Exome sequencing in genuine empty follicle syndrome: Novel candidate genes.

OBJECTIVE(S): Empty follicle syndrome (EFS) is a condition in which no oocytes are retrieved in an IVF cycle despite apparently normal follicular development and meticulous follicular aspiration following ovulation induction. The EFS is called genuine (gEFS) when the trigger administration is correct. The existence of gEFS is a subject of controversy, and it is quite rare with an undetermined etiology. Genetic defects in specific genes have been demonstrated to be responsible for this condition in some patients. Our objective was to identify novel genetic variants associated with gEFS. STUDY DESIGN: We conducted a prospective observational study including 1,689 egg donors from July 2017 to February 2023. WES were performed in patients suffering gEFS. RESULTS: Only 7 patients (0.41 %) exhibited gEFS after two ovarian stimulation cycles and we subsequently performed whole exome sequencing (WES) on these patients. Following stringent filtering, we identified 6 variants in 5 affected patients as pathogenic in new candidate genes which have not been previously associated with gEFS before, but which are involved in important biological processes related to folliculogenesis. These genetic variants included c.603_618del in HMMR, c.1025_1028del in LMNB1, c.1091-1G > A in TDG, c.607C > T in HABP2, c.100 + 2 T > C in HAPLN1 and c.3592_3593del in JAG2. CONCLUSION: As a conclusion, we identified new candidate genes related to gEFS that expand the mutational spectrum of genes related to gEFS.This study show that WES might be an efficient tool to identify the genetic etiology of gEFS and provide further understanding of the pathogenic mechanism of gEFS.

The impact of (very) young donor age on euploid rates: An analysis of 1831 trophectoderm biopsies evaluated with 24-chromosome NGS screening in oocyte donation cycles.

RESEARCH QUESTION: Conflicting data exists regarding whether a younger age of donors has a negative influence on the outcomes of oocyte donation cycles. Is there any correlation between a younger age of donors and the rate of embryonic aneuploidy in oocyte donation cycles? DESIGN: Retrospective study including 515 oocyte donation cycles carried out between February 2017 and November 2022. Comprehensive chromosomal screening was performed on 1831 blastocysts. 1793 had a result which were categorised into groups based on the age of the donor: 18-22 (n = 415), 23-25 (n = 600), 26-30 (n = 488), and 31-35 years (n = 290). The analysis aimed to determine the percentage of biopsy samples that were euploid and the number that were aneuploid, relative to the age group of the oocyte donor. Additionally, linear regression was employed to examine the relationship between age and the proportion of aneuploid embryos, while controlling for relevant variables. RESULTS: Aneuploidy increased predictably with donor age: 18-22 years: 27.5 %; 23-25 years: 31.2 %; 26-30 years: 31.8 %; and 31-35 years: 38.6 %. In the donor group aged 31-35 years, a higher percentage of aneuploid embryos was observed compared to younger donors in univariate analysis (OR: 1.66, 95 % CI: 1.21-2.29, p = 0.002) and multivariate logistic analysis (OR: 2.65, 95 % CI: 1.67-4.23, p < 0.001). The rates of embryonic mosaicism revealed no significant differences. CONCLUSION: The lowest risk of embryonic aneuploidy was found among donors aged <22 years. Conversely, an elevated prevalence was evident within the donor group aged 31-35 years, in contrast to the younger cohorts. The incidence of mosaic embryos remained consistent across all age groups.

To transfer or not to transfer: the dilemma of mosaic embryos - a narrative review.

A frequent finding after preimplantation genetic diagnostic testing for aneuploidies using next-generation sequencing is an embryo that is putatively mosaic. The prevalence of this outcome remains unclear and varies with technical and external factors. Mosaic embryos can be classified by the percentage of cells affected, type of chromosome involvement (whole or segmental), number of affected chromosomes or affected cell type (inner mass cell, trophectoderm or both). The origin of mosaicism seems to be intrinsic as a post-zygotic mitotic error, but some external factors can play a role. As experience has increased with the transfer of mosaic embryos, clinical practice has gradually become more flexible in recent years. Nevertheless, clinical results show lower implantation, pregnancy and clinical pregnancy rates and higher miscarriage rates with mosaic embryo transfer when compared with the transfer of euploid embryos. Prenatal diagnosis is highly recommended after the transfer of mosaic embryos. This narrative review is intended to serve as reference material for practitioners in reproductive medicine who must manage a mosaic embryo result after preimplantation genetic testing for aneuploidies.

