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.

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.

Features of chromosomal abnormalities in relation to consanguinity: analysis of 10,556 blastocysts from IVF/ICSI cycles with PGT-A from consanguineous and non-consanguineous couples.

Consanguineous marriage is defined as marriage between first or second-degree cousins, with high prevalence in many cultures and societies. Descendants from consanguineous unions have an increased risk for genetic diseases. Additionally, in consanguineous couples, chromosomal disjunction during embryogenesis could also be affected, increasing the risk of chromosomal errors. Nowadays, genomic testing allows to identify new genetic syndromes and variants related to copy-number variations (CNV), including whole chromosome, segmental and micro-segmental errors. This is the first study evaluating chromosomal ploidy status on blastocysts formed from consanguineous couples during IVF/ICSI treatments with Preimplantation Genetic Testing for Aneuploidies (PGT-A), compared to non-consanguineous couples. Although consanguine couples were significantly younger, no differences were observed between groups for fertilisation rate, blastulation rate and euploidy rate, once adjusted by age. Nevertheless, the number of blastocysts biopsied on day 5 was lower for consanguine couples. Segmental errors, and aneuploidies of chromosomes 13 and 14 were the most prominent abnormalities in relation to consanguinity, together with errors in chromosome 16 and sex chromosomes when the female partner was younger than 35. Once euploid blastocysts were considered for subsequent frozen embryo transfer, pregnancy outcomes were similar in both groups. The current findings point toward the fact that in consanguine unions, not only the risk of having a child with genetic disorders is increased, but also the risk of specific chromosomal abnormalities seems to be increased. Premarital counselling and tailored reproductive treatments should be offered to these couples.

Clinical and laboratory parameters associated with cycle outcomes in patients undergoing euploid frozen blastocyst transfer.

RESEARCH QUESTION: Which factors impact on clinical pregnancy rate (CPR) and live birth rates (LBR) in euploid frozen embryo transfer (eFET) cycles? DESIGN: Retrospective observational study including 1660 eFET cycles with 2439 euploid blastocysts, from November 2016 to December 2020. The impact of clinical and laboratory parameters on CPR, biochemical miscarriage rate (BMR), clinical miscarriage rate (CMR) and LBR was evaluated. RESULTS: CPR per transfer was 63.4%, LBR per transfer 51.6%. CPR and LBR were significantly higher when double embryo transfer (DET) was performed (71.6% versus 57.7%, P < 0.001; 55.2% versus 49.1%, P = 0.016, respectively). However, pregnancy loss was significantly higher in the DET group (28.8% versus 22.8%, P = 0.02). When patients were classified by body mass index (BMI), no differences were observed for CPR, but CMR was lower (P < 0.001) and LBR higher (p = 0.031) for the normal BMI group. The natural cycle protocol revealed lower CMR (P < 0.001) and lower pregnancy loss (P < 0.001); subsequently, higher LBR (57.6%, 48.8%, 45.0%, P = 0.001) compared with hormonal replacement protocol and stimulated cycle. Day of trophectoderm biopsy affected CPR (P < 0.001) and LBR (P < 0.001), yet no differences were observed for BMR, CMR or pregnancy loss. The multivariate analysis showed that day 6/7 embryos had lower probabilities for pregnancy; overweight and obesity had a negative impact on LBR, and natural cycle improved LBR (adjusted odds ratio 1.445, 95% confidence interval 0.519-0.806). CONCLUSIONS: Day of biopsy affected CPR, while BMI and endometrial preparation protocol were associated with LBR in eFET. DET should be discouraged as it will increase the risk of pregnancy loss. Women with higher BMI should be aware of the higher risk of pregnancy loss and lower LBR even though a euploid blastocyst is transferred.

Effect of the endometrial thickness on the live birth rate: insights from 959 single euploid frozen embryo transfers without a cutoff for thickness.

