Purpose The purposes of this study were to determine whether biomechanical properties of mature oocytes could predict usable blastocyst formation better than morphological information or maternal factors, and to demonstrate the safety of the aspiration measurement procedure used to determine the biomechanical properties of oocytes. Methods A prospective split cohort study was conducted with patients from two IVF clinics who underwent in vitro fertilization. Each patient’s oocytes were randomly divided into a measurement group and a control group. The aspiration depth into a micropipette was measured, and the biomechanical properties were derived. Oocyte fertilization, day 3 morphology, and blastocyst development were observed and compared between measured and unmeasured cohorts. A predictive classifier was trained to predict usable blastocyst formation and compared to the predictions of four experienced embryologists. Results 68 patients and their corresponding 1252 oocytes were included in the study. In the safety analyses, there was no significant difference between the cohorts for fertilization, while the day 3 and 5 embryo development were not negatively affected. Four embryologists predicted usable blastocyst development based on oocyte morphology with an average accuracy of 44% while the predictive classifier achieved an accuracy of 71%. Retaining the variables necessary for normal fertilization, only data from successfully fertilized oocytes were used, resulting in a classifier an accuracy of 81%. Conclusions To date, there is no standard guideline or technique to aid in the selection of oocytes that have a higher likelihood of developing into usable blastocysts, which are chosen for transfer or vitrification. This study provides a comprehensive workflow of extracting biomechanical properties and building a predictive classifier using these properties to predict mature oocytes’ developmental potential. The classifier has greater accuracy in predicting the formation of usable blastocysts than the predictions provided by morphological information or maternal factors. The measurement procedure did not negatively affect embryo culture outcomes. While further analysis is necessary, this study shows the potential of using biomechanical properties of oocytes to predict embryo developmental outcomes.

Whole-genome sequencing of preimplantation human embryos (PGT-WGS) is not currently performed due to the insufficient quality of amplified DNA from embryo biopsies. Here we present a PGT-WGS approach that takes advantage of the improved genome coverage and uniformity of primary template-directed amplification (PTA) to call almost all early embryo genetic variants accurately and reproducibly from a preimplantation biopsy. In a research sibling cohort, we identified clonal and mosaic chromosomal aneuploidy, de novo mitochondrial variants, and variants predicted to cause mendelian and non-mendelian diseases. In addition, we utilized the genome-wide data to compute polygenic risk scores for common diseases. Finally, we performed a clinical study on the parents and 8 sibling embryos of an infant with neonatal-onset seizures and severe developmental delay. We first used low-pass whole genome sequencing to identify embryos most likely to successfully implant based on aneuploidy. We then performed PGT-WGS and evenly covered a mean of 99.1% of the embryo genomes at least 10X, resulting in the detection of 99.5% of parental variants and enabling us to screen genome-wide for inherited and de novo genetic variants in each embryo using ACMG rules. Although numerous computational, interpretive, and ethical challenges remain for widespread implementation, this study establishes the technical feasibility of screening for and preventing numerous debilitating genetic diseases with PGT-WGS.

To observe if embryology outcomes differ between controlled ovarian hyperstimulation initiated in the follicular or luteal phase of the menstrual cycle.

