Reproductive outcomes with delayed blastocyst development: the clinical value of day 7 euploid blastocysts in frozen embryo transfer cycles.

Embryos of optimal development reach blastocyst stage 116 ± 2 h after insemination. Usable D7 blastocysts represent nearly 5% of embryos in IVF with acceptable pregnancy and live birth rates, however data are still limited. Therefore, this study aimed to analyze the ongoing pregnancy rate (OPR) of D7 blastocysts in single euploid frozen embryo transfer (FET) cycles. An observational study was performed including 1527 FET cycles with blastocysts biopsied on D5 (N = 855), D6 (N = 636) and D7 (N = 36). Blastocysts were classified as good (AA/AB/BA), fair (BB) or poor (AC/BC/CC/CA/CB) (Gardner scoring). FETs were performed in natural cycles (NC) or hormone replacement therapy (HRT) cycles. Patient's age differed significantly between D5, D6 and D7 blastocysts FET cycles (33.2 ± 5.6, 34.4 ± 5.3 and 35.9 ± 5.2, P < 0.001). OPRs were higher when D5 euploid blastocysts were transferred compared with D6 and D7 (56.0% vs. 45.3% and 11.1%, P < 0.001). Poor quality blastocysts were predominant in D7 blastocyst FET cycles (good quality: 35.4%, 27.2%, 5.6%; fair quality: 52.1%, 38.5%, 11.1%; poor quality: 12.5%, 34.3%, 83.3%, P < 0.001 for D5, D6 and D7 blastocysts; respectively). OPR was significantly reduced by D7 blastocyst FETs (OR = 0.23 [0.08;0.62], P = 0.004), patient's BMI (OR = 0.96 [0.94;0.98], P < 0.001), HRT cycles (OR = 0.70 [0.56;0.88], P = 0.002) and poor quality blastocysts (OR = 0.33 [0.24;0.45], P < 0.001). OPR is significantly reduced with D7 compared with D5/D6 euploid blastocysts in FET cycles. The older the patient, the more likely they are to have an FET cycle with blastocysts biopsied on D7, therefore culturing embryos until D7 can be a strategy to increase OPR outcomes in patients ≥38 years.

The Ratio of Serum Progesterone (P4) to the Number of Follicles (P4/follicle) is a More Objective Parameter for Euploidy Rate as Compared to Systemic Progesterone Levels.

Does the late follicular phase progesterone (P4) and the P4-to-follicle-ratio affect the ploidy state of the biopsied embryos? A retrospective observational study conducted at ART Fertility Clinics Abu Dhabi and Muscat, including all stimulation cycles performed between January 2015 and December 2019. In total, 975 cycles were considered for this study. Inclusion criteria were ovarian stimulation due to primary/secondary infertility, patient's age between 18 and 45 years, ICSI as fertilization method, and patients undergoing preimplantation genetic testing for aneuploidies (PGT-A). Patients with testicular sperm extraction (TESE) and warmed oocytes were excluded. Our results have shown that progesterone had no effect on the euploid rate (p = 0.371). However, when adding the ratio of P4 to the number of follicles that were bigger than 10 mm in the last scan, a negative effect on the euploid rate (p < 0.05) was observed. This study was able to show that the use of only P4 is unable to predict ploidy outcomes. However, by including the number of follicles > 10 mm, a clear association was observed between P4/Foll ratio and euploid rate per cycle. The use of both parameters could aid clinicians in their decision to trigger a patient or continue stimulation. Further prospective studies are warranted to confirm those results.

Embryo Culture Medium Has No Impact on Mosaicism Rates: a Sibling Oocyte Study.

Human embryos cultured in vitro can contain two or more cytogenetically distinct cell lineages known as "chromosomal mosaicism". Since mosaicism is produced by mitotic errors after fertilization occurs, culture conditions might contribute to mosaicism origins. Many studies demonstrated that euploidy rates are not affected by culture media; however, whether oocytes cultured under continuous culture media (CCM) or sequential culture media (SCM) has a higher risk of mosaicism occurring remains unsolved. Therefore, this study aims to determine whether mosaicism rates differ when sibling oocytes are cultured in CCM or SCM. A single center observational study was performed including 6072 sibling oocytes. Mature oocytes (MII) were inseminated and cultured in CCM (n = 3,194) or SCM (n = 2,359) until blastocyst stage for trophectoderm (TE) biopsy on day (D) 5, D6, or D7 for preimplantation genetic testing analysis with a semi-automated next-generation sequencing. Mosaicism was classified as low (30-50%) or high (50-80%) based on the percentage of abnormal cells constitution detected in TE samples. As a result, 426 women with a mean age of 34.7 ± 6.4 years were included in the study. Fertilization rates were comparable between CCM and SCM (74.0% vs 72.0%, p = 0.091). Although total blastulation rate and usable blastocyst rate (biopsied blastocysts) were significantly higher in CCM than SCM (75.3 % vs. 70.3%, p < 0.001 and 58.0% vs. 54.5%, p = 0.026), euploidy rates did not differ significantly (45.2% vs. 45.7%, p = 0.810, respectively). Mosaicism rate was not significantly different for blastocysts cultured in CCM or SCM (4.7% vs. 5.1%, p = 0.650), neither the proportion of low or high mosaic rates (3.7% vs. 4.4%, p = 0.353 and 1.0% vs. 0.7%, p = 0.355, respectively). Hence, it was concluded that CCM or SCM does not have an impact on mosaicism rate of embryos cultured until the blastocyst stage.

