Faculty Bibliography

OBJECTIVE: Morphologic evaluation of embryos after fertilization is the first step in embryo assessment, with two pronuclei (2PN) indicating normal fertilization. Deviations from 2PN are considered consistent with abnormal fertilization and genetic ploidy, and may be discarded instead of cultured. The aim of this study was to determine the genetic ploidy of three pronuclei (3PN) embryos diagnosed by morphology. MATERIALS AND METHODS: Sixty-two 3PN embryos donated to research that underwent IVF with either insemination or ICSI were collected from January - April, 2021. 3PN embryos were identified at time of fertilization check and vitrified. Batched 3PN embryos were subsequently warmed and cultured. Embryos were assessed for development to the blastocyst stage on days 5, 6 and 7 of culture, and embryos that developed into blastocysts underwent two separate trophectoderm (TE) biopsies. TE biopsies, along with maternal and paternal samples were sent to a pre-implantation genetic testing (PGT) lab to determine the genetic ploidy composition of the morphological based 3PN embryos. Testing included PGT for aneuploidy (PGT-A) using the PGTseq platform that routinely includes triploidy detection via single nucleotide polymorphism (SNP) B allele ratio. Testing was also performed using a second method, SNP allele sharing, with the maternal and paternal DNA samples. This method can detect both triploidy and parental of origin of abnormalities.
RESULT(S): Of the 62 3PN embryos cultured, 17 (27%) developed into blastocysts that underwent TE biopsy. In all cases paired biopsies were concordant. Three of the 17 biopsied embryos were diploid (18%) and 14 were triploid (82%). All 3 diploid embryos were the result of insemination and were aneuploid on PGT-A; no euploid embryos were identified. The overall rate of diploid tested blastocysts was 4.8% (3/62) among all 3PNs collected. Of the 14 triploid embryos, 10 were the result of IVF with traditional insemination and 4 were from ICSI. All triploid embryos from insemination were consistent with paternal origin while all triploid embryos from ICSI were consistent with maternal origin. Both methods for detecting triploidy were concordant.
CONCLUSION(S): Embryos morphologically diagnosed as 3PN are typically discarded as they are likely the result of abnormal fertilization consistent with triploidy. This study demonstrates that a small percentage of 3PN embryos have the potential to develop into blastocysts with a diploid genetic complement. While none of the diploid 3PN embryos in this study were found to be euploid, this could be due to the small sample size and it is possible that a larger number of embryos may result in 3PN euploid embryos which could impact a labs decision on what tissue to discard or culture. It should be noted that due to the inherently subjective component of morphologic assessment, these findings may not translate to other laboratories. IMPACT STATEMENT: While 3PN embryos are typically discarded in IVF after both insemination and ICSI, our study shows that a small proportion have the potential to develop into diploid blastocysts where reproductive potential remains to be seen

OBJECTIVE: To compare telomere length (TL) and telomerase gene expression in human euploid and aneuploid blastocysts generated from IVF treatment. MATERIALS AND METHODS: TL and telomerase gene expression were measured in cryopreserved aneuploid (N=115) and euploid (N=4) human blastocysts donated by 26 patients who consented research under approval of IRB study #16-00154. Blastocysts were classified according to number of aneuploid chromosomes (A1-one segmental error, A2-one whole chromosome error, A3-two chromosomal errors and A4- >= 3 chromosomal errors). Genomic DNA and messenger RNA were separated simultaneously from individual blastocysts after thawing in vitrification-warming media. Telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) mRNA levels were determined by RT-qPCR with GAPDH as internal control, and TL was measured by qPCR with 5s rDNA as internal control. Relative gene expression and TL were calculated by DELTADELTA^Ct method, and GraphPad Prism 8 software was used for statistical analysis.
RESULT(S): TL and telomerase gene expression were not normally distributed, so nonparametric tests were used to compare the medians among groups (Table 1). Median TL, TERTand TERC levels didn't differ by number of chromosome errors nor between aneuploid and euploid groups. Intriguingly, TL, TERT and TERC levels in aneuploid blastocysts tended to be greater compared to euploid blastocysts. TL in blastocysts correlated with telomerase TERT expression (R² =0.054, P = 0.011), but not TERC expression (R² =0.0002, P = 0.865).
