Primary sex ratio in euploid embryos of consanguine couples after IVF/ICSI.

PURPOSE: To assess the primary sex ratio (males-to-females at time of conception) in blastocysts from consanguine couples undergoing IVF/ICSI treatments and its correlation with chromosomal constitution. METHOD: A total of 5135 blastocysts were analyzed by preimplantation-genetic testing for aneuploidy (PGT-A) with next-generation sequencing (NGS) from November 2016 to December 2020. From those, a total of 1138 blastocysts were from consanguine couples (CS) and 3997 from non-consanguine couples (NCS). Only blastocysts presenting normal sex chromosome constitution with or without autosomal aneuploidies were included. Primary sex ratio (PSR) of biopsied blastocysts was compared between CS and NCS couples. RESULTS: Expanded blastocysts derived from CS had 47.7% XY versus 52.3% XX constitutions, presenting a PSR of 0.91. In NCS, 48.9% of expanded blastocysts were XY and 51.2% XX, with a less pronounced PSR of 0.95. When stratifying embryos by ploidy, euploid embryos from CS had the lowest PSR (0.87) with 46.6% XY versus 53.4% XX blastocysts (OR 0.89, 95% CI 0.70-1.14; NS), but it did not achieve statistical significance. The lower PSR seemed rather related to euploid embryos from first-degree cousins (PSR = 0.80 versus 0.98 in second-degree cousins, NS). Euploid embryos from NCS presented a PSR of 0.96, with 49.1% XY versus 50.9% XX blastocysts (OR 0.98, 95% CI 0.79-1.22; NS). Significant differences in prevalence of euploidy of specific chromosomes were encountered between CS and NCS. CONCLUSIONS: The primary sex ratio was generally similar in expanded blastocysts from consanguine and non-consanguine couples, with a slight decrease in primary sex ratio of euploid blastocysts from consanguine couples.

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.

ART outcome after euploid frozen embryo transfer is not affected by previous Cesarean section delivery in the absence of intracavitary fluid.

PURPOSE: To evaluate the impact of a cesarean section (CS) on the chance of clinical pregnancy and live birth (LB) in frozen embryo transfer (FET) cycles in the setting of euploid embryos and the absence of intracavitary fluid (ICF) as causes of implantation failure were excluded. METHODS: Retrospective study, including patients with at least one previous CS or at least one previous vaginal delivery, who underwent a euploid FET cycle. RESULTS: A total of 412 euploid embryo transfer cycles had been included. Patients' mean age was 34.5 years and 42.48% of patients have had at least one previous CS. A clinical pregnancy was seen in 69.42% and 60.19% of the patients had a LB. Positive pregnancy test, clinical pregnancy, and LB rate were not significantly different between the groups without/with a history of a previous CS (p = 0.6/0.45/0.94, respectively). LB rate was significantly reduced by the presence of mucus on the ET catheter (OR: 0.413; p = 0.010), the BMI (OR: 0.946; p = 0.006), the combined embryo quality (embryo quality fair: OR: 0.444; p = 0.001; embryo quality low: OR: 0.062; p < 0.001), and by the HRT endometrial preparation approach (OR: 0.609; p = 0.023). CONCLUSION: The possible negative impact of a CS can be overcome when a euploid FET after exclusion of ICF is performed.

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.

Does blastocyst mitochondrial DNA content affect miscarriage rate in patients undergoing single euploid frozen embryo transfer?

PURPOSE: To determine whether the blastocyst mitochondrial DNA (mtDNA) content is related to the miscarriage rate in patients undergoing single euploid frozen embryo transfer (SEFET). METHODS: A total of 355 single euploid frozen embryo transfer cycles were studied retrospectively between April 2017 and December 2018. A trophectoderm biopsy was performed on day 5/6 blastocysts. Post next-generation sequencing (NGS), the mtDNA content was calculated as the ratio of mitochondrial DNA over nuclear DNA, and the association between blastocyst mtDNA content and miscarriage rate was evaluated. RESULT(S): Three hundred fifty-five euploid blastocysts were selected for SEFET in 314 patients with an average age of 33.7 ± 5.6 years; 255 were biopsied on day 5 (71.8%) and 100 on day 6 (28.2%). Frozen embryo transfer (FET) was performed either in a hormone replacement therapy (HRT) cycle (71.8%; n = 255) or in a natural cycle (NC) (28.2%; n = 100). A pregnancy rate of 66.2% (235/355) was obtained with clinical pregnancy and miscarriage rates of 52.4% (n = 186) and 5.6% (n = 20), respectively. There was no significant difference neither between the blastocyst mtDNA content of pregnant and nonpregnant patients (27.7 ± 9.2 vs. 29.4 ± 8.6, P = 0.095) nor between patients with a clinical pregnancy and miscarriage (30.5 ± 9.3 vs. 27.3 ± 9.2, P = 0.136). Multivariate logistic regression analysis showed the same nonsignificant relationship, except for the miscarriage rate and BMI (OR 1.149, 95% CI 1.03-1.28; P = 0.012). CONCLUSION(S): Mitochondrial DNA content is unable to predict the miscarriage of implanted human euploid blastocysts.