Assessment of mitochondrial DNA viability ratio in day-4 biopsied embryos as an add-in to select euploid embryos for single embryo transfer.

The aim of this study was to assess mitochondrial DNA analysis as a predictor of the pregnancy potential of biopsied preimplantation embryos. The study included 78 blastomeres biopsied from day 4 cleavage stage euploid embryos. The embryo karyotype was confirmed by 24-chromosome preimplantation genetic testing for aneuploidies using the Illumina Next-Generation Sequencing (NGS) system. Mitochondria viability ratios (mtV) were determined from BAM files subjected to the web-based genome-analysis tool Galaxy. From this cohort of patients, 30.4% of patients (n = 34) failed to establish pregnancy. The mean mtV ratio [mean = 1.51 ± 1.25-1.77 (95% CI)] for this group was significantly (P < 0.01) lower compared with the embryo population that resulted in established pregnancies [mean = 2.5 ± 1.82-2.68 (95% CI)]. mtV multiple of mean (MoM) values were similarly significantly (P < 0.01) lower in blastocysts failing to establish pregnancy. At a 0.5 MoM cut-off, the sensitivity of mtV quantitation was 35.3% and specificity was 78.2%. The positive predictive value for an mtV value > 0.5 MoM was 41.4%. This study demonstrates the clinical utility of preimplantation quantification of viable mitochondrial DNA in biopsied blastomeres as a prognosticator of pregnancy potential.

Marginal differences in preimplantation morphokinetics between conventional IVF and ICSI in patients with preimplantation genetic testing for aneuploidy (PGT-A): A sibling oocyte study.

OBJECTIVE: This study aimed to analyze the morphokinetic behaviour between conventional IVF and ICSI, in cycles with preimplantation genetic testing for aneuploidies (PGT-A). MATERIALS: A randomized controlled trial (NCT03708991) was conducted in a private fertility center. Thirty couples with non-male factor infertility were recruited between November 2018 and April 2019. A total of 568 sibling cumulus oocyte complexes were randomly inseminated with conventional IVF and ICSI and cultured in an Embryoscope time-lapse system. The morphokinetic behaviour of IVF/ICSI sibling oocytes was analysed as primary endpoint. As secondary endpoints, morphokinetic parameters that predict blastocysts that will be biopsied, the day of biopsy, gender and euploid outcome was assessed. RESULTS: When comparing IVF to ICSI, only the time to reach the 2-cell stage (t2) was significantly delayed for IVF embryos: OR: 1.282 [1.020-1.612], p = 0.033. After standardizing for tPNf (ct parameters), only Blast(tStartBlastulation-t2) remained significant: OR: 0.803 [0.648-0.994], p = 0.044. For the analysis of zygotes that will be biopsied on day 5/6 versus zygotes without biopsy, only early morphokinetic parameters were considered. All parameters were different in the multivariate model: ct2: OR: 0.840 [0.709-0.996], p = 0.045; ct6: OR: 0.943 [0.890-0.998], p = 0.043; cc2(t3-t2): OR: 1.148 [1.044-1.263], p = 0.004; cc3(t5-t3): OR: 1.177 [1.107-1.251], p<0.0001. When comparing the development between blastocysts biopsied on day 5 versus day 6, only three morphokinetic parameters were significant: cc2(t3-t2): OR: 1.394 [1.010-1.926], p = 0.044; ctBlastocyst: OR: 0.613 [0.489-0.768], p<0.0001 and ctExpandedBlastocyst: OR: 0.913 [0.868-0.960], p = 0.0004. Multivariate analysis of gender and ploidy did not reveal differences in morphokinetic behaviour. CONCLUSION: Minor morphokinetic differences are observed between IVF and ICSI. Early in the development, distinct cleavage patterns are observed between embryos that will be biopsied or not.

Day 5 vs day 6 single euploid blastocyst frozen embryo transfers: which variables do have an impact on the clinical pregnancy rates?