OBJECTIVE: To investigate whether endometrial thickness (ET) independently affects the live birth rate (LBR) after embryo transfer. DESIGN: Retrospective study. SETTING: Private assisted reproductive technology center. PATIENT(S): A total of 959 single euploid frozen embryo transfers. INTERVENTION(S): Vitrified euploid blastocyst transfer. MAIN OUTCOME MEASURE(S): Live birth rate per embryo transfer. RESULT(S): The conditional density plots did not demonstrate either a linear relationship between the ET and LBR or a threshold below which the LBR decreased perceivably. Receiver operating characteristic curve analyses did not suggest a predictive value of the ET for the LBR. The area under the curve values were 0.55, 0.54, and 0.54 in the overall, programmed, and natural cycle transfers, respectively. Logistic regression analyses with age, embryo quality, day of trophectoderm biopsy, body mass index, and ET did not suggest an independent effect of the ET on the LBR. CONCLUSION(S): We did not identify a threshold of the ET that either precluded live birth or under which the LBR decreases perceivably. Common practice of cancelling embryo transfers when the ET is <7 mm may not be justified. Prospective studies, in which the management of the transfer cycle would not be altered by ET, would provide higher-quality evidence on the subject.

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.

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.

Female parental consanguinity is associated with a reduced ovarian reserve.

RESEARCH QUESTION: Is parental consanguinity associated with a reduced ovarian reserve in women from the Arabian Peninsula, comparing anti-Müllerian hormone (AMH) and antral follicle count (AFC)? DESIGN: Retrospective large-scale observational study including 2482 women from the Arabian Peninsula, aged 19-49 years, who had their serum AMH and AFC measured as part of their fertility assessment, from May 2015 to November 2019. Consanguinity was defined as women whose parents were first-degree or second-degree cousins. Serum AMH was measured for all participants. RESULTS: A total of 2198 women were included: 605 in the consanguine group (27.53%), 1593 (72.47%) in the non-consanguine group. There were no significant differences between groups in terms of body mass index, years of infertility or smoking status. Women from the consanguine group were significantly younger (mean age 33.74 ± 6.64 years) compared with the non-consanguine group (mean age 34.78 ± 6.64 years, P < 0.0001). Median AMH and AFC for the consanguine group were 1.90 ng/ml (min-max: 0.01-23.8) and 11 (0-80), respectively, and for the non-consanguine group 1.84 ng/ml (min-max: 0.01-23.0) and 11 (0-60), respectively. AMH and AFC exhibit an age-dependent decline. As both parameters are age-dependent, the multivariate analysis showed that women from the consanguine group presented significantly lower AMH (coefficient of variation [CV] -0.07 ± 0.03, P = 0.036) and AFC (CV -0.16 ± 0.06, P = 0.003) compared with non-consanguine women, and the highest differences were found for women below 35 years of age (AMH median [min-max]: 2.82 ng/ml (0.01-23.80) versus 2.92 ng/ml (0.01-23.00); P = 0.035; AFC median [min-max]: 15 (0-80) versus 14 (0-80); P = 0.001). CONCLUSION: The adjusted analysis by age indicates that female parental consanguinity is associated with reduced ovarian reserve in the studied population. Clinical evaluation should include extensive family history and subsequent counselling of the affected couples.

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.

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.

Anti-Müllerian hormone is an independent marker for oocyte survival after vitrification.

RESEARCH QUESTION: This study explored the relationship between anti-Müllerian hormone (AMH) and oocyte survival after vitrification. The association between AMH and blastocyst formation after oocyte vitrification was also assessed. DESIGN: A retrospective observational analysis was performed in a private IVF centre. A total of 4507 metaphase-II warmed oocytes were included from 450 couples, predominantly of Arab ethnicity. Between August 2015 and August 2018, couples underwent 484 intracytoplasmic sperm injection (ICSI) treatments using vitrified-warmed oocytes. RESULTS: Patients' median age ± SD was 36.2 ± 6.1 years, AMH concentration 2.6 ± 3.4 ng/ml and body mass index (BMI) 26.5 ± 4.6 kg/m2. The oocyte survival rate after vitrification was 87.37 ± 20.42%. AMH concentration showed a significant correlation (Kendall's tau 0.087, P = 0.0079) with oocyte survival rate independent of oocyte yield. Correlation was significant (odds ratio 1.041, 95% confidence interval 1.007-1.077, P = 0.018) when a multivariant model was applied that included AMH, age and BMI. The receiver operating characteristic curve showed an AMH cut-off value of 1.09 ng/ml that could obtain at least a 70% survival rate, with an area under the curve of 0.669. Regarding embryo development in ICSI cycles including fresh and warmed oocytes for the same patient, blastocyst formation rate was higher in fresh compared with warmed oocytes (P < 0.001). In this subgroup no significant correlation was seen between fertilization or blastocyst rate and AMH concentration. CONCLUSIONS: AMH concentration showed a significant correlation with oocyte survival. Blastocyst formation was significantly lower after oocyte vitrification, but no correlation was found with AMH. Clinicians should carefully evaluate oocyte vitrification for patients with AMH below 1.09 ng/ml and consider embryo accumulation for these patients in preference to oocyte accumulation.

