryos/blastomeres may result in a form DNA degradation that interferes with the accuracy or interpretation of results during genetic assessment. Further analysis of similar cases may shed light into the relationship between difficult biopsies and inconclusive PGS results. Disclosures Nothing to disclose.