Identifying risk variants for embryo aneuploidy using ultra-low coverage whole-genome sequencing from preimplantation genetic testing.

Aneuploidy frequently arises during human meiosis and is the primary cause of early miscarriage and in vitro fertilization (IVF) failure. Individuals undergoing IVF exhibit significant variability in aneuploidy rates, although the exact genetic causes of the variability in aneuploid egg production remain unclear. Preimplantation genetic testing for aneuploidy (PGT-A) using next-generation sequencing is a standard test for identifying and selecting IVF-derived euploid embryos. The wealth of embryo aneuploidy data and ultra-low coverage whole-genome sequencing (ulc-WGS) data from PGT-A have the potential to discover variants in parental genomes that are associated with aneuploidy risk in their embryos. Using ulc-WGS data from ∼10,000 PGT-A biopsies, we imputed genotype likelihoods of genetic variants in embryo genomes. We then used the imputed variants and embryo aneuploidy calls to perform a genome-wide association study of aneuploidy incidence. Finally, we carried out functional evaluation of the identified candidate gene in a mouse oocyte system. We identified one locus on chromosome 3 that is significantly associated with meiotic aneuploidy risk. One candidate gene, CCDC66, encompassed by this locus, is involved in chromosome segregation during meiosis. Using mouse oocytes, we showed that CCDC66 regulates meiotic progression and chromosome segregation fidelity, especially in older mice. Our work extended the research utility of PGT-A ulc-WGS data by allowing robust association testing and improved the understanding of the genetic contribution to maternal meiotic aneuploidy risk. Importantly, we introduce a generalizable method that has potential to be leveraged for similar association studies that use ulc-WGS data.

Identifying risk genes for embryo aneuploidy using ultra-low coverage whole-genome sequencing.

Background: Aneuploidy, the state of a cell containing extra or missing chromosomes, frequently arises during human meiosis and is the primary cause of early miscarriage and maternal age-related in vitro fertilization (IVF) failure. IVF patients exhibit significant variability in aneuploidy rates, although the exact genetic causes of the variability in aneuploid egg production remain unclear. Preimplantation genetic testing for aneuploidy (PGT-A) using ultra-low coverage whole-genome sequencing (ulc-WGS) is a standard test for identifying and selecting IVF-derived embryos with a normal chromosome complement. The wealth of embryo aneuploidy data and ulc-WGS data from PGT-A has potential for discovering variants in paternal genomes that are associated with aneuploidy risk in their embryos. Methods: Using ulc-WGS data from ∼10,000 PGT-A biopsies, we imputed genotype likelihoods of genetic variants in parental genomes. We then used the imputed variants and aneuploidy calls from the embryos to perform a genome-wide association study of aneuploidy incidence. Finally, we carried out functional evaluation of the identified candidate gene in a mouse oocyte system. Results: We identified one locus on chromosome 3 that is significantly associated with maternal meiotic aneuploidy risk. One candidate gene, CCDC66, encompassed by this locus, is involved in chromosome segregation during meiosis. Using mouse oocytes, we showed that CCDC66 regulates meiotic progression and chromosome segregation fidelity, especially in older mice. Conclusions: Our work extended the research utility of PGT-A ulc-WGS data by allowing robust association testing and improved the understanding of the genetic contribution to maternal meiotic aneuploidy risk. Importantly, we introduce a generalizable method that can be leveraged for similar association studies using ulc-WGS data.

The correlation between morphology and implantation of euploid human blastocysts.