CONCLUSION(S): To our knowledge, this is the largest study to measure telomere length and telomerase gene expression in human blastocysts. Our data indicated that telomeres are lengthened and telomerase is activated in aneuploid embryos at blastocyst stage. Moreover, telomere length and telomerase gene TERT in human blastocysts correlate regardless of ploidy status. Like cancer cells, TERT is highly expressed in aneuploid blastocysts. IMPACT STATEMENT: Robust TERT expression and telomere maintenance in aneuploid human blastocysts may explain why extended in vitro culture alone is insufficient to cull out aneuploidy embryos during IVF (Table Presented)

OBJECTIVE: The rise of the SARS-CoV-2 pandemic and temporary closures of fertility centers made the effect on POC cycles uncertain but garnered national attention1,2. We sought to assess the impact of the pandemic on POC cycles in a pandemic epicenter. MATERIALS AND METHODS: This is a retrospective cohort study of all POC cycles at an academic fertility center in New York City from 1/1/2019- 12/31/2020. Primary outcomes were number of POC patients (pts) and cycles. Secondary outcomes were pt relationship status, payment method, AMH, and cycle parameters; with subgroup analyses by age groups. We also examined the relationship between monthly number of POC cycles and national SaRS-CoV-2 cases. Statistical analyses included z-score analysis, Mann-Whitney, and Chi-squared, with p<0.05 significant.
RESULT(S): Despite a 5.5 week center closure in 2020, POC pts increased 14% and POC cycles increased 16% from 2019 to 2020 (Table), with a 32% increase seen between June-Dec, 2020 . There was a 28% increase in POC pts <37yo in 2020 (252 pts vs. 323 pts, p<0.04) and no change in pts >37yo in 2020 (p=0.9). Relationship status did not differ between years (16% partnered, 76% single, 8% unknown in 2019 vs. 16% partnered, 73% single, 11% unknown in 2020; p=0.6). Fewer patients in 2020 had insurance coverage (16% vs. 24%, p<0.001). AMH was higher in 2020 (2.3 vs. 2.1, p<0.03), but days of stimulation, oocytes retrieved, oocytes frozen, total gonadotropins, and maximum estradiol (E2) were not different (Table). While national SARS-CoV-2 cases peaked in April, July, and November 2020, monthly POC cycles at our center did not decrease with surges in SARS-CoV-2 after our center reopened in May (p=0.24). In 2020 there were 23 cycles cancelled, none due a positive SARS-CoV-2 test.
CONCLUSION(S): POC volume increased at our center in 2020, especially in young patients, despite center closures and SARS-CoV-2 surges. IMPACT STATEMENT: More young people pursued POC despite the SARS-CoV-2 pandemic. Further research is needed to understand POC pt motivations and experiences during a pandemic. (Table Presented)

OBJECTIVE: With increased availability of genetic testing, particularly expanded carrier screening (ECS) and hereditary cancer (HC) testing, the scope of conditions for which PGT-M is performed is expanding. Our aim was to report on indications for PGT-M from the past decade in our large academic practice. MATERIALS AND METHODS: All PGT-M cases occurring between January 2010 and April 2021 were reviewed.
RESULT(S): A total of 331 patients were identified for which PGT-M was performed for 124 different genes over 582 cycles. Eighteen patients tested for two genes and one patient tested for three genes; therefore, there were a total of 351 unique PGT-M cases. Of the 124 genes tested, 82 (66.1%) were of childhood onset while 16 (12.9%) were of adult onset, and the remaining 26 (21.0%) were of variable onset. Over the entire study period, 70/351 patients (19.9%) tested for 16 genes related to HC syndromes; between 2010-2017, HC-related PGT-M accounted for 12.6% (20/159) of our total PGT-M volume, and since 2018, it rose to 26.0% (50/192). Overall, BRCA1 was the most common gene tested in our practice, and hereditary breast and ovarian cancer syndrome (BRCA1 and BRCA2) accounted for 15.1% (53/351) of our total PGT-M patient population. 181/351 patients (51.6%) tested for 49 genes that are commonly found on ECS, with cystic fibrosis (CFTR) being the most common (34/351) followed by fragile X (FMR1; 32/351); these represented the second and third most common genes tested in our practice (9.7% and 9.1% respectively). Of all patients doing PGT-M for ECS-related conditions, 46.4% (84/181) tested for 41 genes that are not detected by traditional or ethnicity-based carrier screening, with the most common being GJB2-related nonsyndromic hearing loss (the fourth most common condition tested in our practice, representing 6.6% of our total PGT-M volume), followed by 21-hydroxylase deficient congenital adrenal hyperplasia (CYP21A2) and familial Mediterranean fever (MEFV). There were eight patients (2.3%) who either were or could have been identified on our current 283-disease ECS panel but would have been missed on our prior 176-disease ECS panel. Eight patients did PGT-M for HLA matching, and three did non-disclosure PGT-M (two for Huntington's disease/HTT and one for CADASIL/NOTCH3). 80/124 genes tested (64.5%) were unique to a single patient.
CONCLUSION(S): PGT-M is performed for a wide range of genetic conditions, and nearly two-thirds of genes tested in our clinic were unique to a single patient. While most conditions tested are childhood-onset, BRCA1 is the most common gene tested by our patient population, and the proportion of patients testing for HC syndromes has doubled over the past three years. More than half of patients pursued PGT-M for conditions detectable through ECS but not through traditional carrier screening; however, increasing the ECS panel size by more than 100 conditions has only had a minor effect on PGT-M uptake. IMPACT STATEMENT: This large dataset from a single IVF clinic highlights the impact of HC testing and ECS on PGT-M utilization over the past decade

OBJECTIVE: To compare three different implantation prediction models using artificial intelligence algorithms (AI) that analyze (I) morphology only, (II) morphokinetic events only using an automatic AI-based annotation model, and (III) a combination of both, in a retrospective multi-center study. MATERIALS AND METHODS: The automatic morphokinetic evaluation tool was trained on 36561 annotated embryos obtained between 2014 - 2019 (34132 in training set and 2429 in test set). Morphokinetic annotation and morphology evaluation of 6938 embryos with known implantation data (KID) were used to train and test KID+TM, an AI algorithm. The training set consisted of 6363 embryos (1078 KID-positive and 5285 KID-negative). The blind test set consisted of 575 embryos (171 KID-positive and 404 KID-negative). KID+TM scored the embryos for implantation potential based on an automatic evaluation of morphokinetic and morphology data. We compared our combined morphokinetic and morphology model KID+TM to (I) a model that only considers morphokinetic events and (II) a model that only considers morphology from the last frame of embryo development.
RESULT(S): We aimed to compare the implantation prediction potential of algorithms that analyze morphokinetic features only, morphology features only, and a combination of both. To accomplish this, we trained a convolutional neural network (CNN) to perform automatic annotations of embryo development events (r² =0.95). Analysis of these estimated annotations revealed a robust implantation prediction tool with an area under curve (AUC) continuously increasing from 30 to 116 hours post insemination, reaching a maximal AUC of 0.65 at 116 hours. However, single image analysis of the last frame of the embryo video after the morula stage demonstrated better prediction with AUC of 0.68 at 116 hours. Thus, the combined morphokinetic and morphology algorithm was valuable for implantation prediction until the start of blastulation (~90 hours). After the start of blastulation, the combined algorithm did not demonstrate a superior AUC relative to the morphology only algorithm with an AUC of 0.68 at 116 hours.
CONCLUSION(S): The combined morphokinetic and morphology algorithm was more effective for implantation prediction at the cleavage stage than the morphology only algorithm. Thus, the combined algorithm has the potential to improve the embryo selection process for day 3 transfers. In the later stages of embryo development, the morphology only algorithm was as effective for implantation prediction as the combined algorithm. IMPACT STATEMENT: AI models have the potential to eliminate the high degree of inter- and intra-observer variability associated with embryo assessment by automating morphokinetic and morphology evaluation to accurately predict embryo outcomes, thereby improving IVF outcomes. In addition, AI models leveraging morphokinetic and morphology data may be able to accurately evaluate embryos in earlier stages of development to improve selection for day 3 transfers

OBJECTIVE: It is well known that the embryo aneuploidy rate increases with women's age [1], but the effect of age on complex chromosomal abnormality (CCA) is less clear. Here, we addressed the relationship between maternal age and CCA with a retrospective cohort study. MATERIALS AND METHODS: We reviewed results of preimplantation genetic testing (PGT) by aCGH or NGS of embryo biopsies performed in an academic IVF unit between 2010 and 2019. We excluded PGT results from single gene disorder and egg donation cycles. CCA was defined as>=3 chromosome abnormalities (whole, partial and/or mosaic). Maternal age was categorized according to SART age groups: <35, 35-37, 38-40, 41-42, and >42 years. Statistical analyses were conducted using GraphPad Prism 8.