Blastocyst mitochondrial DNA (mtDNA) is not affected by oocyte vitrification: a sibling oocyte study.

PURPOSE: To evaluate whether mtDNA content at the blastocyst stage differs between embryos derived from fresh or vitrified sibling oocytes. MATERIAL AND METHODS: A retrospective analysis was performed between March 2017 and September 2018, including 504 blastocysts from 94 couples undergoing preimplantation genetic testing for aneuploidies (PGT-A), using fresh oocytes together with previously vitrified oocytes. Trophectoderm biopsies were performed and subjected to next generation sequencing. RESULTS: On average, 1.8 ± 1.0 oocyte vitrification cycles were performed per patient. Between fresh and vitrified cycles, no difference was observed between the number of fertilized oocytes (5.3 ± 4.2 versus 5.5 ± 3.0). Blastulation rate on day 5 per fertilized oocyte was significantly higher in the fresh group (62% ± 29% versus 44% ± 31%; p < 0.001). For the 504 biopsied blastocysts, 294 fresh versus 210 vitrified, no significant differences were found in the euploid rate, 40.5% versus 38.6% (p = 0.667), and mtDNA content, 30.1 (± 10.6) versus 30.0 (± 12.5) (p = 0.871), respectively. Regardless of the origin of the oocytes, aneuploid blastocysts contained significantly higher mtDNA values compared with the euploid ones (31.4 versus 28.0; p = 0.001). Furthermore, top-quality blastocysts had a significantly lower mtDNA content compared with moderate and poor-quality blastocysts (p < 0.001) and blastocysts biopsied on day 5 showed significantly lower mtDNA content compared with day 6 or day 7 blastocysts (p < 0.001). However, when analyzing the blastocyst mtDNA content according to the ploidy state, no differences were found for blastocyst quality or day of biopsy between blastocysts originating from fresh or vitrified oocytes. CONCLUSION: Oocyte vitrification does not affect the mtDNA content of trophectoderm biopsies.

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.

Vitamin D in Follicular Fluid Correlates With the Euploid Status of Blastocysts in a Vitamin D Deficient Population.

Context: The widespread distribution of the Vitamin D (VitD) receptor in reproductive tissues suggests an important role for VitD in human reproduction. The assessment of patient´s VitD is based on the 25-hydroxyvitamin D (25(OH)D) metabolite measurement. However, most of the circulating 25(OH)D is bound to either VitD-binding protein (VDBP) (88%) or albumin (12%) and less than 1% circulates free. Objective: To determine a possible correlation between VitD levels in serum (S) and follicular fluid (FF) and blastocyst ploidy status in patients undergoing infertility treatment. Methods: A prospective observational study was performed including couples planned for preimplantation genetic testing for aneuploidies (PGT-A) from ART Fertility Clinics. Patients were classified according to their 25(OH)D-Serum levels: VitD deficient group <20 ng/ml and insufficient/replete ≥20 ng/ml defined as VitD non-deficient group. Results: Serum samples and 226 FF from individual follicles were collected for 25(OH)D, bioavailable 25(OH)D, free 25(OH)D, and % free 25(OH)D measurement. 25(OH)D-Serum in VitD deficient and non-deficient were 13.2±4.0 ng/ml vs 32.3±9.2 ng/ml; p<0.001. FF from 40 and 74 biopsied blastocysts was analysed of which 52.5 and 60.8% were euploid (p = 0.428), respectively. In VitD deficient patients, mean 25(OH)D-FF, bioavailable 25(OH)D-FF, and free 25(OH)D-FF were higher in euploid vs aneuploid blastocysts (18.3±6.3 ng/ml vs 13.9±4.8 ng/ml; p = 0.040; 1.5±0.5 ng/ml vs 1.1±0.4 ng/ml; p = 0.015; 0.005±0.002 ng/ml vs 0.003±0.001 ng/ml; p = 0.023, respectively), whilst no differences were found in VitD non-deficient patients (37.9±12.3 ng/ml vs 40.6±13.7 ng/ml; p = 0.380; 3.1±1.1 ng/ml vs 3.3±1.2 ng/ml; p = 0.323; 0.01±0.003 ng/ml vs 0.01±0.004 ng/ml; p = 0.319, respectively). Conclusion: VitD non-deficient patients have a significantly higher probability of obtaining a euploid blastocyst compared to VitD deficient patients (OR:33.36, p = 0.002).