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.

Intracytoplasmic sperm injection is not superior to conventional IVF in couples with non-male factor infertility and preimplantation genetic testing for aneuploidies (PGT-A).

STUDY QUESTION: Does the insemination method impact the euploidy outcome in couples with non-male factor infertility? SUMMARY ANSWER: Conventional IVF can be applied in cycles with preimplantation genetic testing for aneuploidies (PGT-A), as both IVF and ICSI generate equal numbers of euploid blastocysts. WHAT IS KNOWN ALREADY: Ever since its introduction, the popularity of ICSI has increased tremendously, even in couples with non-male factor infertility. The use of conventional IVF is a contraindication for couples undergoing PGT to ensure monospermic fertilisation and to eliminate potential paternal contamination from extraneous sperm attached to the zona pellucida. Despite this, it has recently been shown that sperm DNA fails to amplify under the conditions used for trophectoderm biopsy samples. STUDY DESIGN, SIZE, DURATION: This single-centre prospective pilot study included 30 couples between November 2018 and April 2019. PARTICIPANTS/MATERIALS, SETTING, METHOD: Arab couples, with a female age between 18-40 years, body mass index ≤30 kg/m2, at least 10 cumulus oocyte complexes (COCs) following oocyte retrieval (OR) and normal semen concentration and motility (WHO) in the fresh ejaculate on the day of OR, were eligible for the study. Half of the sibling oocytes were assigned to conventional IVF, and the other half were assigned to ICSI. All embryos were cultured in a time-lapse imaging system in Global Total LP media. Blastocysts were subjected to trophectoderm biopsy on Day 5, 6 or 7 and next-generation sequencing (NGS) to determine blastocyst ploidy status. The primary objective was to determine the euploid rate in blastocysts from sibling oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 568 COCs were randomly allocated between IVF (n = 283; 9.4 ± 4.0) and ICSI (n = 285; 9.5 ± 4.1). While the incidence of normal fertilisation per cycle (6.1 ± 3.8 (64.0%) vs 6.3 ± 3.5 (65.4%); P = 0.609) was distributed equally between IVF and ICSI, the degeneration rate (0.1 ± 0.3 vs 0.7 ± 0.8; P = 0.0003) was significantly higher after ICSI and the incidence of abnormal fertilisation (≥3 pronuclei) was significantly higher after IVF (0.9 ± 1.2 vs 0.2 ± 0.4; P = 0.005). For all fertilised oocytes, there were no differences in the number of good-quality embryos on Day 3 (74% vs 78%; P = 0.467), nor in the blastulation rate on Day 5 (80.4% vs 70.8%; P = 0.076). The total number of blastocysts biopsied per cycle on Days 5, 6 and 7 was not significantly different between IVF or ICSI (4.0 ± 2.8 vs 3.9 ± 2.5; P = 0.774). With euploid rates of 49.8 and 44.1% (P = 0.755; OR: 1.05664 [0.75188-1.48494), respectively, there was no significant difference identified between IVF and ICSI (2.0 ± 1.8 vs 1.9 ± 1.7; P = 0.808) and all couples had at least one euploid blastocyst available for transfer. When considering only euploid blastocysts, the male/female ratio was 61/39 in IVF and 43/57 in ICSI (P = 0.063). LIMITATIONS, REASON FOR CAUTION: This is a pilot study with a limited patient population of 30 couples (and 568 COCs) with a normal ovarian response. The results of our study should not be extrapolated to other patient populations. WIDER IMPLICATIONS OF THE FINDINGS: It is safe to apply conventional IVF in couples with non-male factor infertility undergoing PGT-A. STUDY FUNDING/COMPETING INTEREST(S): No funding was obtained. There are no competing interests. TRIAL REGISTRATION NUMBER: NCT03708991.

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.