RESULT(S): 27,423 embryos were biopsied from 3,501 women aged 23 to 48 years. 4,740 embryos (16%) has CCA. Consistent with prior study [2], the most frequent chromosomes involved in CCA were 22, 16, 21 and 15, with incidences of 30.6%, 29.1%, 26.1% and 25.8% respectively. The number of chromosomal errors (from 3 to 42) involved in CCA did not correlate with maternal age (Spearman r = -0.0149, P = 0.3352). However, the rate of complex abnormal embryos tended to increase with advancing maternal age (9.7%, 11.2%, 10.9%, 24.8% and 43.6% in women aged < 35, 35-37, 38-40, 41-42, and > 42 years, respectively). Women over 40 years old had significantly higher rates of CCA compared to those under 40 years (Chisquare test, P < 0.0001). Surprisingly, the relationship between maternal age and CCA followed a U-shaped curve, decreasing from the 25 to 30 year old group (Pearson r = -0.831, P = 0.04) to the 30 to 35 year old group (Pearson r = 0.093, P = 0.861), then increased markedly in the 35 to 48 year old group (Pearson r = 0.921, P < 0.0001).
CONCLUSION(S):We found that CCA embryos share common features of aneuploidy, such as association with maternal age and preferential involvement of shorter chromosomes i.e. 22, 16, 21 and 15. Unexpectedly, our data showed that the relationship between CCA and maternal age assumes a U shape with increased rates at very young and very old ages. Both meiotic and mitotic errors contribute to chromosomal abnormality, and the contribution of each to CCA merits further investigation. IMPACT STATEMENT: The complex relationship between maternal age and embryo aneuploidy, which approximates a U-shape, may inform optimal timing of elective oocyte freezing and oocyte donation

OBJECTIVE: Oocyte cryo is widely used for fertility preservation, but the value of M1 cryo remains unclear. We evaluated the utility and efficiency of M1 compared to M2 cryo. MATERIALS AND METHODS: Patients (pts) who thawed autologous oocytes at our academic center from 2004-2020 were reviewed. Pts were excluded if cryo was performed for a medical indication, as research, due to no sperm or a natural disaster, in combination with embryos or for use with a gestational carrier. At our center, all M1s retrieved from 2004-2015 were cryopreserved; after 2015, M1s were only cryopreserved if <15 M2s were retrieved during the same cryo cycle. Outcomes included survival rate, useable embryo rate and embryo transfer (ET) results.Auseable embryo was defined as an embryo that was transferred, biopsied or cryopreserved for future use. Statistics included Fisher's exact test.
RESULT(S): 543 pts (median age at 1st cryo 38y, interquartile range 37-40y) underwent 800 cryo, 605 thaw and 416 ET cycles. Cryo was performed with vitrification for 72%, slow freezing for 4% and both technologies for 24% of pts. In total, 8511 oocytes (1019M1s + 7492 M2s)were thawed.All pts thawed >=1 M2, and 60% (n=327) thawed >=1 M1. See table for thaw outcomes of M1s vs. M2s. For 30 pts, >=1 M1 led to a useable embryo (n=32 useable embryos). Vitrification was used for 69% of these M1s (n=22) and slow freezing was used for 31% (n=10). Of the 32 useable embryos from M1s, 69% (n=22) underwent PGTand 4were euploid (17 aneuploid, 1 mosaic). Therewere 3 single ETs of euploid embryos from M1s, which led to 1 spontaneous abortion (SAB) and 2 biochemical pregnancies. Therewere 3 single ETs of untested embryos from M1s, which led to 1 negative result, 1 SAB and 1 singleton ongoing pregnancy. The ongoing pregnancy is from an ETof a day 5 morula and is now in the third trimester. There were 6 ETs in which untested embryos from M1s were transferred alongwith untested embryos fromM2s, resulting in 3 negative results, 1 SAB, 1 singleton live birth and 1 unknown outcome (ongoing singleton pregnancy at last contact).