OBJECTIVE: To determine which variables affect most the clinical pregnancy rate with positive fetal heartbeat (CPR FHB+) when frozen embryo transfer (FET) cycles are performed with day 5 (D5) or day 6 (D6) euploid blastocysts. Design and method A single center retrospective study was performed from March 2017 till February 2021 including all single FET cycles with euploid D5 or D6 blastocysts and transferred in natural cycles (NC) or hormone replacement therapy (HRT) cycles. Trophectoderm (TE) and inner cell mass (ICM) qualities were recorded before biopsy. RESULTS: A total of 1102 FET cycles were included, 678 with D5 and 424 with D6 blastocysts. Pregnancy rate (PR), clinical PR (CPR), and CPR FHB+ were significantly higher with D5 blastocysts (PR: 70.7% vs 62.0%, OR = 0.68 [0.53-0.89], p = 0.004; CPR: 63.7% vs 54.2%, OR = 0.68 [0.52-0.96], p = 0.002 and CPR FHB+: 57.8% vs 49.8%, OR = 0.72 [0.53-0.96], p = 0.011). However, miscarriage rate (12.5% vs 11.4%, OR = 0.78 [0.48-1.26], p = 0.311) did not differ. From a multivariate logistic regression model, endometrial thickness (OR = 1.11 [1.01-1.22], p = 0.028), patient's age (OR = 1.03 [1.00-1.05], p = 0.021), BMI (OR = 0.97 [0.94-0.99], p = 0.023), and ICM grade C (OR = 0.23 [0.13-0.43], p < 0.001) were significant in predicting CPR FHB+. CONCLUSION: Although clinical outcomes are higher with D5 blastocysts, CPR FHB+ is more affected by endometrial thickness, patient age, BMI, and ICM grade C rather than biopsy day or endometrial preparation protocol.

The position of the euploid blastocyst in the uterine cavity influences implantation.

RESEARCH QUESTION: Does the position of the euploid blastocyst in the uterine cavity upon transfer, measured as distance in millimetres (mm) from the fundus (DFF) to the air bubble, influence implantation potential? DESIGN: A total of 507 single/double euploid frozen embryo transfer (FET) cycles at blastocyst stage were included retrospectively between March 2017 and November 2018 at a single centre. The patients were on average 33.3 years old. The FET were performed in natural cycles (n = 151) or hormone replacement therapy cycles (n = 356). RESULTS: Of the 507 transfers, 370 (73.0%) resulted in a pregnancy, defined as human chorionic gonadotrophin concentration over 15 mIU/ml, and 341 (67.3%) in a clinical pregnancy, with an implantation rate of 62.0% and ongoing pregnancy rate of 59.6% (302/507). When comparing the number of embryos transferred, the pregnancy rate, clinical pregnancy rate and ongoing pregnancy rate were significantly higher after double-embryo transfer (DET) (P = 0.002: P < 0.001 and P = 0.002). The quality of the blastocyst in the single-embryo transfer group had a positive effect on the pregnancy rate (A versus B, P = 0.016; A versus C, P = 0.003) and clinical pregnancy rate (A versus C, P = 0.013). After performing a multivariate logistic regression analysis to consider the effect of all explanatory variables, a negative effect between DFF and pregnancy (P = 0.001), clinical pregnancy (P = 0.001) and ongoing pregnancy (P = 0.030) was found. When all variables remained constant, an increase of 1 mm of DFF changed the odds of pregnancy by 0.882, of clinical pregnancy by 0.891 and of ongoing pregnancy by 0.925. No significant effect of DFF was found on the miscarriage outcome (P = 0.089). CONCLUSIONS: The depth of blastocyst replacement inside the uterine cavity may influence the pregnancy, clinical pregnancy and ongoing pregnancy rates and should be considered as an important factor to improve the success of IVF cycles.

Different CO2 settings (6.0% vs 7.0%) do have an impact on extracellular pH of culture medium (pHe) and euploidy rates rather than on blastocyst development: a sibling oocyte study.