BACKGROUND: Preimplantation genetic testing is commonly performed by removing cells from the trophectoderm, the outer layer of the blastocyst, which subsequently forms the placenta. Because preimplantation genetic testing removes the cells that are destined to form the placenta, it is possible that preimplantation genetic testing could be associated with an increased risk for adverse outcomes associated with abnormal placentation. Despite the increasing utilization of preimplantation genetic testing, few studies have investigated the perinatal outcomes, with published studies yielding contradictory findings and using small sample sizes. OBJECTIVE: This study aimed to compare the perinatal outcomes of singleton pregnancies conceived following frozen embryo transfer of a single, autologous blastocyst either with or without preimplantation genetic testing. STUDY DESIGN: This was a retrospective analysis of autologous frozen embryo transfer cycles that led to singleton live births per the Society for Assisted Reproductive Technology Clinical Outcomes Reporting System, including cycles initiated between 2014 and 2015. The perinatal outcomes, including birthweight, Z-score, small for gestational age, large for gestational age, macrosomia, and preterm birth, were compared between pregnancies with or without preimplantation genetic testing. We conducted multivariable linear regression analyses for the birthweight and Z-score and logistic regression for the binary outcomes. A false discovery rate was adjusted to decrease the type I error from multiple hypothesis testing. RESULTS: Of the 16,246 frozen embryo transfers resulting in singleton births included in this analysis, 6244 involved the transfer of a single blastocyst that had undergone preimplantation genetic testing, and the remainder (n=10,002) involved the transfer of a single blastocyst that had not undergone a biopsy. When compared with the women from the nonpreimplantation genetic testing group, the average maternal age (35.8 +/- 4.1 vs 33.7 +/- 3.9; P<.001) and prevalence of prior spontaneous abortion (37.3% vs 27.7%; P<.001) were higher among women from the preimplantation genetic testing group. Bivariate analysis revealed a higher prevalence of small-for-gestational-age newborns (4.8% vs 4.0%; P=.008) and premature delivery (14.1% vs 12.5%; P=.005) and a lower prevalence of large-for-gestational-age newborns (16.3% vs 18.2%; P=.003) and macrosomia (11.1% vs 12.4%; P=.013) among the preimplantation genetic testing pregnancies. Multivariate regression analyses, adjusting for the year of transfer, maternal age, maternal body mass index, smoking status (3 months before the treatment cycle), obstetrical histories (full-term birth, preterm birth, and spontaneous abortion), infertility diagnosis, and infant sex suggested a significantly increased odds of preterm birth (adjusted odds ratio, 1.20; 95% confidence interval, 1.09-1.33; P<.001) from preimplantation genetic testing blastocysts. Birthweight (-14.63; 95% confidence interval, -29.65 to 0.38; P=.056), birthweight Z-score (-0.03; 95% confidence interval, -0.06 to 0.00; P=.081), and odds of small-for-gestational-age newborns (adjusted odds ratio, 1.17; 95% confidence interval, 0.99-1.38; P=.066), largefor-gestational-age newborns (adjusted odds ratio, 0.96; 95% confidence interval, 0.88-1.06; P=.418), and macrosomia (adjusted odds ratio, 0.96; 95% confidence interval, 0.85-1.07; P=.427) did not differ between the frozen transfer cycles with or without preimplantation genetic testing in the analysis adjusted for the confounders. Subgroup analysis of the cycles with a stated infertility diagnosis (n=14,285) yielded consistent results. CONCLUSION: Compared with frozen embryo transfer cycles without preimplantation genetic testing, the frozen embryo transfer cycles with preimplantation genetic testing was associated with a small increase in the likelihood of preterm birth. Although the increase in the risk for prematurity was modest in magnitude, further investigation is warranted.

Objective: To determine whether trophectoderm (TE) grade or inner cell mass (ICM) grade have predictive value after euploid frozen Patient(s): Women with >= 2 prior pregnancy losses with >= 1 euploid embryo for transfer undergoing preimplantation genetic testing for aneuploidy. Intervention(s): Intracytoplasmic sperm injection, TE biopsy, blastocyst grading and vitrification, and single euFET, with first transfer outcome recorded. Main Outcome Measure(s): Live birth and clinical miscarriage rates. Result(s): The study included 660 euFET cycles. In a binomial logistic regression analysis accounting for age, body mass index, antimullerian hormone level, and day of blastocyst biopsy, or ICM grade C was not significantly associated with odds of live birth, miscarriage, or biochemical pregnancy loss. TE grade C was significantly associated with odds of live birth and was not associated with odds of miscarriage or biochemical pregnancy loss. Blastocyst grade CC had significantly lower live birth rate compared with Conclusion(s): Embryo grade CC and TE grade C are associated with decreased odds of live birth after euFET in RPL patients. Embryo grade is not associated with odds of clinical miscarriage in this cohort of RPL patients, suggesting that additional embryonic or uterine factors may influence the risk of pregnancy loss.

