Does recurrent implantation failure exist? Prevalence and outcomes of five consecutive euploid blastocyst transfers in 123 987 patients.

STUDY QUESTION: What are the clinical pregnancy and live birth rates in women who underwent up to two more euploid blastocyst transfers after three failures in the absence of another known factor that affects implantation? SUMMARY ANSWER: The fourth and fifth euploid blastocyst transfers resulted in similar live birth rates of 40% and 53.3%, respectively, culminating in a cumulative live birth rate of 98.1% (95% CI = 96.5-99.6%) after five euploid blastocyst transfers. WHAT IS KNOWN ALREADY: The first three euploid blastocysts have similar implantation and live birth rates and provide a cumulative live birth rate of 92.6%. STUDY DESIGN, SIZE, DURATION: An international multi-center retrospective study was conducted at 25 individual clinics. The study period spanned between January 2012 and December 2022. A total of 123 987 patients with a total of 64 572 euploid blastocyst transfers were screened for inclusion. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients with a history of any embryo transfer at another clinic, history of any unscreened embryo transfer at participating clinics, parental karyotype abnormalities, the use of donor oocytes or a gestational carrier, untreated intracavitary uterine pathology (e.g. polyp, leiomyoma), congenital uterine anomalies, adenomyosis, communicating hydrosalpinx, endometrial thickness <6 mm prior to initiating of progesterone, use of testicular sperm due to non-obstructive azoospermia in the male partner, transfer of an embryo with a reported intermediate chromosome copy number (i.e. mosaic), preimplantation genetic testing cycles for monogenic disorders, or structural chromosome rearrangements were excluded. Ovarian stimulation protocols and embryology laboratory procedures including trophectoderm biopsy followed the usual practice of each center. The ploidy status of blastocysts was determined with comprehensive chromosome screening. Endometrial preparation protocols followed the usual practice of participating centers and included programmed cycles, natural or modified natural cycles. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 105 (0.085% of the total population) patients met the criteria and underwent at least one additional euploid blastocyst transfer after failing to achieve a positive pregnancy test with three consecutive euploid blastocyst transfers. Outcomes of the fourth and fifth euploid blastocyst transfers were similar across participating centers. Overall, the live birth rate was similar with the fourth and fifth euploid blastocysts (40% vs 53.3%, relative risk = 1.33, 95% CI = 0.93-1.9, P value = 0.14). Sensitivity analyses excluding blastocysts biopsied on Day 7 postfertilization, women with a BMI >30 kg/m2, cycles using non-ejaculate or donor sperm, double-embryo transfer cycles, and cycles in which the day of embryo transfer was modified due to endometrial receptivity assay test result yielded similar results. Where data were available, the fourth euploid blastocyst had similar live birth rate with the first one (relative risk = 0.84, 95% CI = 0.58-1.21, P = 0.29). The cumulative live birth rate after five euploid blastocyst transfers was 98.1% (95% CI = 96.5-99.6%). LIMITATIONS, REASONS FOR CAUTION: Retrospective design has its own inherent limitations. Patients continuing with a further euploid embryo transfer and patients dropping out from treatment after three failed euploid transfers can be systematically different, perhaps with regard to ovarian reserve or economic status. WIDER IMPLICATION OF THE FINDINGS: Implantation failure seems to be mainly due to embryonic factors. Given the stable and high live birth rates up to five euploid blastocysts, unexplained recurrent implantation failure should have a prevalence of <2%. Proceeding with another embryo transfer can be the best next step once a known etiology for implantation failure is ruled out. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Calcifediol (25OHD) Deficiency and Its Treatment in Women's Health and Fertility.

Currently, there is abundant scientific evidence showing that the vitamin D endocrine system (VDES) is a highly complex endocrine system with multiple actions in different regions of the body. The unequivocal presence of vitamin D receptors in different tissues related to fertility, and to specific aspects of women's health such as pregnancy, undoubtedly implies functions of this steroid hormone in both male and female fertility and establishes relationships with different outcomes of human gestation. In order to review the role of the VDES in human fertility, we evaluated the relationships established between 25-hydroxyvitamin D (calcifediol) deficiency and in vitro fertilization, as well as aspects related to ovarian reserve and fertility, and commonly diagnosed endocrinopathies such as polycystic ovary disease. Likewise, we briefly reviewed the relationships between calcifediol deficiency and uterine fibroids, as well as the role that treatment may have in improving human fertility. Finally, the best scientific evidence available on the consequences of calcifediol deficiency during pregnancy is reviewed in relation to those aspects that have accumulated the most scientific literature to date, such as the relationship with the weight of the newborn at the time of delivery, the appearance of preeclampsia, and the risk of developing gestational diabetes and its final consequences for the pregnancy. To date, there is no definitive consensus on the necessary dose for treatment of calcifediol deficiency in the therapeutic management of infertility or during pregnancy. Large prospective clinical intervention studies are needed to clarify the benefits associated with this supplementation and the optimal dose to use in each situation. Although most intervention studies to date have been conducted with cholecalciferol, due to its much longer history of use in daily care, the use of calcifediol to alleviate 25-hydroxyvitamin D deficiency seems safe, even during pregnancy. The unequivocal presence of vitamin D receptors in very different tissues related to human fertility, both male and female, as well as in structures typical of pregnancy, allows us to investigate the crucial role that this steroid hormone has in specific aspects of women's health, such as pregnancy and the ability to conceive. Well-designed clinical studies are needed to elucidate the necessary dose and the best form of treatment to resolve the very common calcifediol deficiency in women of reproductive age.

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.

Ethnic and Sociocultural Differences in Ovarian Reserve: Age-Specific Anti-Müllerian Hormone Values and Antral Follicle Count for Women of the Arabian Peninsula.

Background: Anti-Müllerian hormone (AMH) and antral follicle count (AFC) age-specific reference values form the basis of infertility treatments, yet they were based upon studies performed primarily on Caucasian populations. However, they may vary across different age-matched ethnic populations. This study aimed to describe age-specific serum AMH and AFC for women native to the Arabian Peninsula. Methods: A retrospective large-scale study was performed including 2,495 women, aged 19 to 50 years, native to the Arabian Peninsula. AMH and AFC were measured as part of their fertility assessment at tertiary-care fertility centres. Age-specific values and nomograms were calculated. Results: 2,495 women were evaluated. Mean, standard deviation and median values were calculated for AMH and AFC by 1-year and 5-years intervals. Median age was 34.81 years, median AMH was 1.76ng/ml and median AFC was 11. From the total group, 40.60% presented with AMH levels below 1.3ng/mL. For women <45 years old, the decrease in AFC was between -0.6/-0.8 per year. Up to 36 years old, the decrease of AMH was 0.1ng/ml. However, from 36 to 40 years old, an accelerated decline of 0.23ng/ml yearly was noted. In keeping with local customs, 71.23% of women wore the hijab and 25.76% the niqab. AMH and AFC were significantly lower for niqab group compared with hijab group (p=0.02 and p=0.04, respectively). Conclusion: This is to-date the largest data set on age-specific AMH and AFC values in women from the Arabian Peninsula aiming to increase clinical awareness of the ovarian reserve in this population.

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.

Clinical reassessment of human embryo ploidy status between cleavage and blastocyst stage by Next Generation Sequencing.

One of the most important limitations of genetic testing in preimplantation embryos is embryonic mosaicism, especially when performed on D3 with only a single blastomere evaluated. Previous publications, using Array-Comparative Genomic Hybridization (a-CGH) to compare day 3 (D3) biopsies versus trophectoderm biopsies for the analysis of aneuploid embryos, showed similar high concordance rates per embryo diagnosis for D3 biopsies and trophectoderm biopsies. Next generation sequencing (NGS) was introduced lately as a new technique for preimplantation genetic testing for aneuploidies (PGT-A). Using this technique, this retrospective descriptive study evaluated the degree of the concordance of the diagnosis between preimplantation human cleavage stage (D3) and blastocyst stage (D5) embryos. Double biopsies on D3 and D5 were performed on 118 embryos, reaching blastocyst stage on D5 and had not been selected for transfer. As the fertilization law of the United Arab Emirates does not allow embryo freezing, also surplus euploid embryos after D 3 biopsy were included. Analysis of the NGS results from D3 and D5 embryo biopsies showed a total concordance rate per embryo diagnosis of 85.6% for euploid and aneuploid embryos. The concordance rates per embryo chromosomal pattern for embryo diagnosed as aneuploid at both biopsy stages was 82.2%. However, the status regarding the affected chromosomes was not identical on D3 and D5. Hence, the total concordance rate between D3 biopsy and D5 biopsy was limited to 67.8%. This current study clearly demonstrated that the concordance rates between D3 and D5 biopsies in aneuploid and euploid embryos are lower than previously reported.