Random-start ovarian stimulation in an oocyte donation programme: a large, single-centre, experience.

RESEARCH QUESTION: Do live birth rates differ between recipients matched with donors using conventional ovarian stimulation compared with those using random-start protocols? DESIGN: Retrospective analysis of 891 ovarian stimulations in egg donors (January-December 2018) and clinical outcomes in matched recipients (n = 935). Donors commenced ovarian stimulation on day 1-3 of the menstrual cycle (n = 223) or in the mid/late-follicular (n = 388) or luteal phase (n = 280) under a conventional antagonist protocol. Live birth rate of matched recipients was the main outcome. RESULTS: Duration of stimulation and total gonadotrophin dose were comparable between conventional versus random-start groups. The number of collected eggs were similar (17.6 ± 8.8 versus 17.2 ± 8.5, P = 0.6, respectively). Sub-group analysis showed that stimulation length (10.2 ± 1.8 versus 9.8 ± 1.7 versus 10.4 ± 1.7, P < 0.001) and gonadotrophin consumption (2041.5 ± 645.3 versus 2003.2 ± 647.3 versus 2158.2 ± 685.7 IU, P = 0.01) differed significantly between the conventional, mid/late follicular and luteal phase groups, respectively. In matched recipients receiving fresh oocytes and undergoing fresh embryo transfer, the biochemical pregnancy (63.8% and 63.3%; P = 0.9), clinical pregnancy (54.6% and 56.1%; P = 0.8) and live birth rates (47.7% and 46.6%; P = 0.7) per embryo-transfer were similar between conventional versus random groups. Similar results were obtained in recipients receiving vitrified eggs. Euploidy rate was also comparable. CONCLUSIONS: No notable variations were found in clinical outcomes using oocytes obtained from random-start protocols and those proceeding from conventional ovarian stimulation in oocyte donation treatments. Luteal-phase stimulation seems to require longer stimulation and higher FSH consumption. Random-start stimulation strategy does not impair the potential of the oocyte yield or clinical outcomes in oocyte donation cycles.

Implicit bias in diagnosing mosaicism amongst preimplantation genetic testing providers: results from a multicenter study of 36 395 blastocysts.