We sought to examine current use of, and indications for, progesterone supplementation in the luteal phase of non-in vitro fertilization (non-IVF) infertility treatments among Obstetrician Gynaecologists (OB/GYN) compared to Reproductive Endocrinology and Infertility (REI) Subspecialists. Using a web-based survey, the practices of U.S. REI and OB/GYN physicians practicing infertility from 2014-2016 were assessed. The main outcome measures were frequency of use and indications for progesterone supplementation for luteal-phase support in non-IVF infertility treatments. Comparisons between physicians groups by indication and treatment type were performed using Chi-square and Fisher's exact tests. Sixty-four REIs and 49 OB/GYNs completed the survey. One hundred per cent of REI and 73.5% of OB/GYN respondents prescribed progesterone for luteal-phase support as part of non-IVF infertility treatment. The majority of all respondents utilized progesterone supplementation for one or more indications in clomiphene citrate and letrozole treatment cycles. Treatment type was the primary decisional factor reported by REIs (56%) for prescription of luteal-phase progesterone support. Serum progesterone level was reported as the leading decisional factor for luteal-phase supplementation (66.7%) by OB/GYNs. Luteal-phase progesterone supplementation in non-IVF treatments appears common for both physician groups in the United States in spite of lack of evidence supporting its effectiveness.

The impacts of aneuploidy on gene expression arise via multiple mechanisms of dysregulation. These include not only the direct (i.e., primary) effects of dosage alteration—which are predicted to alter expression in the direction of copy number change—but also complex indirect (i.e., secondary or trans-acting) effects that cascade through regulatory networks, including genes on other chromosomes (1FitzPatrick D.R. Transcriptional consequences of autosomal trisomy: primary gene dosage with complex downstream effects.Trends Genet. 2005; 21: 249-253Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). So-called tertiary effects may additionally arise in response to the phenotypic effects of aneuploidy (e.g., growth defects). Reported in this issue of Fertility and Sterility, Sanchez-Ribas et al. generated some of the first RNA-seq data to quantify these effects at a critical stage of human preimplantation development (2Sanchez-Ribas I. Transcriptomic behavior of genes associated with chromosome 21 aneuploidies in early embryo development.Fertil Steril. 2019; 111: 991-1001Abstract Full Text Full Text PDF Scopus (8) Google Scholar). By means of testing for differential expression between blastocysts with putative trisomy 21 (T21), monosomy 21 (M21), and euploid controls, the authors added to the set of candidate genes, previously compiled from later developmental stages, that may contribute to development of Down syndrome phenotypes. This complements similar data recently published for monosomic and trisomic blastocysts of every individual chromosome (3Licciardi F. Lhakhang T. Kramer Y.G. Zhang Y. Heguy A. Tsirigos A. Human blastocysts of normal and abnormal karyotypes display distinct transcriptome profiles.Sci Rep. 2018; 8: 14906Google Scholar). Future work will be necessary to refine such lists of differentially expressed genes both through further bioinformatic analysis (e.g., layering with relevant functional annotations) and, ultimately, through detailed mechanistic work in model organisms or human cell lines. Notably, T21, M21, and control groups of blastocysts were assigned based on array comparative genomic hybridization testing of a single blastomere biopsied at the day-3 cleavage stage. This approach assumes meiotic origins of detected chromosome abnormalities and that a single blastomere biopsy would therefore be representative of the subsequently tested blastocyst—a major point of contention in the field in light of the phenomenon of mitotic-origin chromosomal mosaicism. Interestingly, a subset of putative aneuploid embryos exhibited gene expression patterns, both when analyzed globally and when restricted to the affected chromosome, that clustered with control embryos. Does this reflect mosaic status or other factors driving expression heterogeneity among embryos classified as aneuploid? Single-cell studies of disaggregated embryos will be vital for resolving this question. Recent evidence supports widespread compensation of copy number variation at the post-transcriptional level, mitigating protein aggregation and maintaining stoichiometry of multisubunit protein complexes (4Ishikawa K. Makanae K. Iwasaki S. Ingolia N.T. Moriya H. Post-translational dosage compensation buffers genetic perturbations to stoichiometry of protein complexes.PLoS Genet. 2017; 13: e1006554Crossref PubMed Scopus (44) Google Scholar). In contrast, studies in both humans and model organisms (5Gasch A.P. Hose J. Newton M.A. Sardi M. Yong M. Wang Z. Further support for aneuploidy tolerance in wild yeast and effects of dosage compensation on gene copy-number evolution.Elife. 2016; 5: e14409Crossref PubMed Scopus (24) Google Scholar) have arrived at conflicting conclusions about the existence of autosomal “dosage compensation” at the level of transcription. Consistent with some earlier reports from other developmental stages (as well as limited aforementioned data from the blastocyst stage [3]), Sanchez-Ribas et al. found that only a minority (5.4% at a false discovery rate of 0.05) of genes on chromosome 21 are differentially expressed in M21 blastocysts compared with euploid control samples. Somewhat more surprisingly, they detected no differentially expressed genes for T21 embryos compared with control samples. For both M21 and T21, chromosome 21 harbored a smaller proportion of differentially expressed genes compared with other chromosomes, further indicating that at least at this stage, the indirect effects of chromosome 21 aneuploidy outweigh the primary dosage effects. It is important to caution, however, that absence of evidence of differential expression does not constitute evidence of absence, because factors such as sample size, expression variability, and baseline expression level all influence statistical power. Similarly, the discovery of more differentially expressed genes for M21 than T21 could either reflect a biological distinction—potentially consistent with the relative viability of trisomy versus monosomy—or a statistical consequence of the fact that monosomy constitutes a proportionally greater change in copy number (−1/2 vs. +1/3 for trisomy). Nevertheless, these findings raise important questions about the possibility of transcriptional “dosage compensation” and the global or gene-specific regulatory mechanisms underlying such a phenomenon. A translational objective of this study was to identify transcriptional biomarkers to distinguish viable aneuploid embryos, nonviable aneuploid embryos, and euploid embryos based on RNA-seq of trophectoderm biopsies. Given the limited evidence of differential expression reported by the authors, it remains unclear whether this goal can be achieved or how it would improve on DNA-based tests. Moreover, even when robust differential expression is detected among groups of samples, accurate classification of an individual sample may not be trivial. Despite these challenges, the rich functional genomic data generated by this and other recent studies are providing intriguing new perspectives on the biology of early human development. Transcriptomic behavior of genes associated with chromosome 21 aneuploidies in early embryo developmentFertility and SterilityVol. 111Issue 5PreviewTo analyze how chromosome 21 (HSA21) ploidy affects global gene expression of early human blastocysts. Full-Text PDF