CONCLUSION(S): Cryopreserved M1s can result in useable embryos and pregnancies, but are less likely to survive or form useable embryos than cryopreserved M2s. To our knowledge, this is the first report of an ongoing third trimester pregnancy from a cryopreserved M1. This information may be helpful for pt counselling and designing oocyte cryo protocols for embryology labs. IMPACT STATEMENT: Cryopreserved M1s may be a viable option for pts with a low M2 yield. (Table Presented)

OBJECTIVE: Mosaic embryo transfer (MET) following preimplantation genetic testing for aneuploidy (PGT-A) has become more commonplace, particularly among patients who do not have any euploid embryos available for transfer. Our aim was to report on uptake and results of prenatal diagnosis (PND) following MET. MATERIALS AND METHODS: All MET cases occurring at our clinic between September 2015 and February 2021 in which an ongoing pregnancy was documented were reviewed. Patients received genetic counseling prior to MET, including discussion of prenatal testing options, and a summary letter was provided upon discharge. Medical records were reviewed to determine whether PND was performed, and the types of analyses performed on amniotic fluid (AF) or chorionic villi (CV).
RESULT(S): Sixty-five patients had an ongoing pregnancy following MET. Eight patients were excluded from the analysis due to gestational age too early for PND, and we were able to obtain PND results for 33 of the 57 patients (57.9%). Of the remaining patients, 9/57 (15.8%) declined PND, and we were unable to obtain information about whether PND was performed for 15/57 (26.3%). Since 2/33 patients were carrying twins both originating from MET, there were a total of 35 conceptuses with PND results; 34 on AF and 1 on CV. In one case, spontaneous abortion (SAB) occurred following attempted amniocentesis and was attributed to an infection caused by the procedure. In 35/35 cases (100%), karyotype results were normal (46,XY or 46,XX). In 28/35 cases (80.0%), chromosomal microarray was also performed, and there were no cases in which segmental mosaicism identified in the embryo was detected. In 6/28 cases (21.4%), a variant of uncertain significance (VUS) was identified on a different chromosome; 5/6 of which were inherited from a parent and were below the resolution of PGT-A (mean size 206.6 kb). Two out of 35 cases (5.7%) involved additional uniparental disomy (UPD) analysis; one result was normal while the other was inconclusive due to lack of informative parental markers.
CONCLUSION(S): There were no cases identified in which PND results confirmed mosaicism detected by PGT-A; therefore, this risk appears to be low. Genetic counseling about MET should address benefits, limitations, and risks of PND, including potential of an unrelated VUS and procedurerelated risk of SAB. Currently, it remains unclear as to whether the benefits of PND outweighs these risks, and whether additional analyses such as UPD testing are warranted. Future studies are necessary to determine whether specific types of mosaic results are associated with different risks, as well as the psychological impact of PND after MET on patients. IMPACT STATEMENT: Our study provides evidence that the risks associated with METare likely overestimated, as we did not identify any cases of abnormal PND consistent with the mosaic PGT-A result. These data are necessary for accurate patient counseling and informed consent, as well as evidence-based clinical policy development

Study question: To explore the effect of chromosomal mosaicism detected in preimplantation genetic testing (PGT-A) on prenatal and postnatal outcome of mosaic embryo pregnancies Summary answer: No significant difference between euploid and mosaic embryos was observed in terms of weeks of gestation, average weight, and developmental defect of the babies born What is known already: Mosaic embryos have the potential to implant and develop into healthy babies.Transfer of these embryos is now offered as an option for women who undergo IVF resulting in no euploid embryos. While, prenatal diagnosis has shown the depletion of chromosomal mosaicism in mosaic embryos, several concerns remain. For instance, the direct effects of different kind of mosaicism on prenatal/postnatal outcome and the possibility that intra-biopsy mosaicism in the TE is a poor predictor of the ploidy status of the ICM. Thus, there is certainly a need for comprehensive analyses of obstetrical and neonatal outcome data of transferred mosaic embryos. Study design, size, duration: Compiled analysis from multicenter data on transfers of mosaic embryos (n=1,000) and their outcome, with comparison to a euploid control group (n=5,561). To