B. Behr

Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage

We report studies of preimplantation human embryo development that correlate time-lapse image analysis and gene expression profiling. By examining a large set of zygotes from in vitro fertilization (IVF), we find that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA). These parameters can be reliably monitored by automated image analysis, confirming that successful development follows a set of carefully orchestrated and predictable events. Moreover, we show that imaging phenotypes reflect molecular programs of the embryo and of individual blastomeres. Single-cell gene expression analysis reveals that blastomeres develop cell autonomously, with some cells advancing to EGA and others arresting. These studies indicate that success and failure in human embryo development is largely determined before EGA. Our methods and algorithms may provide an approach for early diagnosis of embryo potential in assisted reproduction.

Genome-wide Single-Cell Analysis of Recombination Activity and De Novo Mutation Rates in Human Sperm

Meiotic recombination and de novo mutation are the two main contributions toward gamete genome diversity, and many questions remain about how an individual humans genome is edited by these two processes. Here, we describe a high-throughput method for single-cell whole-genome analysis that was used to measure the genomic diversity in one individuals gamete genomes. A microfluidic system was used for highly parallel sample processing and to minimize nonspecific amplification. High-density genotyping results from 91 single cells were used to create a personal recombination map, which was consistent with population-wide data at low resolution but revealed significant differences from pedigree data at higher resolution. We used the data to test for meiotic drive and found evidence for gene conversion. High-throughput sequencing on 31 single cells was used to measure the frequency of large-scale genome instability, and deeper sequencing of eight single cells revealed de novo mutation rates with distinct characteristics.

An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells

An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti–stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs—the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.

Preclinical validation of a microarray method for full molecular karyotyping of blastomeres in a 24-h protocol

BACKGROUND Preimplantation genetic screening (PGS) has been used in an attempt to determine embryonic aneuploidy. Techniques that use new molecular methods to determine the karyotype of an embryo are expanding the scope of PGS. METHODS We introduce a new method for PGS, termed ‘parental support’, which leverages microarray measurements from parental DNA to ‘clean’ single-cell microarray measurements on embryonic cells and explicitly computes confidence in each copy number call. The method distinguishes mitotic and meiotic copy errors and determines parental source of aneuploidy. RESULTS Validation with 459 single cells of known karyotype indicated that per-cell false-positive and false-negative rates are roughly equivalent to the ‘gold standard’ metaphase karyotype. The majority of the cells were run in parallel with a clinical commercial PGS service. Computed confidences were conservative and roughly concordant with accuracy. To examine ploidy in human embryos, the method was then applied to 26 disaggregated, cryopreserved, cleavage-stage embryos for a total of 134 single blastomeres. Only 23.1% of the embryos were euploid, though 46.2% of embryos were mosaic euploid. Mosaicism affected 57.7% of the embryos. Counts of mitotic and meiotic errors were roughly equivalent. Maternal meiotic trisomy predominated over paternal trisomy, and maternal meiotic trisomies were negatively predictive of mosaic euploid embryos. CONCLUSIONS We have performed a major preclinical validation of a new method for PGS and found that the technology performs approximately as well as a metaphase karyotype. We also directly measured the mechanism of aneuploidy in cleavage-stage human embryos and found high rates and distinct patterns of mitotic and meiotic aneuploidy.

Incidence of monozygotic twinning with blastocyst transfer compared to cleavage-stage transfer

OBJECTIVE To evaluate the incidence of monozygotic twinning (MZT) in pregnancies conceived after blastocyst transfer compared to cleavage-stage transfer. DESIGN Retrospective study. SETTING University IVF program. PATIENT(S) All IVF patients with viable pregnancies conceived during a 4-year period. INTERVENTION(S) Blastocyst transfer or day 3 ET. MAIN OUTCOME MEASURE(S) Incidence of MZT assessed by transvaginal ultrasound. RESULT(S) There were 11 incidences of MZT in 197 viable pregnancies (5.6%) with blastocyst transfer compared to 7 of 357 viable pregnancies (2%) with day 3 ET. In 10 of 18 pregnancies, MZT was observed in the setting of a higher order multiple gestation (6 of 11 for blastocyst transfer and 4 of 7 for day 3 ET). In the day 3 ET group, assisted hatching or intracytoplasmic sperm injection (ICSI) did not increase MZT (4 of 213, 1.9%) compared to cycles without zona breaching (3 of 144, 2.1%). Similarly, in the blastocyst-transfer group, ICSI did not increase the incidence of MZT (4 of 74, 5.5% for ICSI and 7 of 123, 5.7% for non-ICSI IVF). CONCLUSION(S) Compared to day 3 ET, blastocyst transfer appears to significantly increase the incidence of gestations with MZT. This information should be taken into account when counseling patients about the pros and cons of extended culture.