OBJECTIVE:To determine if trophectoderm (TE) grade or inner cell mass (ICM) grade have predictive value after euploid frozen embryo transfer (euFET) among RPL patients.DESIGN:Retrospective cohort study.SETTING:Single fertility center, 2012-2018.PATIENTS:Patients with ≥ 2 prior pregnancy losses performing PGT-A with ≥1 euploid embryo for transfer.INTERVENTIONS:All patients underwent ICSI, trophectoderm biopsy, blastocyst grading and vitrification, and single euFET. Outcome of the first transfer was recorded.MAIN OUTCOME MEASURES:Live birth (LB) and clinical miscarriage (CM) rates.RESULTS:660 euFET were included. In a binomial logistic regression analysis accounting for age, BMI, AMH and day of blastocyst biopsy, ICM grade C was not significantly associated with odds of live birth (aOR 0.50, 95% CI 0.24-1.02 p=0.057), miscarriage (aOR 1.67, 95% CI 0.56-5.00, p=0.36) or biochemical pregnancy loss (aOR 1.58, 95% CI 0.53-4.75, p=0.42). TE grade C was significantly associated with odds of live birth (aOR 0.49, 95% CI 0.28-0.86, p=0.01) and was not associated with odds of miscarriage (aOR 2.00, 95% CI 0.89-4.47, p=0.09) or biochemical pregnancy loss (aOR 1.85, 95% CI 0.77-4.44, p=0.17). Blastocyst grade CC had significantly lower LB rate compared to all other blastocyst grades (p<0.05, chi-square analysis).CONCLUSION:Embryo grade CC and TE grade C are associated with decrease in odds of LB after euFET in RPL patients. Embryo grade is not associated with odds of CM in this cohort of RPL patients, suggesting that additional embryonic or uterine factors may influence risk of pregnancy loss.

Research question: To investigate the association between anti-Mullerian hormone (AMH) concentration and maternal age with single euploid cryopreserved embryo transfer. Design: Retrospective cohort study from 2014 to 2018 at an academic medical centre, including 389 cycles of IVF with 24-chromosome Day 5/6 preimplantation genetic testing for aneuploidies (PGT-A). Multivariate logistic regression was used to study AMH and age in relation to IVF outcomes (positive beta human chorionic gonadotrophin [bHCG], ongoing pregnancy and pregnancy loss rates) for patients with at least one euploid embryo for transfer, controlling for patient and cycle confounders. Results: In this cohort the overall unadjusted positive bHCG rate was 69.2% and ongoing pregnancy rate was 52.7% per transfer, while the pregnancy loss rate was 23.4% per cycle with positive bHCG. Multivariate analysis found that compared with the reference group of AMH 1 to <5 ng/ml, AMH <1 and 5+ did not have any significant difference in positive bHCG (odds ratio, OR 0.65 [0.30-1.44] and 1.27 [0.61-2.65] for AMH <1 and AMH 5+, respectively) or ongoing pregnancy (OR 0.80 [0.43-1.50] and 1.41 [0.68-2.90]). However, AMH <1 had statistically significant lower euploid miscarriage rates compared with the reference group with OR 0.32 (0.12-0.85, P = 0.022); AMH 5+ did not have any statistical difference in miscarriage rate. Neither age at retrieval nor age at transfer were significantly associated with transfer outcomes. Conclusions: AMH concentration was not associated with positive bHCG or ongoing pregnancy for euploid embryo transfers after adjustment for potential confounders. Maternal age was not associated with euploid transfer outcomes. Further study is warranted in larger cohorts.

Recurrent pregnancy loss (RPL) is a diverse syndrome with many causes, the most common being embryonic aneuploidy. PGT-A should thus improve outcomes in RPL patients, but studies conflict as to whether PGT-A reduces clinical losses or improves live birth. This study seeks to determine if PGT-A improves outcomes in RPL patients. Retrospective cohort study. Patients undergoing their first IVF cryo-all cycle followed by single embryo transfer (SET) from 2012 to 2018 were reviewed. Patients with ≥2 losses were considered to have RPL independent of other diagnoses. Outcomes from first cryo-SETs were recorded. Clinical pregnancy was defined as having a gestational sac. Ongoing pregnancy was defined as delivery or ongoing gestation beyond 8 weeks. There were 3 patient groups: Infertile using PGT-A, RPL using PGT-A, and RPL not using PGT-A. These divisions empower fundamental questions: 1. Does PGT-A use in RPL reduce clinical loss rates and/or raise delivery rates; 2. Does it normalize outcomes to the same level as infertile controls? 3975 infertile and 660 RPL patients had euploid SETs. An additional 101 RPL patients underwent frozen SET without PGT-A. Clinical pregnancy rates were higher in RPL patients using PGT-A (73%) than those not (61%) demonstrating a positive impact of selection. In fact, they normalized to that of infertile controls (72%). Most importantly, PGT-A reduced clinical loss risk amongst RPL patients. (32% vs 15%). The risk remained higher than infertile controls (15% vs 12%) indicating that aneuploidy is not the lone source of RPL. Ultimately the higher initial pregnancy rate and lower loss risk increased live birth rates in the RPL PGT-A group to 62% compared to those not using PGT-A (41%). PGT-A use in RPL patients significantly raises clinical pregnancy rates while reducing loss rates and provides a 50% relative increase in delivery rates (62% vs 41%). While clinical loss rates are reduced, they remain 3% higher than in infertile controls. These findings reflect that much of RPL may be attributed to aneuploidy, but that other factors also lead to RPL. PGT-A is a useful adjunct in the care of patients with RPL. While our large sample size provides powerful insight into this question, definitive resolution awaits class I data.Tabled 1Infertile w/ PGT-A N=3975RPL w/ PGT-A N=660RPL w/o PGT-A N=101P-valueMean oocyte age in years (SD)*36.2 (3.4)36.7 (3.4)34.9 (4.8)<.01Outcome †Clinical Pregnancy per ET2859 (72%)480 (73%)62 (61%)0.01Ongoing Pregnancy per ET2670 (67%)431 (65%)56 (55%)0.03Live birth per ET2524 (63%)408 (62%)42 (41%)<.01Clinical loss per pregnancy335 (12%)72 (15%)20 (32%)<.01Total loss per pregnancy641 (20%)124 (23%)34 (44%)<.01*ANOVA analysis.†Chi-squared analysis. Open table in a new tab

The hatching of the developing blastocyst through the zona pellucida, the glycoprotein coat that facilitates fertilization and protects the embryo during early growth and transport through the reproductive tract, is critical for the direct embryo–endometrium interface needed for implantation. An abnormally thickened or rigid zona pellucida may be a cause for some patients' reproductive failures, and assisted hatching—a micromanipulation technique that artificially breaks or weakens the zona—may improve clinical pregnancy rates for specific patient populations with particularly poor prognoses.

To investigate if history of miscarriage or recurrent pregnancy loss (RPL, two or more miscarriages) is associated with euploid frozen embryo transfer outcomes. Retrospective cohort study from 2014 to 2018 of patients undergoing first cycle of in-vitro fertilization with 24-chromosome day 5/6 preimplantation genetic screening (IVF-PGS) at an academic medical center. We used multivariate logistic regression to investigate the relationship between history of miscarriage and euploid single embryo frozen transfer outcomes (ongoing pregnancy, miscarriage). Ongoing pregnancy was defined as continued pregnancy at 8-12 weeks with transfer to obstetrical care; miscarriage included both biochemical and clinical miscarriage. The analysis controlled for the following patient and cycle potential confounders: IVF indication, gravidity, parity, age at retrieval, age at transfer, antimullerian hormone (AMH), body mass index (BMI), ICSI, oocytes retrieved, cohort size, morphology (expansion, inner cell mass, and trophectoderm), medicated versus natural frozen embryo transfer, and endometrial thickness. In our cohort of 283 patients, the overall ongoing pregnancy rate was 51.2% and the miscarriage rate was 17.3%. 35.7% of patients had a history of at least one prior miscarriage, while 14.5% of patients had a history of RPL. For patients with a history of miscarriage, we found that the adjusted odds ratio (OR, 95% confidence interval CI) of ongoing pregnancy was OR 0.93 (0.43-2.02, p=0.85) and OR for miscarriage was 1.94 (0.71-5.26, p=0.20), when compared to patients without a history of miscarriage. We also found that when compared to women without a history of miscarriage, the OR for ongoing pregnancy was 0.89 (0.40-2.01, p=0.88) for history of 1 miscarriage, and 0.91 (0.26-3.17, p=0.69) for RPL patients. For miscarriage as an outcome, the OR was 2.10 (0.74-5.93, p=0.16) for history of 1 miscarriage, and OR 1.47 (0.35-6.28, p=0.60) for RPL patients. In our cohort, there was no significant association between miscarriage history and euploid transfer outcomes (ongoing pregnancy, miscarriage) after adjustment for potential confounders. However, women with a history of miscarriage had elevated odds of having a miscarriage after euploid embryo transfer (though not statistically significant); further study in larger data sets is warranted.Tabled 1Euploid transfer outcomes in relation to miscarriageNumber of patientsUnadjusted ongoing pregnancy (OP) rateAdjusted odds ratio OP, OR (95% CI)p-valueUnadjusted miscarriage rateAdjusted odds ratio miscarriage, OR (95% CI)p-valueNo miscarriage23453.8%Reference14.3%ReferenceHistory of miscarriage15549.5%0.93 (0.43-2.02)0.8522.8%1.94 (0.71-5.26)0.20History of 1 miscarriage9053.3%0.89 (0.40-2.01)0.8823.3%2.10 (0.74-5.93)0.16History of 2 or more miscarriages (RPL)6543.9%0.91 (0.26-3.17)0.6922.0%1.47 (0.35-6.28)0.60 Open table in a new tab

The objective of our study is to assess the relationship of embryo ploidy status in relation to embryo sex, morphological characteristics, and transfer parameters.