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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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02380-1.

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% CO(2), 5% O(2), and 89% N(2). 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02187-0.

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-020-02050-8.

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

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)?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.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).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.

Study question Does the limited exposure of embryos outside the incubator, during evaluation and changeover, have an impact on the blastocyst development, blastocyst quality and euploid outcomes? Summary answer Exposure of embryos outside the incubator, negatively impacts the number, quality and euploidy rate of day 5 blastocysts. What is known already The laboratory environment with its culture conditions is one of the crucial elements of the delicate equation to a successful ART outcome. It has been shown that increased fluctuations in the culture conditions have a considerable impact on the number of blastocysts obtained and cycle outcomes. Compared to conventional benchtop incubators, Time Lapse Technology (TLT) incubators capture images of the embryo and allow morphologic and morphokinetic assessment without disturbance during incubation. Several studies have been published comparing the efficiency, safety and outcome performance between conventional and TLT incubators, however, none of them explored the euploid outcomes. Study design, size, duration An observational sibling oocyte study was performed at ART Fertility Clinics, Abu Dhabi between March 2018 and April 2020 and included data of 796 mature oocytes injected from 42 stimulation cycles. Sibling oocytes were randomly split between 2 different incubators: 12 oocytes were assigned to the twelve wells of the EmbryoscopeTM (ES) and the remaining oocytes were cultured in a conventional benchtop incubator, G185 K-System (KS). Participants/materials, setting, methods Embryos from patients with primary or secondary infertility, who underwent ovarian stimulation for ICSI and PGT-A through NGS on trophectoderm biopsies, were eligible. All patients had at least 16 fresh mature oocytes, randomly allocated to two different incubators after ICSI: 503 (63.2%) oocytes were cultured in ES and 293 (36.8%) in KS. The fertilization, cleavage, useable blastocyst and euploid rates, as well as embryo/blastocyst qualities were assessed to evaluate each incubator’s performance. Main results and the role of chance The fertilization and cleavage rates were similar between incubators. Total useable blastocyst rate (64.8% vs 49.6%, p Limitations, reasons for caution The current observational study needs confirmation in a prospective trial and should also include the implantation potential of the euploid blastocysts, which was not followed in the current study. A good prognosis population (≥16 mature oocytes) was studied and may not reflect the outcomes in patients with lower oocyte numbers. Wider implications of the findings: This work builds evidence to the solid introduction of the TLT incubators to the clinical routine, as the reduced exposure of embryos outside the incubator – and hence decreased stress - improves the blastocyst development. Trial registration number NA

Study question Is parental consanguinity associated with reduced ovarian reserve in women from the Arabian Peninsula? Summary answer: Women descending from consanguineous unions have a reduced ovarian reserve compared with daughters of non-consanguine couples. What is known already Consanguineous marriage is defined as marriage between second-degree cousins or closer, with high prevalence in the Arabian Peninsula societies. An increased incidence of autosomal recessive diseases has been described in consanguineous marriages compared with non-consanguineous marriages. Despite the known adverse genetic impact of consanguinity, most available studies focus on the fertility of the consanguine couple. Only few publications, including low number of women, evaluated the impact of consanguinity on the fertility of their offspring, suggesting that daughters of consanguine parents might have reduced fertility associated to reduced ovarian reserve. Study design, size, duration A retrospective large-scale observational study was performed including 2482 women from the Arabian Peninsula who had their serum AMH and AFC measured as part of their fertility assessment at ART Fertility Clinics (UAE and Oman), from May 2015 to November 2019. Participants/materials, setting, methods 2482 women from the Arabian Peninsula, aged 19–50 years, were assessed. Consanguinity was defined as women whose parents were first-degree or second-degree cousins. Ovarian reserve was evaluated by Antral Follicle Count (AFC) with transvaginal ultrasound and serum AMH, measured by Elecsys (Cobas, Roche®) for all participants. Women with adnexal surgery history or/and hormonal treatment within previous three months (n = 284) were excluded. Ethical approval was obtained from the Research Ethics Committee (REFA040). Main results and the role of chance After excluding women with previous adnexal surgeries, 2198 women were included for analysis. A total of 605 participants (27.53%) were descendants from consanguineous unions and 1593 (72.47%) reported non-consanguineous kinship of their parents. AMH and AFC (mean±SD) for the consanguineous group were 2.62±2.88 ng/mL and 12.78±9.73 antral follicles, respectively; and AMH and AFC (mean±SD) for the non-consanguineous group were 2.65±2.91 ng/mL and 13.07±9.39 antral follicles, respectively. Women from the consanguinity group were significantly younger (mean±SD: 33.74±6.64 years old) compared with the non-consanguinity group (mean±SD: 34.78±6.64 years old, p Limitations, reasons for caution Despite the large number of women included, the retrospective study design is a limitation. Results have to be treated with caution before translating into other populations, as these data are obtained from women native to the Arabian Peninsula, with high sociocultural/religious/ethnical similarities, which might differ to other consanguine populations. Wider implications of the findings: Female parental consanguinity is associated with reduced ovarian reserve in the studied population, that might contribute to infertility. Future studies should examine the genetic and epigenetic basis of the current findings. Comprehensive clinical evaluation should include extensive family history and subsequent counselling of the affected couples. Trial registration number Not applicable

Study question Do delayed mature oocytes result in similar euploid blastocyst rates as their immediate mature sibling oocytes? Summary answer Once a blastocyst is obtained, delayed mature oocytes have similar euploid rates compared to immediate mature oocytes. What is known already Intracytoplasmic sperm injection (ICSI) of metaphase II oocytes few hours post oocyte retrieval is standard practice in IVF laboratories. Immature metaphase I (MI) and prophase I (GV) oocytes are usually discarded. Immature oocytes may mature overnight, after which ICSI can be performed. Studies demonstrated lower fertilization and blastulation rates for these delayed mature oocytes. However, live births have been reported from blastocysts transferred. The evidence available is not compelling, since most of the studies had either low sample size, no preimplantation genetic testing for aneuploidies (PGT-A), or the outcome was not compared to sibling MII oocytes at time of denudation. Study design, size, duration A single-center retrospective sibling oocyte study was performed between January 2019 and December 2020 at ART Fertility clinics Abu Dhabi, UAE. A total of 345 PGT-A cycles, with at least one delayed mature oocyte inseminated by ICSI, were included: 2506 immediate mature oocytes and 669 delayed mature oocytes. Participants/materials, setting, methods Following controlled ovarian stimulation, MII oocytes at the time of denudation were inseminated by ICSI/IVF (immediate mature). Immature oocytes (MI/GV) were cultured for 16–24 hours in fertilization medium and injected the next day if matured (delayed mature). Trophectoderm biopsy was performed on day 5/6/7 and samples were subjected to Next Generation Sequencing to screen the ploidy state of the blastocyst. Main results and the role of chance The 345 controlled ovarian stimulation cycles resulted in the insemination of 2506 MII oocytes on the day of oocyte retrieval (Day0) and 669 delayed mature oocytes on day 1. Normal fertilization rate was significantly higher in the immediate mature oocytes compared to delayed mature oocytes (68% vs 56%, p Fisher’s Exact Test was performed to compare the euploid rate of blastocysts biopsied on day 5/6/7 originating from immediate mature oocytes or sibling delayed mature oocytes. The euploid potential of blastocyst biopsied showed no significant difference between the two groups (p = 0.388). Limitations, reasons for caution The timing of MI/GV oocytes transition to MII stage was not recorded since the incubation was done in a benchtop incubator. Furthermore, the same sperm sample was used to inseminate immediate and delayed mature oocytes, which might contribute to the compromised embryo development due to increased sperm DNA fragmentation. Wider implications of the findings: Insemination of delayed mature oocytes by ICSI, should be considered as a tool to increase patients’ chances of obtaining a euploid embryo. Especially in cases where low yield of euploid embryos is expected. Trial registration number Not applicable

Study question Is the live birth rate (LBR) in euploid frozen embryo transfer (FET) cycles affected by the endometrial thickness (EMT)? Summary answer A significantly higher LBR was observed in patients with an endometrial thickness of at least 7.5mm (46.24% vs. 54.63%) What is known already Parameters assessing the endometrium prior planning a FET include endometrial thickness, pattern and blood flow. The impact of the endometrial thickness on ART outcomes is controversial, with conflicting results published. A recent meta-analysis evaluated whether EMT could predict pregnancy outcomes and suggested that lower EMT was associated with lower incidence of clinical pregnancy rate (CPR), implantation rate (IR) and LBR. Due to heterogeneity of parameters evaluated between different publications, where embryos with unknown ploidy status were transferred, in conjunction with variability of stimulation protocols and the number of embryos transferred, the real effect of the EMT was difficult to infer. Study design, size, duration This was a two-center retrospective observational study including a total of 1522 euploid FET cycles between March 2017 and March 2020 at ART Fertility Clinics Muscat, Oman and Abu Dhabi, UAE. Participants/materials, setting, methods Trophectoderm biopsies were analyzed with Next Generation Sequencing (NGS). Vitrification/warming of blastocysts was performed using Cryotop method (Kitazato). EMT was measured by vaginal ultrasound prior initiating the progesterone administration (± 1 day) and LBR was recorded. Multivariate analysis was performed between LB outcomes and median EMT while controlling for confounding factors. Main results and the role of chance A total of 1522 FET cycles were analyzed: 975 single embryo transfer (SET) and 547 double embryo transfer (DET). The mean age of the patients was 33.38 years with a mean BMI of 27.1 kg/m2. FET were performed in EMT ranging from 3 to 15 mm and 50.52% resulted in a live birth. Though potentially all ranges of EMT were associated with LB, the median EMT in patients with LB was significantly higher than the median EMT of patients without LB (7.6mm vs. 7.4mm; p The dataset was stratified into two groups based on the median EMT (7.5mm): < 7.5mm (n = 744 cycles) and ≥ 7.5mm (n = 778 cycles). A significantly higher live birth rate was observed in ≥ 7.5mm group (46.24% vs. 54.63%. p = 0.0012). In multivariate analysis, EMT, FET endometrial preparation protocol, and number of embryos transferred were the main parameters influencing the chance to achieve LB: OR 1.10 [1.01–1.19], p Limitations, reasons for caution Besides the retrospective nature of the study, the inter-observer variability in EMT assessment between different physicians is a limitation. The physician and embryologist performing the embryo transfer could not been standardized due to the multicenter design of the study. Wider implications of the findings: The EMT in FET may influence the LBR and should be considered as an important factor for the success of embryo transfer cycles. Whether these results can be extrapolated to fresh embryo transfer and to blastocysts with unknown ploidy status, needs further investigation. Trial registration number Not applicable

Study question Do euploid blastocysts biopsied on day (D) 5 or D6 differ in clinical pregnancy rates when single FET are performed in NC or HRT cycles? Summary answer In single FET cycles, euploid D5 blastocysts have higher clinical pregnancy rates than D6 in NC, while outcomes are comparable in HRT cycles. What is known already: The synchronization between the endometrium and the embryo development is fundamental for a successful implantation. When performing FET with euploid blastocysts biopsied on D5 or D6, higher clinical pregnancy rates have been reported with D5 blastocysts, however contradictory findings were described due to the study design heterogeneity and endometrial preparation (EP) protocol variabilities. In FET cycles, no consensus has been defined of the superiority of NC over HRT cycles when euploid blastocysts are transferred. Consequently, the question remains unanswered if the clinical pregnancy rates of single euploid FET with D5 or D6 blastocysts differ when the EP protocol remains constant. Study design, size, duration A single center observational study was performed between June 2017 and November 2020, including 1027 single euploid FET with blastocysts biopsied on D5 or D6. All patients with primary or secondary infertility who underwent a FET in a NC or HRT EP protocol, with blastocysts graded ≥ BL3CC (Gardner scoring system) prior to biopsy were included. Vitrified-warmed blastocysts that did not re-expand within 1-hour post-warming were excluded from the analysis. Participants/materials, setting, methods In NCs, vaginal progesterone (P4) (Endometrin®) was administrated (3x100mg) after endocrinological confirmation of ovulation until pregnancy test. For HRT cycles, oral estradiol administration was started on day 2 (4 mg) and increased to 6mg on D5 of the cycle. When endometrial thickness was ≥6 mm, P4 was given (3x100mg) until pregnancy test. All FET were performed on D5 after start of P4 administration. Clinical pregnancy was recorded as the presence of an intrauterine gestational sac. Main results and the role of chance Women’s mean age was 33.8 ± 5.5 years. A total of 651 FETs were performed with D5 euploid blastocysts (37.6% in NC and 62.4% in HRT) and 376 with D6 (43.1% in NC and 56.9% in HRT). Clinical pregnancy rate in NC was higher with D5 blastocysts compared to D6 (66.9% vs 50.0%; OR = 0.494, 95% CI = 0.322–0.758; p Limitations, reasons for caution The current results are based on an observational retrospective study. Live birth and perinatal outcomes should be considered in a further analysis to evaluate the performance of the NC vs HRT protocols when D5 or D6 euploid blastocysts are transferred in FET cycles. Wider implications of the findings: While the clinical pregnancies of D5 and D6 euploid blastocysts are comparable in HRT protocols only, the miscarriage rates seem to be significantly increased as compared to NC. Further studies are required to personalize EP protocols based on the day of blastocyst biopsy in order to improve clinical outcomes. Trial registration number No

Study question Which factors affect the clinical pregnancy rate (CPR) after single euploid frozen embryo transfers (FET), when the blastocyst is transferred in the upper uterine cavity area? Summary answer Blastocyst quality, embryo transfer difficulty and endometrial thickness affect the CPR in FET. What is known already There is a limited understanding of the factors affecting success rates after FET. The most important factors influencing implantation rates are patient characteristics, type of endometrial preparation, embryo quality and transfer difficulty. It has been shown that the position of the euploid blastocyst, measured as distance from the fundus (DFF) of the uterine cavity (mm), affects the implantation potential. Although the ideal location within the uterine cavity is still being debated in very heterogeneous patient populations, most studies have found that the highest pregnancy rates are obtained when the embryo is placed in the upper area of the uterine cavity. Study design, size, duration This single center retrospective cohort study included a total of 603 single euploid FET cycles, in the upper half of the uterine cavity, between January 2019 and November 2020 in ART Fertility Clinic Abu Dhabi, UAE. Participants/materials, setting, methods Trophectoderm biopsy samples were subjected to Next Generation Sequencing to screen the ploidy state. Vitrification and warming were performed using the Cryotop method (Kitazato, Biopharma). The full length of the uterine cavity and the longitudinal distance between the fundal endometrial surface and the air bubble after transfer were measured. Main results and the role of chance The patients were on average 33.9 (19–46) years old. The FET was performed in a natural cycle (NC) (n = 278) or hormone replacement therapy (HRT) (n = 325). Of the 603 transfers which had been performed in the upper half of the uterus, 412 (68.3%) resulted in a pregnancy and 311 (51.5%) in a clinical pregnancy. After bivariate analysis, the clinical pregnancy rate was significantly higher for high quality blastocysts (grade 1–2 versus 3–4) (p After performing a multivariate logistic regression analysis to consider the effect of all explanatory variables (age, Anti Müllerian hormone, body mass index, endometrial thickness, quality of the blastocyst, difficulty of the transfer [requirement of additional instrumentation], presence of mucus or blood on the transfer catheter, day 5 or day 6 biopsy, FET endometrial preparation), the clinical pregnancy was affected by the endometrial thickness: OR 1.20 [1.05–1.37], p = 0.007; transfer difficulty: OR 0.44 [0.25–0.79], p = 0.006; blastocyst quality 3: OR 0.38 [0.18–0.79], p = 0.01 and blastocyst quality 4: OR 0.15 [0.06–0.37], p Limitations, reasons for caution The limitation of this study was its retrospective nature and the small sample size. Other parameters may be important in live birth outcomes. Wider implications of the findings: Optimization of clinical pregnancy outcomes after FET depends on multiple factors. Even after transfer of euploid blastocysts in the upper uterine cavity, the endometrial thickness, transfer difficulty and blastocyst quality will still affect the clinical pregnancy outcomes. Trial registration number NA

Study question Do woman with diminished ovarian reserve exhibit poor blastocyst formation and ploidy outcomes, irrespective of age? Summary answer Patients with extreme diminished ovarian reserve (AMH≤0.65ng/ml) have a lower chance to have at least one euploid blastocyst compared to their age-related reference population (AMH=1.3–6.25ng/ml). What is known already AMH is an established marker of the ovarian reserve for predicting ovarian response to ovarian stimulation and it is strongly correlated with female age. However, it has been suggested that AMH is not only a quantitative, but also a qualitative biomarker of oocyte/embryo competence. Previous studies show conflicting outcomes as to whether reduced ovarian reserve per se is associated with decreased oocyte developmental competence, leading to increased aneuploidy rates in embryos independent of the patient’s age. Study design, size, duration A retrospective analysis was performed between March 2017 and July 2020 at ART Fertility Clinics (Abu Dhabi) including all couples that were triggered for final oocyte maturation and planned for Preimplantation Genetic Testing for Aneuploidies (PGT-A). Patients were stratified into four age categories [≤30, 31–35, 36–40, >40 years]. For each age category patients were further divided into three AMH groups: ≤0.65ng/ml, 0.65–1.3ng/ml and 1.31–6.25ng/ml (reference group). Participants/materials, setting, methods Trophectoderm biopsy samples were subjected to Next Generation Sequencing. AMH serum levels (ng/ml) were determined using the commercial fully automated Elecsys® (Roche) assay. Patients with a Progesterone rise of > 1.5ng/ml on the day of final oocyte maturation and patients with AMH values >6.25ng/ml were excluded from the analysis. Per patient that was triggered, the chance to have at least one euploid blastocyst in that cycle, was calculated. Main results and the role of chance A total of 1.300 couples were included with an mean maternal age of 35.6±6.2 years, AMH of 2.1 ±1.5ng/ml and body mass index of 27.5±5.0 kg/m2. The chance to have at least one blastocyst biopsied per cycle was affected in all patients with extreme low AMH (≤0.65ng/ml), irrespective of age; ≤30 years: 58.33%–100.00%–94.84% (p 40 years from 29.42% (n = 102) to 6.45% (n = 155) (OR 0.16 [0.08–0–36], p Limitations, reasons for caution The main limitation of this study is its retrospective design. Wider implications of the findings: AMH is a clear biomarker of oocyte-embryo competence. Incorporation of AMH-specific counseling recommendations into clinical practice guidelines, could lead to a more informed guidance on cycle ploidy outcomes, rather than age alone. Trial registration number Not applicable

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-019-01544-4) contains supplementary material, which is available to authorized users.

To evaluate whether the mitoscore of cleavage stage embryos might correlate with developmental kinetics and the ploidy status. 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). 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). 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.

Study question How reliable are cleavage stage and trophectoderm (TE) biopsies compared to inner cell mass (ICM) biopsies? Summary answer The reliability of TE biopsy compared to ICM biopsy is almost perfect, but only substantial between cleavage stage biopsy and ICM biopsy. What is known already One of the prevailing reasons for implantation failure is presumed to be chromosomal aneuploidy in human preimplantation embryos. Preimplantation genetic testing for aneuploidies (PGT-A) has been introduced into assisted reproduction in an effort to increase pregnancy rates. Increasing evidence indicates that genetic results obtained following blastomere or TEbiopsy may not accurately reflect the true genetic status of the embryo due to the presence of embryonic mosaicism, and therefore the reliability of PGT is highly controversial. Study design, size, duration This was an observational descriptive study, performed in a private infertility centre from August 2016 to January 2017. Participants/materials, setting, methods The mean female age was 33.9 years, ranging from 24 to 46 years, and the mean number of biopsied embryos per couple was 2.2 (range 1-7 embryos). Blastomere biopsies had been performed at cleavage stage on Day 3 (D3) due to the turnover time of genetic testing and the inability to cryopreserve embryos in accordance with the local law governing ART. To confirm the genetic results in embryos not chosen for transfer, additional biopsies of the TE at blastocyst stage (BLASTO-TE) as well as of the ICM (BLASTO-ICM) were performed on D5. Only surplus blastocysts, which had not been selected for transfer and were not cryopreserved in accordance with the law governing ART, had been included. Main results and the role of chance Comparison of all biopsies (D3/BLASTO-ICM/BLASTO-TE) per embryo demonstrated that 50 (59.5%) out of 84 embryos showed concordance in all three results (= full concordance). Thirty-four (40.4%) embryos had at least two discordant results between the three biopsies, regardless of whether the embryo diagnosis (aneuploid/euploid) was discordant or not, or in aneuploid embryos, whether the chromosomal patterns were inconsistent. Nine (= 10.7%) embryos had complete discordance between all three biopsies. False positive results between D3/BLASTO-TE, D3/BLASTO-ICM and BLASTO-TE/BLASTO-ICM were 26.4%/30.2% and 7.5%, respectively, while the Kappa agreement between the different approaches was 0.647, 0.553 and 0.857, respectively. Therefore the reliability of D3/BLASTO-TE, D3/BLASTO-ICM and BLASTO-TE/BLASTO-ICM can be interpreted as substantial, as moderate and as almost perfect. Limitations, reasons for caution The limitation of this study is the possible bias in the concordance/discordance rate because embryos that had been selected for transfer did not undergo biopsy on D5. Wider implications of the findings The obvious discordance between the three different approaches for PGT-A underlines the limitations of genetic testing and highlights the importance of ongoing research in order to improve the accuracy of PGT-A results. Until then reproductive specialists will continue to make challenging decisions on whether to transfer or discard an embryo in light of current evidence questioning the reliability of genetic results. Study funding/competing interest(s) This study was supported by Igenomix. The funder provided support in the form of salary for R.C. The co-author R.C. is an employee of Igenomix. She participated in the blinded analysis of the samples; however the final data collection and statistical analysis of the results, as well as the decision to publish, was taken by B.L, I.E. and H.F. The authors B.L., I.E., A.L., A.B., A.A., N.D. and H.F. have no competing interests. The funder did not have any additional role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. The commercial affiliation of R.C. did not play any role in the study. Trial registration number This study was approved by the Ethics Committee of IVIRMA Middle East Fertility Clinic, Abu Dhabi, UAE (Research Ethics Committee IVI-MEREFA009a/2017).