Comparison of blastocyst transfer with day 3 embryo transfer in similar patient populations

OBJECTIVE To compare implantation and pregnancy rates (PRs) achieved with blastocyst transfer (BT) and day 3 ET in similar patient populations. DESIGN Retrospective analysis. SETTING Academic infertility center. PATIENT(S) One hundred consecutive patients <40 years undergoing IVF, each with more than three eight-cell embryos on day 3. INTERVENTION(S) Patients used their own eggs for IVF or IVF and intracytoplasmic sperm injection. Embryos were cultured in P1 medium (Irvine Scientific, Santa Ana, CA) until day 3, when they were either transferred or, in the case of embryos for BT, incubated in Blastocyst Medium (Irvine Scientific), followed by transferring on day 5. MAIN OUTCOME MEASURE(S) Implantation and PRs. RESULT(S) There were no statistically significant differences in patient age, FSH level, or number of oocytes or zygotes. The BT group had fewer embryos transferred (mean, 2.4) compared with the day 3-ET group (mean, 4.6). The viable PR (cardiac activity at 6-7 weeks was considered indicative of a viable pregnancy) was higher with BT (68%, 34/50) than with day 3 ET (46%, 23/50). The implantation rate was increased with BT (47%, 56 sacs/120 embryos) compared with day 3 ET (20%, 46 sacs/231 embryos). CONCLUSION(S) The BT group in our study had higher implantation and PRs compared with the day 3-ET group. Better embryo selection, improved embryo-uterine synchrony, and decreased cervical mucus on day 5 may have accounted for the enhanced outcome. Our data support the use of BT to limit the number of embryos transferred while improving PRs.

Dynamic blastomere behaviour reflects human embryo ploidy by the four-cell stage

Previous studies have demonstrated that aneuploidy in human embryos is surprisingly frequent with 50–80% of cleavage-stage human embryos carrying an abnormal chromosome number. Here we combine non-invasive time-lapse imaging with karyotypic reconstruction of all blastomeres in four-cell human embryos to address the hypothesis that blastomere behaviour may reflect ploidy during the first two cleavage divisions. We demonstrate that precise cell cycle parameter timing is observed in all euploid embryos to the four-cell stage, whereas only 30% of aneuploid embryos exhibit parameter values within normal timing windows. Further, we observe that the generation of human embryonic aneuploidy is complex with contribution from chromosome-containing fragments/micronuclei that frequently emerge and may persist or become reabsorbed during interphase. These findings suggest that cell cycle and fragmentation parameters of individual blastomeres are diagnostic of ploidy, amenable to automated tracking algorithms, and likely of clinical relevance in reducing transfer of embryos prone to miscarriage.

Blastocyst-ET and Monozygotic Twinning

AbstractPurpose: To examine the rate of monozygotic twinning associatedwith blastocyst transfer using commercially available,cell-free culture systems with unmanipulated blastocysts. Methods: A retrospective analysis was conducted in multipleprivate and academic infertility centers throughout theUnited States, of 199 pregnant patients following in vitrofertilization (IVF) blastocyst embryo transfer (ET). Humanembryos obtained through standard IVF stimulation protocolswere cultured in commercially available, cell-free mediasystems and transferred as blastocysts. The main outcomemeasure was the rate of monozygotic twinning. Results: A total of 199 blastocyst-ET pregnancies wereachieved during the study period at the fertility centersexamined. Monozygotic twinning was noted in 10/199 (5;pc)of these pregnancies. All were monochorionic diamnionic. Conclusions: Monozygotic twinning previously has beenreported following IVF, especially in relation to assistedhatching. While blastocyst transfer has been available formany years using coculture, there have been no publishedmulticenter reports of monozygotic twinning associated withunmanipulated blastocysts. In a multicenter analysis, a definiteincrease in monozygotic twinning was seen followingblastocyst-ET. We believe this phenomenon is real and thatthis information should be considered when counselingpatients for treatment.

Two-blastocyst transfer has similar pregnancy rates and a decreased multiple gestation rate compared with three-blastocyst transfer

OBJECTIVE To examine the effect of the number of blastocysts transferred on pregnancy and multiple gestation rates. DESIGN Retrospective study. SETTING Academic infertility center. PATIENT(S) Patients < 40 years undergoing IVF, with FSH levels of < 15 mIU/mL and more than three eight-cell embryos. INTERVENTION(S) Embryos were cultured in P1 until day 3 and then transferred to blastocyst medium. A maximum of three blastocysts were transferred. MAIN OUTCOME MEASURE(S) Pregnancy, multiple gestation, and implantation rates. RESULT(S) All 55 patients developed blastocysts and underwent ET. Twenty-four patients had three embryos transferred and 29 patients had two embryos transferred. Two patients had only one embryo each for transfer. There was no difference in the viable pregnancy rate between the two-blastocyst transfer and three-blastocyst transfer groups (62% vs. 58%). In the two-blastocyst transfer group, 39% of pregnancies were multiple gestations (all twin gestations), compared with 79% of pregnancies in the three-blastocyst transfer group (50% twin gestations, 29% triplet gestations). The implantation rate was 47% in both groups. CONCLUSION(S) A commercially available, sequential culture system is highly effective for producing viable blastocysts. Two-blastocyst transfer eliminated the risk of triplets while maintaining the same high success rates seen with three-blastocyst ET.

Altered subcellular localization of transcription factor TEAD4 regulates first mammalian cell lineage commitment

In the preimplantation mouse embryo, TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP sequencing to define genomewide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation toward the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification.