Low Implantation Potential Research Articles

O-040 Clinical competence of oocytes with developmental abnormalities

Abstract For decades we have looked for marker of embryo competence. And while we have come a long way in terms of identifying markers related to embryonic competence, a lot of research is still focused on finding this simple measurable indicator predicting a positive outcome of the individual treatment. Many potential indicators have been evaluated. This includes the cumulus oocyte complex, zona pellucida, perivitelline space, polar bodies, vacuoles, refractile bodies, SER clusters, cytoplasmic granularity, and many others. For some of these there are data suggesting potential prediction of negative outcome (for review see Bartolacci et al. 2022). Embryo autonomy may result in cleavage and development despite the lack of competence to establish a pregnancy. And pregnancies may occur without the competence to go all the way to a live birth. In a study by Lundin et al. we looked into chromosomal constitution after rescuing immature (developmentally abnormal) human oocytes. And while 15% of the oocytes developed into an embryo only very few appeared chromosomally normal after cytogenetic analysis. The study concluded that the majority of hCG exposed oocytes that have not reached the MII stage at time of pick-up are defect and consequently that GV or MI oocytes should not be used in ART programs. As reproductive specialists our main task is to assist our patients in achieving their dream of having a child. We are supposed to use all our combined expertise, knowhow, craftmanship, routine and experience to optimize the chance for the patients to have a baby. However, in our ambition to help our patients we sometimes lose touch of the underlying biology of human reproduction. Oocytes from a stimulated ovary are not like those we buy in the supermarket of equal quality. The depressing fact is that only 5.0 % of the oocytes in a first cycle is biologically competent and in around 2/3 of all cycles, none of the oocytes have the potential to result in the birth of a child (Lemmen et al 2016). We also have very limited ability to determine competence of individual sperm cells – the strongest prognosticator being if they look and move like a “normal” sperm sample. Are we getting a bit desperate when we start to look for normality in the abnormal as suggested in the title of this talk? Have we reached a point were patient expectations overrides our biological training and education resulting in low willingness to accept a negative outcome dictated by our biology? And is this reinforced by patient perceptions like “we did not pay a lot of money for the oocytes not being fertilized/developed/implanted”. Embryo competence evaluation is based on markers of what have happened rather than indicators of what will happen. At the end of the day, we as health care professionals as well as our patients needs to accept the fact that there is no such thing as “CLINICAL COMPETENCE OF OOCYTES WITH DEVELOPMENTAL ABNORMALITIES”. And what will be the consequence of a marker indicating a lower implantation potential in an otherwise ok embryo? It might affect the ranking of the embryos from a particular cycle, but we would be willing transfer it anyway if needed. Embryo evaluation don’t increase pregnancy chance per se. But it may decrease time to pregnancy by improved selection between the embryos available. Maybe it is time to re-focus our efforts and move from a binary normality approach identifying “developmental abnormalities” toward a more biological oriented approach mapping “developmental variation” in embryos and their impact on pregnancy potential and thus recognizing the multi-factorial nature of embryo maturation and ongoing developing competence.

O-062 MicroRNAs in Blastocoel Fluid: a molecular signature for predicting human embryo implantation potential