OBJECTIVE: To determine whether euploidy rates and blastocyst development differ in a continuous culture medium under different CO(2) 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) CO(2), 5.0% O(2), and 89.0% or 88.0% N(2). 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% CO(2) (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% CO(2) (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% CO(2), respectively. The mean blastocyst mtDNA content was significantly lower in 6.0% CO(2) (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% CO(2), while euploidy rates are significantly decreased at a higher CO(2) concentration, therefore at a lower pHe. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02303-0.

PURPOSE: To verify which genetic abnormalities prevent embryos to blastulate in a stage-specific time. METHODS: A single center retrospective study was performed between April 2016 and January 2017. Patients requiring Preimplantation Genetic Testing for Aneuploidies (PGT-A) by Next Generation Sequencing (NGS) were included. All embryos were cultured in a time-lapse imaging system and single blastomere biopsy was performed on day 3 of development. Segmental duplications and deletions as well as whole chromosome monosomies and trisomies were registered. Embryo arrest was defined if the embryo failed to blastulate 118 h post-injection. A logistic regression model was applied using the time to blastulate as the response variable and the different mutations as explanatory variables. A p value < 0.05 was considered significant. RESULTS: Of the 285 biopsied cleavage stage embryos, 103 (36.1%) were euploid, and 182 (63.9%) were aneuploid. There was a significant difference in the developmental arrest between euploid and aneuploid embryos (8.7% versus 42.9%; p = 0.0001). Segmental duplications and whole chromosome monosomies were found to have a significant effect on developmental arrest (p = 0.0163 and p = 0.0075), while trisomies and segmental deletions had no effect on developmental arrest. In case of segmental duplications, an increase of one extra segmental duplication increases the odd of arrest by 159%. For whole chromosome monosomies, the odd will only increase by 29% for every extra chromosomal monosomy. Both chromosomal abnormalities remained significant after adding age as an explanatory variable to the model (p = 0.014 and p = 0.009). CONCLUSION: Day 3 cleavage stage embryos with segmental duplications or monosomies have a significantly decreased chance to reach the blastocyst stage.

ContextThe 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.ObjectiveTo determine a possible correlation between VitD levels in serum (S) and follicular fluid (FF) and blastocyst ploidy status in patients undergoing infertility treatment.MethodsA 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 ResultsSerum 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; pConclusionVitD non-deficient patients have a significantly higher probability of obtaining a euploid blastocyst compared to VitD deficient patients (OR:33.36, p = 0.002).

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?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).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: P0.001 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).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.

Research question This study explored the relationship between anti-Mullerian 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 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.

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.