mtDNA dynamics between cleavage-stage embryos and blastocysts.

PURPOSE: The aim was to evaluate mtDNA content and its dynamics in euploid and aneuploid embryos from cleavage to blastocyst stage following consecutive biopsies. The effect of female age on mtDNA content was evaluated by comparing reproductively younger (≤ 37 years) with older (> 37 years) women. METHODS: A retrospective single-centre descriptive study was performed between August 2016 and January 2017. Forty patients, with 112 embryos, undergoing preimplantation genetic testing for aneuploidies (PGT-A) by next-generation sequencing (NGS) were included. Embryos that reached the blastocyst stage and were not selected for fresh embryo transfer were included following consecutive biopsies of a single blastomere on day 3 and trophectoderm biopsy of day 5 blastocysts. RESULTS: Cleavage-stage mtDNA was significantly lower in fast cleaving embryos (p = 0.016). Based on the concordance between day 3 and day 5 biopsies, a difference was identified in blastocyst mtDNA content between groups (p = 0.019); true euploid blastocysts presented a lower mtDNA content. No association was identified between cleavage-stage mtDNA content and ploidy status (OR 1.008 [0.981-1.036], p = 0.565) nor between blastocyst mtDNA content and ploidy outcome (OR 0.954 [0.898-1.014], p = 0.129). No difference was found when comparing mtDNA content and ploidy outcome between the two reproductive age groups (p = 0.505 (cleavage stage) and p = 0.774 (blastocyst)). CONCLUSION: Mitochondrial DNA content of cleavage-stage embryos and blastocysts is unable to predict ploidy status. Subgroup analysis based on ploidy concordance between day 3 and day 5 revealed a significantly lower mtDNA content for true euploid blastocysts. Reproductive ageing does not affect mtDNA content.

Cleavage stage mitochondrial DNA is correlated with preimplantation human embryo development and ploidy status.

PURPOSE: To evaluate whether the mitoscore of cleavage stage embryos might correlate with developmental kinetics and the ploidy status. MATERIALS: This retrospective single-center study involved all cycles between April 2016 and April 2018 in which preimplantation genetic testing for aneuploidy (PGT-A) on day 3 was performed. The mitochondrial DNA (mtDNA) content and embryo ploidy were determined on 375 single blastomere biopsies by next generation sequencing (NGS). After intracytoplasmic sperm injection, a time-lapse imaging system (embryoscope) was used to follow the development. The median mtDNA content of cleavage stage embryos (49.4) was used to stratify the embryos into two groups to compare embryo development and ploidy status: low mitoscore group (≤ 49.4) and high mitoscore group (> 49.4). RESULTS: The total number of euploid embryos was equal between both mitoscore groups (32.1% versus 33.5%; p = 0.854). However, embryos in the low mitoscore group had a significantly higher cell number on day 3 (8.13 ± 1.59 versus 7.62 ± 1.5; p = 0.0013) and showed a significantly faster development up until the 8-cell stage. Mitoscore was not different between euploid and aneuploid embryos, with the same blastomere number at the time of biopsy. Furthermore, absence of cavitation within 118 h after insemination was correlated with higher mitoscore values (60.22 ± 42.23 versus 50.97 ± 13.37; p = 0.006) and a lower chance of being euploid (17.1% versus 47.4%; p = 0.001). CONCLUSION: mtDNA content of cleavage stage embryos correlates with time-lapse parameters. Early blastulation is correlated with a lower mtDNA content and a higher chance of euploidy.