RESEARCH QUESTION: Does the composite morphology score or a particular developmental component (expansion stage, inner cell mass [ICM] or trophectoderm [TE]) of euploid blastocysts undergoing single frozen embryo transfer (FET) impact ongoing pregnancy/live birth (OP/LB) rates? DESIGN: Retrospective cohort study including a total of 2236 embryos from 1629 patients who underwent single euploid FET between 2012 and 2017. RESULTS: Embryos with an ICM grade of A compared with C had a higher OP/LB rate (55.6% versus 32.3%, P < 0.001). Blastocysts with a TE grade of A or B compared with C had a higher likelihood of OP/LB (A versus C: odds ratio [OR] 1.6, 99% confidence interval [CI] 1.1-2.3, B versus C: OR 1.5, 99% CI 1.1-2.1), and blastocysts with a developmental stage of 4 or 5 compared with 6 had higher odds of OP/LB (4 versus 6: OR 1.6, 99% CI 1.2-2.2, 5 versus 6: OR 1.6, 99% CI 1.2-2.3). CONCLUSIONS: Among euploid embryos, ICM morphology is the best predictor of sustained implantation; however, a composite score may provide additional guidance. While there is a known benefit in genomic screening prior to embryo selection, morphology provides individualized, prognostic information about implantation potential.

Reproductive outcome is optimized by genomic embryo screening, vitrification, and subsequent transfer into a prepared synchronous endometrium.

PURPOSE: The aim of this study is to compare implantation and live birth rates (LBR) between fresh euploid embryo transfers versus cryo-all cycles with a subsequent embryo transfer into a prepared endometrium. MATERIAL AND METHODS: This is a retrospective cohort study. Patients who underwent an IVF cycle with PGS with trophectoderm biopsy from January 2011 to July 2015 were included. Patients were divided into three groups: "Fresh Only," "Frozen Embryo Transfer ('FET) Only," and "Fresh ET then FET." For "Fresh Only" group (n = 345), PGS results were received within 24 h. For "FET Only" group (n = 514), results were expected after 24 h, and embryos were cryopreserved after biopsy; only FET was performed in this group (no fresh transfer). For "FET with a previous fresh ET" (n = 139) group, patients underwent a fresh ET with a subsequent FET, in which the same cohort of embryos was utilized. The main outcome measures were pregnancy rate (PR), clinical PR, implantation rate (IR), LBR, and early pregnancy loss rate. RESULTS: IRs were statistically higher in the "FET Only" group when compared to the "Fresh Only" group (59.5 vs. 50.6%, p < 0.01) and the "FET with a previous fresh ET" (59.5 vs. 50.6%, p < 0.05). LBR was statistically significant in the "FET Only" group when compared to the "Fresh Only" group (57.6 vs. 46.5 %, p < 0.005) but not when compared to "FET with a previous fresh ET" group (57.6 vs. 47.7%, p = 0.07). CONCLUSIONS: This analysis suggests euploid embryos to be more likely to implant and achieve a LBR in a synthetic FET cycle than in a fresh cycle.

Embryo selection versus natural selection: how do outcomes of comprehensive chromosome screening of blastocysts compare with the analysis of products of conception from early pregnancy loss (dilation and curettage) among an assisted reproductive technology population?

OBJECTIVE: To compare the incidence of numerical chromosomal abnormalities (NCAs) reported after preimplantation genetic screening (PGS) analysis compared with that reported after cytogenetic analysis of products of conception after spontaneous abortion. DESIGN: Retrospective study. SETTING: Private academic in vitro fertilization center. PATIENT(S): Cytogenetic reports of patients who underwent an IVF cycle with PGS of at least one biopsied embryo were compared with cytogenetic analysis reported from patients who had dilation and curettage (D&C) for the treatment of a spontaneous abortion after assisted reproductive technology (ART) treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Frequencies for each numerical chromosomal abnormality from both groups were compared. RESULT(S): A total of 1,069 NCAs were reported after PGS (trisomy 54.3%, monosomy 45.7%, no polyploidies), resulting in a trisomy/monosomy ratio of 0.82. A total of 447 NCAs was reported after D&C (trisomy 83%, polyploidy 10.7%, monosomy 6.3%). The aneuploidies most frequently identified were similar in both groups and included 15, 16, 18, 21, and 22. Monosomies (n = 28, 6.3%) were rarely observed in the group that underwent D&C after ART. CONCLUSION(S): This review provides an analysis of the most commonly identified NCAs after PGS and in first-trimester D&C samples in an infertile population utilizing ART. Although monosomies comprised >50% of all cytogenetic anomalies identified after PGS, there were very few identified in the post-D&C samples. This suggests that although monosomies occur frequently in the IVF population, they commonly do not implant. Despite this difference, this study demonstrated that the specific NCAs observed after PGS analysis and D&C were comparable.