Abstract Study question Can microRNA expression in blastocoel fluid (BF) predict embryo implantation potential? Summary answer Up-regulation of miR-106, miR-373, miR-301, miR-320 and miR-525-3p represents the molecular signature characterizing the embryos with high implantation capability. What is known already The discovery of DNA within the BF and in spent embryo culture media, has caused increased interest in the non-invasive preimplantation testing application for monogenic disorders and aneuploidies. Aneuploid embryos have a lower implantation potential, however, chromosomal abnormalities represent only a tiny percentage of the causes of implantation failure. MiRNAs can control all cellular pathways and are involved in pluripotency, self-renewal, and stemness, and their altered regulation affects different human diseases. Study design, size, duration From September 2018 to March 2022, 112 BFsampleswere collected from human embryos on the fifth day of development, before the blastocyst cryopreservation. The samples were classified according to blastocyst grade and the data on implantation outcome and term births were collected for the transferred embryos. We compared the expression profiles of 89 miRNAs, previously identified in BF, between 33 BF from implanted embryos and 30 from non-implanted ones, regardless of blastocyst grade. Participants/materials, setting, methods By custom-designed TaqMan Low-Density Array card (TLDA), we analyzed the expression of 89 miRNAs in 4 different BF samples simultaneously. Differentially expressed (DE) miRNAs were identified by Volcano plot and Significance Analysis of Microarrays (SAM) tests. Bioinformatic analysis was performed to identify the biological role of the DE miRNAs. To evaluate miRNA’s ability for predicting implantation, Pearson’s correlation analyses, classical univariate Receiver Operator Characteristic (ROC) curve analysis, and the optimal cut-off value determination were performed. Main results and the role of chance We found five miRNAs, miR-106, miR-373, miR-301, miR-320 and miR-525-3pup-regulated in BF from implanted blastocysts. The identified miRNAs perform an important role during the first phases of embryo development suggesting that their up-regulation may reflect embryo health. Moreover, four of the five miRNAs showed significant correlation coefficients in both implanted and non-implanted blastocysts, indicating that their expression changes in the same way in the single sample and reflects the potentiality of the embryo to implant. Finally, ROC curve analysis confirmed that our miRNAs could be considered potential biomarkers for implantation. Limitations, reasons for caution Successful implantation requires a close dialogue between the embryo and the endometrium, mediated by different proteins and miRNAs produced by both the embryo and maternal tissues. Determining the quality of the embryo and its implantation potential is not sufficient to predict successful pregnancy outcomes. Wider implications of the findings This study represents the first report correlating miRNA profiles in BFand implantation and suggests that miRNA signature could become an accurate tool to evaluate embryo quality. It could be associated or replaced with the PGT-A to choose the most competent embryo and improve the outcome of assisted reproduction cycles. Trial registration number The study has been approved by theEthical Committee of Azienda Ospedaliero Universitaria Policlinico "G.Rodolico -San Marco" Catania.

Recent clomiphene citrate exposure does not impact subsequent clinical outcomes in single euploid frozen embryo transfer cycles

Do infertile couples who recently utilized clomiphene citrate (CC) for ovulation induction or ovarian stimulation (<90 days previously) followed by a single euploid embryo transfer (SEET) have lower implantation potential compared with patients who were not exposed to CC within 90 days before embryo transfer (ET)? There does not appear to be an association between recent CC exposure and lower implantation potential in patients who undergo a frozen embryo transfer (FET) of euploid embryos. Clomiphene has been found to be associated with lower pregnancy rates when compared against other ovarian stimulation medications. The majority of published research about the effects of CC on implantation potential suggest an anti-estrogenic effect on the endometrium. Quality evidence and information about utilization of CC and its effect on implantation potential after euploid ETs is lacking in the literature. A retrospective cohort study with propensity score matching was carried out. We included all patients that underwent an autologous SEET from September 2016 to September 2022 at a single academic-private ART center. The study group included patients that had utilized CC during either ovulation induction cycles and/or controlled ovarian stimulation at least 90 days before FET. A propensity score-matched control group of patients that were unexposed to CC within 90 days prior to SEET was used for comparisons. The primary outcome was positive pregnancy test (defined as a positive serum β-hCG measured 9 days after ET), with other outcomes including clinical pregnancy, ongoing pregnancy, biochemical pregnancy loss, and clinical pregnancy loss rates per SEET. Multivariate regression analyses fitted with generalized estimating equations were utilized to analyze if there was an association between CC utilization and IVF outcomes. Furthermore, the study evaluated the cumulative effect of CC and endometrial receptivity in vivo and subsequent IVF outcomes. A total of 593 patients with utilization of CC in <90 days before ET were compared with 1779 matched controls. Positive pregnancy test rates were comparable among the control group and the CC exposed groups, respectively (74.3% versus 75.7%, P = 0.79), as were clinical pregnancy (64.0% versus 65.0%, P = 0.60), ongoing pregnancy (51.8% versus 53.2%, P = 0.74), biochemical pregnancy loss (15.7% versus 14.03%, P = 0.45), and clinical pregnancy loss rates were also comparable among cohorts (17.1% versus 18.1%, P = 0.71). No association was found between utilization of clomiphene and lower implantation rates (adjusted odds ratio 0.95, 95% CI 0.76-1.18). Also, no differences were observed in sub-analyses based on multiple CC utilization periods. Finally, no association was found between the number of consecutive cumulative clomiphene cycles and sub-optimal IVF outcomes. The study has inherent bias that originated from its retrospective design. Serum levels of CC were not measured and sample size for the sub-analyses was small. There does not appear to be an association between recent CC exposure and lower implantation potential in patients who undergo a FET of euploid embryos. This finding remains consistent, even in patients who undergo multiple, consecutive clomiphene cycles prior to ET. There were no long-term effects of CC on endometrial development and clinical characteristics examined in this study. Patients that utilized CC medication prior to a SEET cycle for either ovarian stimulation or ovulation induction, can be assured that there is no evidence of a residual effect of recent CC administration that could jeopardize their pregnancy probability. No funding was received for the realization of this study. A.C. is advisor and/or board member of Sema4 (stakeholder in data) and Progyny. The other authors have no conflicts of interest to declare. N/A.