STUDY QUESTION: Does the diagnosis of mosaicism affect ploidy rates across different providers offering preimplantation genetic testing for aneuploidies (PGT-A)? SUMMARY ANSWER: Our analysis of 36 395 blastocyst biopsies across eight genetic testing laboratories revealed that euploidy rates were significantly higher in providers reporting low rates of mosaicism. WHAT IS KNOWN ALREADY: Diagnoses consistent with chromosomal mosaicism have emerged as a third category of possible embryo ploidy outcomes following PGT-A. However, in the era of mosaicism, embryo selection has become increasingly complex. Biological, technical, analytical, and clinical complexities in interpreting such results have led to substantial variability in mosaicism rates across PGT-A providers and clinics. Critically, it remains unknown whether these differences impact the number of euploid embryos available for transfer. Ultimately, this may significantly affect clinical outcomes, with important implications for PGT-A patients. STUDY DESIGN, SIZE, DURATION: In this international, multicenter cohort study, we reviewed 36 395 consecutive PGT-A results, obtained from 10 035 patients across 11 867 treatment cycles, conducted between October 2015 and October 2021. A total of 17 IVF centers, across eight PGT-A providers, five countries and three continents participated in the study. All blastocysts were tested using trophectoderm biopsy and next-generation sequencing. Both autologous and donation cycles were assessed. Cycles using preimplantation genetic testing for structural rearrangements were excluded from the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The PGT-A providers were randomly categorized (A to H). Providers B, C, D, E, F, G, and H all reported mosaicism, whereas Provider A reported embryos as either euploid or aneuploid. Ploidy rates were analyzed using multilevel mixed linear regression. Analyses were adjusted for maternal age, paternal age, oocyte source, number of embryos biopsied, day of biopsy, and PGT-A provider, as appropriate. We compared associations between genetic testing providers and PGT-A outcomes, including the number of chromosomally normal (euploid) embryos determined to be suitable for transfer. MAIN RESULTS AND THE ROLE OF CHANCE: The mean maternal age (±SD) across all providers was 36.2 (±5.2). Our findings reveal a strong association between PGT-A provider and the diagnosis of euploidy and mosaicism. Amongst the seven providers that reported mosaicism, the rates varied from 3.1% to 25.0%. After adjusting for confounders, we observed a significant difference in the likelihood of diagnosing mosaicism across providers (P < 0.001), ranging from 6.5% (95% CI: 5.2-7.4%) for Provider B to 35.6% (95% CI: 32.6-38.7%) for Provider E. Notably, adjusted euploidy rates were highest for providers that reported the lowest rates of mosaicism (Provider B: euploidy, 55.7% (95% CI: 54.1-57.4%), mosaicism, 6.5% (95% CI: 5.2-7.4%); Provider H: euploidy, 44.5% (95% CI: 43.6-45.4%), mosaicism, 9.9% (95% CI: 9.2-10.6%)); and Provider D: euploidy, 43.8% (95% CI: 39.2-48.4%), mosaicism, 11.0% (95% CI: 7.5-14.5%)). Moreover, the overall chance of having at least one euploid blastocyst available for transfer was significantly higher when mosaicism was not reported, when we compared Provider A to all other providers (OR = 1.30, 95% CI: 1.13-1.50). Differences in diagnosing and interpreting mosaic results across PGT-A laboratories raise further concerns regarding the accuracy and relevance of mosaicism predictions. While we confirmed equivalent clinical outcomes following the transfer of mosaic and euploid blastocysts, we found that a significant proportion of mosaic embryos are not used for IVF treatment. LIMITATIONS, REASONS FOR CAUTION: Due to the retrospective nature of the study, associations can be ascertained, however, causality cannot be established. Certain parameters such as blastocyst grade were not available in the dataset. Furthermore, certain platform-related and clinic-specific factors may not be readily quantifiable or explicitly captured in our dataset. As such, a full elucidation of all potential confounders accounting for variability may not be possible. WIDER IMPLICATIONS OF THE FINDINGS: Our findings highlight the strong need for standardization and quality assurance in the industry. The decision not to transfer mosaic embryos may ultimately reduce the chance of success of a PGT-A cycle by limiting the pool of available embryos. Until we can be certain that mosaic diagnoses accurately reflect biological variability, reporting mosaicism warrants utmost caution. A prudent approach is imperative, as it may determine the difference between success or failure for some patients. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Torres Quevedo Grant, awarded to M.P. (PTQ2019-010494) by the Spanish State Research Agency, Ministry of Science and Innovation, Spain. M.P., L.B., A.R.L., A.L.R.d.C.L., N.P.P., M.P., D.S., F.A., A.P., B.M., L.D., F.V.M., D.S., M.R., E.P.d.l.B., A.R., and R.V. have no competing interests to declare. B.L., R.M., and J.A.O. are full time employees of IB Biotech, the genetics company of the Instituto Bernabeu group, which performs preimplantation genetic testing. M.G. is a full time employee of Novagen, the genetics company of Cegyr, which performs preimplantation genetic testing. TRIAL REGISTRATION NUMBER: N/A.

Noninvasive preimplantation genetic testing using the embryo spent culture medium: an update.

PURPOSE OF REVIEW: The presence of cell-free DNA (cf-DNA) in the embryo spent culture medium allows to develop a noninvasive PGT-A (niPGTA). Noninvasive PGT-A may provide a simpler, safer and less costly approach to preimplantation genetic testing of aneuploidy (PGT-A). Furthermore, niPGTA would provide wider access to embryo genetic analysis and circumvent many legal and ethical considerations. However, the concordance rate between the results obtained by PGT-A and niPGTA varies among studies and, their clinical utility has not been already demonstrated. This review evaluates the niPGTA reliability based on SCM and adds new knowledge about the clinical relevance of SCM for noninvasive PGT-A. RECENT FINDINGS: The most recent concordance studies evaluating the accuracy of niPGTA using SCM showed a high variation in the informativity rate of SCM and the diagnostic concordance. Also, sensitivity and specificity showed similar heterogeneous results. Therefore, these results do not support the clinical utility of niPGTA. Regarding clinical outcome, the data are initial and further research, including randomized and nonselection studies are needed. SUMMARY: Further research, including randomized and nonselection studies, as well as optimization of embryo culture conditions and medium retrieval, are needed to improve the reliability and clinical utility of niPGTA.

Characterization of the Endometrial Microbiome in Patients with Recurrent Implantation Failure.

An abnormal endometrial microbiota has been associated with implantation failure; therefore, it may be important to evaluate it in order to improve reproductive outcomes in infertile patients. The main objective of our study was to compare the endometrial microbiome of patients with recurrent implantation failure (RIF) and control patients undergoing assisted reproduction treatment (ART). A prospective cohort study including forty-five patients with their own or donated gametes. The endometrial microbiome was analysed by massive sequencing of the bacterial 16S rRNA gene. Different bacterial communities were detected in RIF and control patients. Lactobacillus stands out as the most frequent genus, with 92.27% in RIF patients and 97.96% in control patients, and significant differences were reported between the two groups (p = 0.002). No significant differences were found regarding alpha diversity index. In beta diversity analysis, a significant trend was observed in the separation of the bacterial community between established groups (p < 0.07). Relative abundance analysis identified genera Prevotella (p < 0.001), Streptococcus (p < 0.001), Bifidobacterium (p = 0.002), Lactobacillus (p = 0.002) and Dialister (p = 0.003). Our results demonstrated the existence of an endometrial microbiota characteristic of RIF patients and showed that there might be a relationship between population of the endometrial microbiome and embryo implantation failure, providing us the possibility to improve clinical results in this patients.

Application of machine learning to predict aneuploidy and mosaicism in embryos from in vitro fertilization cycles.

BACKGROUND: The factors associated with embryo aneuploidy have been extensively studied. Mostly maternal age and to a lesser extent male factor and ovarian stimulation have been related to the occurrence of chromosomal alterations in the embryo. On the other hand, the main factors that may increase the incidence of embryo mosaicism have not yet been established. OBJECTIVE: This study aimed to establish a machine learning model that would allow prediction of aneuploidies and mosaicism in embryos conceived via in vitro fertilization, and thus help to determine which variables are associated with these chromosomal alterations. STUDY DESIGN: The study design was observational and retrospective. A total of 6989 embryos from 2476 cycles of preimplantation genetic testing for aneuploidies were included (January 2013 to December 2020). The trophoectoderm biopsies on day-5, -6, or -7 blastocysts were analyzed by preimplantation genetic testing for aneuploidies (PGT-A). The different maternal, paternal, couple, embryo, and in vitro fertilization cycle characteristics were recorded in a database (22 predictor variables) from which predictive models of embryo aneuploidy and mosaicism were developed; 16 different unsupervised classification machine learning algorithms were used to establish the predictive models. RESULTS: Two different predictive models were performed: one for aneuploidy and the other for mosaicism. The predictor variable was of multiclass type because it included the segmental- and whole-chromosome alteration categories. The best predicting models for both aneuploidies and mosaicism were those obtained from the Random Forest algorithm. The area under ROC curve (AUC) value was 0.792 for the aneuploidy explanatory model and 0.776 for mosaicism. The most important variable in the final aneuploidy model was maternal age, followed by paternal and maternal karyotype and embryo quality. In the predictive model of mosaicism, the most important variable was the technique used in preimplantation genetic testing for aneuploidies and embryo quality, followed by maternal age and day of biopsy. CONCLUSION: It is possible to predict embryo aneuploidy and mosaicism from certain characteristics of the patients and their embryos.

FSH receptor genotype and its influence on the results of donor ovarian stimulation using corifollitropin alfa.

RESEARCH QUESTION: Does the FSH receptor (FSHR) genotype influence the results of donor ovarian stimulation using corifollitropin alfa? DESIGN: A prospective cohort study was performed including 152 oocyte donor ovarian stimulations: group 1 (n = 80) using a single dose of 150 µg of corifollitropin alpha; and group 2 (n = 72) using in addition to corifollitropin alpha, continued stimulation using recombinant FSH 225 IU daily. Allelic discrimination was used to genotype the FSHR p.N680S polymorphism. Linear regression analysis was performed to study the differences between groups. RESULTS: No differences in clinical characteristics between genotypes were reported. Overall, the results of ovarian stimulation were better in oocyte donors with SN and NN genotypes compared with SS in terms of the number of retrieved oocytes (15.78 versus 10.83; P = 0.008) and retrieved metaphase II (MII) oocytes (12.34 versus 9.00; P = 0.032). Corresponding differences were also observed in group 1 for the number of retrieved oocytes (13.83 versus 7.50, P = 0.018) and retrieved MII oocytes (10.24 versus 5.42; P = 0.038). However, in group 2 no significant differences were found for oocytes retrieved (17.55 versus 13.06, P = 0.064) or MII oocytes (14.25 versus 11.39; P = 0.12). CONCLUSIONS: This study suggests that ovarian stimulation protocols with corifollitropin alfa in women with the SS genotypes could be associated with fewer oocytes and MII oocytes retrieved. Despite the fact that corifollitropin alfa has a longer half-life, the results for the SS genotype do not match those for the other genotypes, so other factors must be involved. Therefore, to tailor treatments, it would be advisable to genotype women at p.N680S of the FSHR.

Sperm DNA fragmentation on the day of fertilisation is not associated with assisted reproductive technique outcome independently of gamete quality.

The evaluation of sperm DNA fragmentation has been postulated as a predictive molecular parameter of the semen fertilising potential, as well as the ability to give rise to a healthy embryo and an ongoing pregnancy. However, there are controversial results due to oocyte quality, the use of different measurement techniques and interpretation criteria. Our objective is to investigate if sperm DNA fragmentation on the day of fertilisation influences in vitro fertilisation (IVF) outcome in a prospective double-blind study. Three groups of patients were defined: (i) 68 couples undergoing intracytoplasmic sperm injection (ICSI) due to severe male factor with normal ovarian response (NOR); (ii) 113 couples undergoing conventional in vitro fertilisation (IVF) in our oocyte donation programme due to ovarian failure; and (iii) 150 low ovarian response (LOR) patients undergoing ICSI or IVF. TUNEL assay was performed from an aliquot of each capacitated semen sample to detect DNA fragmentation. There was no relationship between blood serum ß-hCG positive test, clinical pregnancy and first trimester miscarriage with DFI levels in NOR (p = 0.41, p = 0.36, p = 0.40), recipient (p = 0.49, p = 0.99 and p = 0.38) and LOR (p = 0.52, p = 0.20, p = 0.64) groups of patients, respectively. Therefore, ART outcomes are not affected by sperm DNA fragmentation independently of gamete quality.

Impact of the Vaginal and Endometrial Microbiome Pattern on Assisted Reproduction Outcomes.

Uterine microbiota may be involved in reproductive health and disease. This study aims to describe and compare the vaginal and endometrial microbiome patterns between women who became pregnant and women who did not after in vitro fertilization. We also compared the vaginal and endometrial microbiome patterns between women with and without a history of repeated implantation failures (RIF). This pilot prospective cohort study included 48 women presenting to the fertility clinic for IVF from May 2017 to May 2019. Women who achieved clinical pregnancy presented a greater relative abundance of Lactobacillus spp. in their vaginal samples than those who did not (97.69% versus 94.63%; p = 0.027. The alpha and beta diversity of vaginal and endometrial samples were not statistically different between pregnant and non-pregnant women. The Faith alpha diversity index in vaginal samples was lower in women with RIF than those without RIF (p = 0.027). The alpha diversity of the endometrial microbiome was significantly higher in women without RIF (p = 0.021). There were no significant differences in the vaginal and endometrial microbiomes between pregnant and non-pregnant women. The relative abundance of the genera in women with RIF was different from those without RIF. Statistically significant differences in the endometrial microbiome were found between women with and without RIF.

Methylenetetrahydrofolate reductase gene polymorphisms are not associated with embryo chromosomal abnormalities and IVF outcomes.

The aim of our study was to investigate the effect of maternal and embryo MTHFR C677T and A1298C polymorphisms on embryo aneuploidies and mosaicism and the correlation between these genetic variants in transferred euploid embryos and IVF outcomes. MTHFR genotype was analyzed in 77 women who performed an IVF/ICSI cycle with PGT-A. Moreover, to evaluate the effect of embryo MTHFR polymorphisms on embryo aneuploidies and mosaicism, the MTHFR genotype was analyzed in 191 biopsied embryos from the PGT-A cycles of these patients. Additionally, 218 DNA samples from trophectoderm biopsies belonging to a different group of patients were also genotyped. MTHFR polymorphisms were analyzed in a total amount of 409 trophectoderm samples. The main parameters analyzed were embryo aneuploidy and mosaicism rates. Finally, the IVF outcomes of 241 single euploid embryo transfers were assessed and compared between different MTHFR embryo genotypes. The aneuploidy rates were similar in embryos from homozygous normal women and women with at least one mutated allele (54.7% vs. 30.2% in 677C>T and 37.8% vs. 42.7% in 1298A>C). Furthermore, no differences were observed in the mosaicism rate (24.0% vs. 13.8% in 677C>T and 17.1% vs. 17.3% in 1298A>C). A similar analysis was performed, taking into account the embryo genotype results. No differences in aneuploidy rate were observed between the study groups. The only significant difference was the mosaicism rate among 677C>T genotype (13.5% in 677CC group vs. 5.4% in 677CT/TT; p = 0.019). Implantation rate, biochemical and clinical miscarriage rates, and ongoing pregnancy rate were compared between different embryo genotypes, and no statistically significant differences were found. In conclusion, the maternal MTHFR genotype did not influence embryo chromosomal abnormalities. Moreover, the embryo MTHFR genotype was not associated with embryo aneuploidy or IVF outcomes such as implantation, pregnancy loss, and ongoing pregnancy when euploid embryos were transferred.Abbreviations: MTHFR: methylenetetrahydrofolate reductase; IVF: in vitro fertilization; PGT-A: preimplantation genetic testing for aneuploidies; SAM: S-adenosyl methionine; SNP: single nucleotide polymorphism; SPSS: Statistical Package for Social Sciences; RIF: recurrent implantation failure; RPL: recurrent pregnancy loss; hCG: human chorionic gonadotropin; PBS: phosphate buffered saline; CGH: comparative genomic hybridization; NGS: next generation sequencing.

The Association between Vaginal Dysbiosis and Reproductive Outcomes in Sub-Fertile Women Undergoing IVF-Treatment: A Systematic PRISMA Review and Meta-Analysis.

Recent advances in molecular microbiology have enabled refined studies of the genital tract microbiota. This constitutes the basis of the present updated systematic review and meta-analysis which investigate vaginal dysbiosis (VD) as defined by either microscopy (e.g., Nugent score for bacterial vaginosis) or molecular methods (qPCR and Next Generation Sequencing) to evaluate the impact of VD on the reproductive outcomes in women undergoing IVF-treatment. A total of 17 studies were included, comprising 3543 patients and with a VD prevalence of 18% (95%CI 17-19). Across all methods, VD is a significant risk factor for early pregnancy loss in IVF (Relative risk (RR) = 1.71 95%CI 1.29-2.27). Moreover, a predefined sub-analysis of studies using molecular methods for VD diagnosis showed a significant reduction in the clinical pregnancy rate when compared to normal vaginal microbiota patients (RR = 0.55 95%CI 0.32-0.93). However, regardless of diagnostic methodology, VD did not significantly influence live birth rate (LBR). In conclusion, molecular tools have provided a more detailed insight into the vaginal microbiota, which may be the reason for the increased adverse effect estimates in IVF patients with molecularly defined VD. However, the quality of evidence was very low across all outcomes according to GRADE and thus, more studies are warranted to understand the impact of VD in IVF.

Consistent results of non-invasive PGT-A of human embryos using two different techniques for chromosomal analysis.