Should ICSI be recommended routinely in patients with four or fewer oocytes retrieved?

PURPOSE: To determine if patients with a low response to controlled ovarian hyperstimulation during IVF benefit from intracytoplasmic sperm injection (ICSI) METHODS: Retrospective analysis of 350 IVF cycles in which four or fewer oocytes were retrieved. Severe male factor cases were excluded from analysis. Conventional insemination (CI) and ICSI were compared, with primary outcome measures of fertilization rate, implantation rate, clinical pregnancy rate per embryo transfer, and pregnancy loss rate. RESULT(S): Fertilization rates per oocyte retrieved for CI and ICSI were comparable (51.5% vs. 51.8%). Parallel implantation rates (22% vs. 25%), clinical pregnancy rates (32.8% vs. 33.3%), and loss rates (26.7% vs. 39.5%) were also noted. No difference in cancelled cycles was reported. CONCLUSION(S): Our results demonstrate that in the presence of normal semen parameters, low egg number is not an indication to perform ICSI.

Human blastocyst morphological quality is significantly improved in embryos classified as fast on day 3 (>or=10 cells), bringing into question current embryological dogma.

OBJECTIVE: To evaluate developmental potential of fast cleaving day 3 embryos. DESIGN: Retrospective analysis. SETTING: Academic reproductive center. PATIENT(S): Three thousand five hundred twenty-nine embryos. INTERVENTION(S): Day 3 embryos were classified according to cell number: slow cleaving: or=10 cells, and further evaluated on day 5. The preimplantation genetic diagnosis (PGD) results of 43 fast cleaving embryos were correlated to blastocyst formation. Clinical outcomes of transfers involving only fast cleaving embryos (n = 4) were evaluated. MAIN OUTCOME MEASURE(S): Blastocyst morphology correlated to day 3 blastomere number. Relationship between euploidy and blastocyst formation of fast cleaving embryos. Implantation, pregnancy (PR), and birth rates resulting from fast embryo transfers. RESULT(S): Blastocyst formation rate was significantly greater in the intermediate cleaving (72.7%) and fast cleaving (54.2%) groups when compared to the slow cleaving group (38%). Highest quality blastocysts were formed significantly more often in the fast cleaving group. Twenty fast cleaving embryos that underwent PGD, formed blastocysts, of which 45% (9/20) were diagnosed as euploid. Aneuploidy was diagnosed in 82.6% (19/23) of arrested embryos. A 50% implantation and 100% PR and birth rate were achieved with embryo transfers involving fast cleaving embryos. CONCLUSION(S): Fast cleaving embryos not only reach the blastocyst stage at a similar rate to intermediate cleaving embryos, but also exceed morphological quality criteria on day 5. Fast cleaving embryo transfers demonstrated a high clinical potential.

The morphology of extracted testicular sperm correlates with fertilization but not pregnancy rates.

OBJECTIVE: To investigate sperm morphology on the day of fresh testicular sperm extraction (TESE) with intracytoplasmic sperm injection (ICSI), and its effect on fertilization and pregnancy rates, as TESE in conjunction with ICSI results in high fertilization and pregnancy rates in most patients, but to our knowledge only one small study has assessed the morphology of retrieved sperm and found no correlation with the success of fertilization. PATIENTS AND METHODS: In a retrospective database analysis in a large academic centre, 68 men had 75 cycles of TESE combined with ICSI from January 2004 until April 2006. Sperm obtained by TESE was morphologically analysed at high (x 400-600) magnification and used for ICSI on the day of tissue retrieval. Sperm were classified as being either normal, having an amorphous head, having a mid-piece defect or having multiple defects. The calculated percentage of abnormal sperm injected was compared with the normal fertilization rate using Pearson's correlation coefficient, and pregnancy rates between groups were compared using chi-square analysis. RESULTS: Fifteen cycles had all morphologically normal sperm; 21 cycles had 50-99% normal forms and 39 cycles had <50% normal sperm. There was a highly significant correlation between the percentage of normal sperm used for ICSI and fertilization rates (P = 0.007). Overall, 43 clinical pregnancies resulted in this series, i.e. three among the group with all normal sperm injected, 12 in the group with 50-99% normal sperm and 28 in the group with <50% normal forms. There were also 11 pregnancies in cycles that used no normal forms. Pregnancy rates did not differ significantly among the groups (P = 0.08). CONCLUSIONS: TESE with ICSI frequently results in successful pregnancy; normal morphology was highly and significantly associated with successful fertilization, but importantly there were still 10 clinical pregnancies in cycles where only abnormal sperm were used. Sperm morphology after TESE should be assessed at the time of the procedure, and whenever possible, morphologically normal sperm chosen for injection. However, it is reassuring that acceptable fertilization and pregnancy rates are still achievable in cases with no morphologically normal sperm available.