P-133 The best of the worst: analyze of outcome in artificial reproductive technologies (ART) fresh cycles with transfers of slow-growing embryos and expanded poor-quality blastocysts

Abstract Study question Fresh transfer of slow-growing embryos versus expanded poor-quality blastocysts, in case of absence of good-quality blastocysts on day 5: which is the optimal approach? Summary answer The clinical pregnancy rate (CPR), live birth rate (LBR) are significantly higher during expanded poor-quality blastocysts transfers. There’s no statistical difference in miscarriage rate (MR). What is known already Several studies show contradictory results about which blastocyst morphological parameter best predicts pregnancy outcomes in ART cycle. However, in clinical practice embryologists encounter in ART protocols with scheduled fresh embryo transfer the absence of expanded good-quality blastocysts on day 5. In this case, slow-growing embryos or poor-quality blastocysts could be chosen for transfer. Day 5 slow-growing embryos have low implantation potential in fresh ART cycles, it could be due to embryo-endometrial asynchrony. Also, the quality of further blastocyst remains unclear. Poor-quality blastocysts have a high aneuploidy rate and lower viability compared to good-quality blastocysts. Study design, size, duration This study included ART cycles with fresh embryo transfer on day 5, with selected group of patients under 40 years of age, between 2014 and 2019. Group A consisted of 619 cycles with slow-growing embryos transfer; group B included 88 cycles with expanded poor-quality blastocysts transfer; the control group counted 1501 cycles with a transfer of good-quality blastocysts. Group A had an average of 1,5 embryos transferred per patient, control group and group B 1,3. Participants/materials, setting, methods Embryos were cultured until day 5 and scored according to Gardner and Schoolcraft’s grading system. In group A, slow-growing embryos were transferred at the stage of morula and early blastocyst with a degree of expansion 1. In group B, expanded poor-quality blastocysts were scored as 3-6CB, 3-6BC, 3-6CC. In control group expanded good-quality blastocysts were scored as 3-6AA, 3-6AB, 3-6BA, 3-6BB.The cycles with donor gametes, surrogacy, and preimplantation genetic testing were excluded. Main results and the role of chance The main women’s age of group A, B and control group was 32,2 ± 4,2, 31,5 ± 4,7 and 32,6 ± 4,6 years respectively. The CPR, the ongoing pregnancy rate (OPR), the LBR and the implantation rate (IR) were significantly increased in the group B compared to the group A [34,0% (30/88) vs 17,7% (110/619); P &amp;lt; 0,01], [21,6% (19/88) vs 11,9% (74/619); P = 0,012], [19,3% (17/88) vs 10,5% (65/619); P = 0,015] and [26,2% (31/118) vs 12,6% (119/946); P &amp;lt; 0,01] respectively. There’s no statistical difference in the MR between group A and B [35,2% (42/119) vs 35,5% (11/31); P = 0,946]. The CPR, the OPR, the LBR and the IR were significantly higher in the control group compared to the group A and B [50,5% (758/1501); P &amp;lt; 0,01], [41,6% (625/1501); P &amp;lt; 0,001], [40,3% (605/1501); P &amp;lt; 0,001] and [40,45% (849/2099); P &amp;lt; 0,01]. The MR in the control group was almost two times lower compared to the group A and B [16,2% (138/849); P &amp;lt; 0,01). Relationships between variables were assessed by Pearson's chi-squared test. Limitations, reasons for caution The study is limited due to the uneven distribution of patients in three groups and by a low number of participants in group B. The grading of blastocysts’ quality is also subjected to a human factor. Wider implications of the findings If in ART protocol with scheduled fresh embryo transfer have no expanded good-quality blastocysts, expanded poor-quality blastocysts could be chosen for transfer and slow-growing embryos should be cultured until day 6 with subsequent cryopreservation of blastocysts. Patients should be informed about elevated MR after the transfer of poor-quality blastocysts. Trial registration number Not applicable