RESEARCH QUESTION: Are discordances in non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) results attributable to the technique used for chromosomal analysis? DESIGN: A prospective blinded study was performed (September 2018 to December 2019). In total 302 chromosomal analyses were performed: 92 trophectoderm PGT-A biopsies and their corresponding spent embryo culture medium (SCM) evaluated by two methods (n = 184), negative controls (n = 8), and trophectoderm and inner cell mass biopsies from trophectoderm-aneuploid embryos (n = 18). Trophectoderm analyses were carried out using Veriseq (Illumina), and SCM was analysed using Veriseq and NICS (Yikon). RESULTS: Genetic results were obtained for 96.8% of trophectoderm samples versus 92.4% for both SCM techniques. The mosaicism rate was higher for SCM regardless of the technique used: 30.4% for SCM-NICS and 28.3% for SCM-Veriseq versus 14.1% for trophectoderm biopsies (P = 0.013, P = 0.031, respectively). No significant differences in diagnostic concordance were seen between the two SCM techniques (74.6% for SCM-NICS versus 72.3% for SCM-Veriseq; P = 0.861). For embryos biopsied on day 6, these rates reached 92.0% and 86.5%, respectively. On reanalysing trophectoderm-aneuploid embryos, the discrepancies were shown to be due to maternal DNA contamination (55.6%; 5/9), embryo mosaicism (22.2%; 2/9) and low resolution in SCM-NICS (11.1%; 1/9) and in both SCM techniques (11.1%; 1/9). CONCLUSIONS: This is the first study evaluating the consistency of different chromosomal analysis techniques for niPGT-A. In conclusion, the diagnostic concordance between PGT-A and niPGT-A seems independent of the technique used. Optimization of culture conditions and medium retrieval provides a potential target to improve the reliability of niPGT-A.

Effect of ovarian stimulation on embryo aneuploidy and mosaicism rate.

There is a high incidence of chromosome abnormalities in human embryos that leads to a failed IVF cycle. Different studies have shown that maternal age is the determining factor in the appearance of chromosomal alterations in the embryo. However, the possible influence of ovarian stimulation on oocyte and embryo aneuploidies and mosaicism is controversial. A retrospective study was carried out in which 835 embryos from 280 couples undergoing reproductive treatment using their oocytes were chromosomally analyzed. A binary logistic regression analysis was performed to evaluate the relationship between different parameters characterizing controlled ovarian stimulation (COS) and the rate of aneuploidy and embryonic mosaicism. The embryo aneuploidy rate showed no association with the use of oral contraceptives, type, total and daily doses of gonadotropins, stimulation protocol type, and drugs used for ovulation trigger (p > 0.05). In contrast, the duration of the ovarian stimulation treatment was correlated with the aneuploidy rate: patients requiring more days of stimulation presented a lower rate of aneuploid embryos (p = 0.015). None of the variables studied showed any association with the rate of embryo mosaicism. However, the duration of COS showed association with the appearance of aneuploidy, suggesting that faster recruitment could be deleterious for those reassuming meiosis, yielding more abnormal karyotype.Abbreviations: IVF: in vitro fertilization; COS: controlled ovarian stimulation; PGT-A: preimplantation genetic test for aneuploidy; hCG: human chorionic gonadotropin; GnRH: gonadotropin-releasing hormone; LH: luteinizing hormone; FSH: follicle-stimulating hormone; NGS: next-generation sequencing; a-CGH: comparative genomic hybridization; TUNEL: Terminal transferase dUTP Nick End Labeling; FISH: fluorescent in situ hybridization.

Luteal phase stimulation versus follicular phase stimulation in poor ovarian responders: results of a randomized controlled trial.