Report of four donor-recipient oocyte cryopreservation cycles resulting in high pregnancy and implantation rates.

OBJECTIVE: To determine the clinical potential of donor-oocyte cryopreservation and thaw techniques for recipient patients. DESIGN: Institutional review board-approved prospective study of donor oocyte cryopreservation. SETTING: A large, private infertility center. PATIENT(S): Four anonymous oocyte donors underwent ovarian hyperstimulation for the purpose of oocyte retrieval and cryopreservation. The oocytes were subsequently thawed, fertilized, and transferred to 4 recipient patients. INTERVENTION(S): Oocytes were obtained from young donor patients and were cryopreserved with a slow freeze/rapid thaw protocol in which 1,2-propanediol (PrOH) and sucrose were used as cryoprotectants. Oocytes that survived were inseminated using intracytoplasmic sperm injection (ICSI). Resulting embryos were replaced into the recipient patients on the third day post-insemination. MAIN OUTCOME MEASURE(S): Post-thaw survival rate, fertilization rate, cleavage rate, implantation and clinical pregnancy rates. RESULT(S): A total of 79 metaphase II oocytes were frozen, stored frozen overnight in liquid nitrogen, and then thawed. The post-thaw survival rate was 86.1%. Normal fertilization following ICSI occurred in 89.7% of the surviving oocytes. Cleavage was observed in 91.8% of normally fertilized oocytes. A total of 23 embryos were transferred to 4 recipient patients. A clinical pregnancy rate of 75% and an implantation rate of 26.1% were achieved. CONCLUSION(S): Human oocyte cryopreservation is an effective technique that can be applied in clinical situations with high oocyte survival and clinical pregnancy rates expected.

Blastocyst embryo transfer is associated with a sex-ratio imbalance in favor of male offspring.

OBJECTIVE: To evaluate the sex ratio of offspring born after blastocyst transfers. DESIGN: Retrospective data analysis. SETTING: A large assisted reproductive technology center. PATIENT(S): We included 1,284 offspring from 937 deliveries during the period August 2003-August 2005. INTERVENTION(S): Tabulation and statistical analysis of all births resulting from fresh IVF cycles. The sex of resulting offspring was compared in both day 3 and blastocyst transfers for all births and for singleton deliveries. In addition, the sex of children conceived with the use of autologous oocytes and donor oocytes was evaluated. MAIN OUTCOME MEASURE: Sex ratio of offspring born following embryo transfers (ETs) after day 3 of culture and sequential blastocycst culture. RESULT(S): The overall sex ratio was significantly shifted toward males when blastocyst transfers were performed. Blastocyst transfers with only the use of autologous oocytes resulted again in a significantly higher proportion of male offspring. An even greater proportional difference was encountered in singleton offspring from donor oocytes. However, significance was not reached because of the limited number of offspring in the subgroup. CONCLUSION(S): This is the first individual-center report of a significant sex-ratio imbalance after the sequential media culture of blastocysts. The large imbalance in singleton births associated with the use of donor oocytes, although not significant, is cautionary in regard to the use of elective single ETs. Observation and publication of phenomena such as the effects of extended culture on the sex ratio of live-borns will allow us a better understanding of early differences in sexual dimorphism of the embryo, and will allow us to counsel our patients more appropriately.