explore the effect of embryonic mosaicism on newborns, we matched mosaic embryos resulting in a birth with a euploid embryo by a series of parameters (maternal age, embryo morphology, and indication for PGT-A). Prenatal tests and birth characteristics of >200 neonates from mosaic embryo transfers were compared to >200 euploid embryos. Participants/materials, setting, methods: PGT-A was performed on blastocyst- stage embryos with 24-Chromosome whole genome amplification (WGA)-based Next Generation Sequencing (NGS). In accordance with established guidelines, embryos were categorized as mosaic when PGT-A results indicated 20-80% aneuploid content. Prenatal testing where performed in 30% of pregnancies with amniocentesis, 4% did an extra analysis for potential UPD for the suspected mosaic chromosome, and an additional 16% performed chorionic villus sampling (CVS) and 9.5% performed noninvasive prenatal testing (NIPT). Main results and the role of chance: Of the 465 mosaic embryos that implanted, about 20% miscarried, and out of those, 75% were early spontaneous abortions. Of the pregnancies, 3 out of 368 were stillborn (2 out of them were twins that were extremely premature at 23 weeks, and the other died during pregnancy from a heart defect). The remaining 99% of those have been born or are late ongoing pregnancies at the time of analysis. Prenatal tests were performed in >200 pregnancies and the vast majority tested normal. All 5 abnormal cases were amniocentesis tests showing microdeletions or insertions of sizes smaller than the resolution used during PGT-A, so they were unrelated to the mosaicism detected with PGT-A. In fact, in none of the cases did the prenatal test reflect the mosaicism detected at the embryonic stage. Matching each of the 162 mosaic embryos resulting in a birth with a euploid embryo, we found that the length of gestation was similar on average, and so was the average weight of the babies at birth. We also gathered information on the routine physical examination performed on babies at birth, and of those 162 babies from mosaic embryo transfers, none had obvious developmental defects or gross abnormalities. Limitations, reasons for caution: Even though newborns resulting from mosaic embryo transfers in this study invariably appeared healthy by routine examination, concerns for long-term health cannot yet be entirely dispelled. The question must therefore be carefully considered by each clinic and patient situation. Wider implications of the findings: Prenatal testing of >200 pregnancies from mosaic embryo transfers showed no incidence of mosaicism that matched the PGT-A findings, indicating the involvement of self-corrective mechanisms. Pregnancy and obstetric data indicates that mosaic embryos prevailing through gestation and birth have similar chromosomal and physiological health compared to euploid embryos

OBJECTIVE:To validate and apply a strategy permitting parallel preimplantation genetic testing (PGT) for mitochondrial DNA (mtDNA) disease and aneuploidy (PGT-A). DESIGN/METHODS:Preclinical test validation and case reports. SETTING/METHODS:Fertility centers. Diagnostics laboratory. PATIENTS/METHODS:Four patients at risk of transmitting mtDNA disease caused by m.8993T>G (Patients A and B), m.10191T>G (Patient C), and m.3243A>G (Patient D). Patients A, B, and C had affected children. Patients A and D displayed somatic heteroplasmy for mtDNA mutations. INTERVENTIONS/METHODS:Embryo biopsy, genetic testing, and uterine transfer of embryos predicted to be euploid and mutation-free. MAIN OUTCOME MEASURES/METHODS:Test accuracy, treatment outcomes, and mutation segregation. RESULTS:Accuracy of mtDNA mutation quantification was confirmed. The test was compatible with PGT-A, and half of the embryos tested were shown to be aneuploid (16/33). Mutations were detected in approximately 40% of embryo biopsies from Patients A and D (10/24) but in none from Patients B and C (n = 29). Patients B and C had healthy children following PGT and natural conception, respectively. The m.8993T>G mutation displayed skewed segregation, whereas m.3243A>G mutation levels were relatively low and potentially impacted embryo development. CONCLUSIONS:Considering the high aneuploidy rate, strategies providing a combination of PGT for mtDNA disease and aneuploidy may be advantageous compared with approaches that consider only mtDNA. Heteroplasmic women had a higher incidence of affected embryos than those with undetectable somatic mutant mtDNA but were still able to produce mutation-free embryos. While not conclusive, the results are consistent with the existence of mutation-specific segregation mechanisms occurring during oogenesis and possibly embryogenesis.