BACKGROUND: In young women with poor ovarian response, luteal-phase ovarian stimulation (LPOS) is a potential method for collecting competent oocytes. The aim of this study was to assess the efficacy of LPOS compared with follicular phase ovarian stimulation (FPOS) in young women with poor ovarian response (POR). METHODS: This single-center, prospective, randomized pilot study compared LPOS and FPOS in women with POR fulfilling Bologna criteria who underwent in vitro fertilization at the Instituto Bernabeu. The primary outcome was the number of metaphase II (MII) oocytes obtained by follicular puncture. RESULTS: Sixty women were included in the study, with 27 women completing LPOS and 30 undergoing FPOS. There was no statistically significant difference in the number of MII oocytes obtained between the LPOS group and the FPOS group (2.1 ± 2.0 vs. 2.6 ± 2.2, p = 0.31). Length of stimulation was also similar in both groups (8.35 ± 2.8 vs. 8.15 ± 4.1 days, p = 0.69). Similarly, there was no significant difference in the follicle-stimulating hormone total dose, number of cumulus-oocyte complexes, survival rate, fertilization rate, or cancellation rate between groups. A significantly higher Ovarian Sensitivity Index was observed in the LPOS group versus the FPOS group (0.96 vs. 0.57, p = 0.037). CONCLUSION: LPOS was comparable with FPOS in terms of efficacy and may improve ovarian responsiveness in young women with POR. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02625532; EudraCT identifier: 2015-003856-31.

Prevalence of candidate single nucleotide polymorphisms on p53, IL-11, IL-10, VEGF and APOE in patients with repeated implantation failure (RIF) and pregnancy loss (RPL).

Recurrent pregnancy loss (RPL; defined as the loss of three or more consecutive pregnancies) and recurrent implantation failure (RIF; when implantation is not achieved after at least three cycles of IVF) are two of the major challenges that reproductive medicine faces. Some polymorphisms have been identified as possible causes of an increased risk of these diseases. This paper studies the prevalence of the polymorphisms in p53, VEGF, IL-10, IL-11 and APOE in RIF and RPL patients that determines the risk for these pathologies. A total of 255 patients were selected (89 RPL patients, 77 RIF patients and 89 controls) and genotyped for p53-R72P; IL-11-1082-AG; VEGF-1154-AG; IL-10; APOE-R112C; APOE-R158C. Statistically significant differences were found in the prevalence of the E4 isoform (R122-R158) of the APOE gene in RPL patients (p < 0.05), and in RIF patients, the R72P polymorphism of the p53 gene and the 1154-AG of the VEGF gene showed different distribution (p < 0.05). Regarding the p53 and IL-11 studied polymorphisms, PP of p53 gene and GG of IL-11 are more prevalent in RPL patients without reaching statistical significance. In conclusion, our results suggest patients carrying variants in p53 and VEGF would be at risk of RIF, and those carrying variants in APOE gene would suffer RPL.

The relevance of the individual screening for genetic variants in predicting ovarian response.

OBJECTIVE: To investigate if polymorphisms of some genes involved in folliculogenesis predict ovarian response. METHODS: This prospective randomized study includes 124 egg donors genotyped for six SNPs ESR1 (rs2234693), AMHR2 (rs2002555), GDF-9 (rs10491279 and rs254286), AMH (rs10407022) and LHCBR (rs229327) genes and four STRs in ESR1 rs3138774), SHBG (rs6761), CYP19A1 (rs60271534) and AR genes (CAG repeats in exon 1). All donors followed standard ovarian stimulation protocol using a daily dose of 225 UI. The genotypes obtained were compared with the ovarian stimulation outcome. RESULTS: Regarding the number of retrieved oocytes, we found statistical differences for the ESR1 SNP and STR (19.3 ± 8.9 for TT vs 15.3 ± 6.2 for CC/CT, P = 0.027; 19.1 ± 8.3 for <17repeats vs 14.7 ± 6.2 for >17repeats, P = 0.020). Moreover, women carrying TT in the ESR1 at position c.-397T>C with ESR1 (TA)n=17 retrieved the highest number of oocytes (20.4 ± 9.3) (P = 0.001). Concerning AMHR2, we observed an association with the length of stimulation (9.1 ± 1.4 d for AA vs 9.7 ± 1.3 d for AG/GG, P = 0.021) and gonadotropin received (2050 ± 319 for AA vs 2188 ± 299 for AG/GG, P = 0.017). No significant differences were observed for the other polymorphisms (P > 0.05). CONCLUSION: The polymorphisms in ESR1 and AMHR2 genes showed a clear association with the number of retrieved oocytes and the stimulation data, respectively. Our results suggest that polymorphisms in the genes for key reproductive hormones receptors could be used to predict the ovarian response and to personalize the stimulation prior the treatment.