OBJECTIVE: To determine whether euploidy rates and blastocyst development differ in a continuous culture medium under different CO2 concentrations. DESIGN AND METHOD: A single-center retrospective study was performed from July 2018 to October 2019 including 44 fresh cycles with at least four fresh mature oocytes (MII) without severe male factor infertility. Sibling MII were injected and cultured in Global®Total®LP under 6.0% (pHe = 7.374 ± 0.014) or 7.0% (pHe = 7.300 ± 0.013) CO2, 5.0% O2, and 89.0% or 88.0% N2. Analyzed variables were normally fertilized oocytes (2PN), cleavage rate, blastulation rate on day 5/2PN, usable blastocyst (blastocysts biopsied/2PN), and euploidy rates. Blastocyst's trophectoderm biopsy was performed on day 5, 6, or 7 for genetic testing and mitochondrial DNA (mtDNA) quantification by next-generation sequencing. RESULTS: Women's mean age was 33.0 ± 6.6 years old. From a total of 604 MII, no differences were found in normal fertilization and cleavage rates on day 3 between 6.0 and 7.0% CO2 (72.3% vs 67.1%, p = 0.169 and 96.6% vs 96.3%, p = 0.897, respectively). Blastulation rate on day 5/2PN was comparable between 6.0 and 7.0% CO2 (68.1% vs 64.2%, p = 0.409). Although usable blastocyst rate was not different (54.3% vs 55.3%, p = 0.922), total euploidy rates differed significantly (58.7% vs 42.8%, p = 0.016) between 6.0% and 7.0% CO2, respectively. The mean blastocyst mtDNA content was significantly lower in 6.0% CO2 (30.4 ± 9.1 vs 32.9 ± 10.3, p = 0.037). CONCLUSION: Blastocyst development is not affected when embryos are cultured in vitro at 6.0% or 7.0% CO2, while euploidy rates are significantly decreased at a higher CO2 concentration, therefore at a lower pHe.

Euploidy rates are not affected when embryos are cultured in a continuous (CCM) or sequential culture medium (SCM): a sibling oocyte study.

PURPOSE: To determine if euploidy rates and embryo development differ when blastocysts are cultured in CCM or SCM. METHOD: A single-center retrospective observational study was performed from September 2018 to March 2019. Patients [23-46 years] with at least four fresh mature oocytes (MII) without severe male factor infertility were included. Sibling MII were injected and cultured in Global®Total®LP (CCM) or Sage Quinn's Advantage® Cleavage and Blastocyst media (SCM) under 6% CO2, 5% O2, and 89% N2. Fertilization, cleavage, day (D) 5 blastulation, usable blastocyst (blastocysts biopsied/normally fertilized oocytes), and euploidy rates were recorded. Blastocysts were graded prior to trophectoderm (TE) biopsy on D5, 6, or 7 for genetic testing and mitochondrial DNA (mtDNA) quantification. RESULTS: According to clinical practice, 1452 MII were randomly distributed: 751 in CCM and 701 in SCM. No differences were observed in fertilization and cleavages rates for CCM and SCM (77.4% vs 75.5%, p = 0.429 and 97.6% vs 99.1%, p = 0.094, respectively). Blastulation rate on D5 was higher in CCM (70.6% vs 62.2, p = 0.009); however, usable blastocyst rates were comparable (CCM: 58.3% vs SCM: 56.7%, p = 0.625). From a Poisson regression model adjusted for confounding factors, euploidy rates were not different between media (aOR = 1.18, [0.94-1.48], p = 0.157). Euploid blastocyst's mtDNA values were similar (CCM: 32.2, [30.5, 34.1] and SCM: 33.5, [31.8, 35.2], p = 0.345) and top-quality blastocysts (AA/BA) were increased in SCM (OR=1.04, [1.00-1.09], p = 0.037). CONCLUSION: Under controlled in vitro conditions, euploidy rates and embryo development are comparable when embryos are cultured in CCM or SCM.

Small extracellular vesicle-encapsulated miR-181b-5p, miR-222-3p and let-7a-5p: Next generation plasma biopsy-based diagnostic biomarkers for inflammatory breast cancer.

Inflammatory breast cancer (IBC) is a rare, but aggressive entity of breast carcinoma with rapid dermal lymphatic invasion in young females. It is either poorly or misdiagnosed as mastitis because of the absence of a distinct lump. Small extracellular vesicles (sEVs) circulating in liquid biopsies are a novel class of minimally invasive diagnostic alternative to invasive tissue biopsies. They modulate cancer progression via shuttling their encapsulated cargo including microRNAs (miRNAs) into recipient cells to either trigger signaling or induce malignant transformation of targeted cells. Plasma sEVs < 200 nm were isolated using a modified cost-effective polyethylene glycol (PEG)-based precipitation method and compared to standard methods, namely ultracentrifugation and a commercial kit, where the successful isolation was verified by different approaches. We evaluated the expression levels of selected sEV-derived miR-181b-5p, miR-222-3p and let-7a-5p using quantitative real PCR (qPCR). Relative to non-IBC, our qPCR data showed that sEV-derived miR-181b-5p and miR-222-3p were significantly upregulated, whereas let-7a-5p was downregulated in IBC patients. Interestingly, receiver operating characteristic (ROC) curves analysis revealed that diagnostic accuracy of let-7a-5p alone was the highest for IBC with an area under curve (AUC) value of 0.9188, and when combined with miR-222-3p the AUC was improved to 0.973. Further, 38 hub genes were identified using bioinformatics analysis. Together, circulating sEV-derived miR-181b-5p, miR-222-3p and let-7a-5p serve as promising non-invasive diagnostic biomarkers for IBC.