P-237 Non-invasive metabolic live cell imaging of early embryo development using adapted confocal microscopy; a safety study

Abstract Study question Is it safe to use metabolic imaging to measure nicotinamide adenine dinucleotide (NADH) associated auto-fluorescence during embryo development using adapted confocal microscopy? Summary answer Non-invasive metabolic imaging is safe as no differences were observed between controls and illuminated embryos in terms of embryo development, blastocyst formation and implantation potential. What is known already Developing non-invasive methods that are reliable to assess oocyte and embryo quality has been a significant aim for assisted reproductive technologies. Changes in metabolic activity could lead to cell death or abnormal embryo development and low implantation potential. This could potentially be predicted by incorporating non-invasive measurements of metabolism. Metabolic imaging in embryos has been investigated through complex methodologies, however, scientific evidence for its utility during embryo development using simple technology remains unexplored. Measurements of metabolic activity could be a useful tool as the auto-fluorescence of molecules such as NADH is a straightforward representation of mitochondrial function. Study design, size, duration Super-ovulated female mice (n = 30) were subjected to mating with 10 males. In-vivo produced embryos collected at the 2-cell stage were divided in control group (n = 151), sham control group (n = 151) and illuminated group (n = 152). Illuminated embryos were assessed for NADH levels during embryo development every 3 hours using arbitrary units of autofluorescence (AU). Produced blastocysts were assessed for total cell and inner-cell-mass (ICM) number (Oct4 immuno-staining) and implantation potential through outgrowth assays in separate experiments. Participants/materials, setting, methods F1 (CBA/C57Bl6) mouse strain was used. NADH auto-fluorescence levels were measured during embryo development using adapted confocal microcopy (Olympus FV1200). A confocal Z-stacking function was used to record 15 focal planes using a 20x/0.95NA air objective of entire embryos, opening the confocal pinhole system completely. Then, images were collected and analysed using FIJI software (version: 2.0.0-rc-69/1.52n;ImageJ). Blastocyst cell number, formation rates and outgrowth rates for 4 days post blastocyst formation were compared between study groups. Main results and the role of chance Embryo culture experiments showed no significant differences in blastocyst formation rates between study groups (Control: 71.7%; Sham: 64.9%; Illuminated 71.7%; p &amp;gt; 0.05). Similarly, the total number of cells (Control: 82.9±5.6; Sham: 76.5±3.3; Illuminated: 77.1±4.2; ± Standard error of mean [SEM]) and ICM cells (Control: 10.8±1.3; Sham: 9.4±0.7; Illuminated: 11.9±0.8; ± SEM) did not differ between groups (p &amp;gt; 0.05). Outgrowth assays presented similar outgrowth areas during day5 to day8 post-blastocyst development between study groups (p &amp;gt; 0.05). Illuminated embryos presented significantly different NADH activity levels during embryo development, particularly between the 2-cell stage (987.1±36.2AU), morulae stage (1226±31.5AU) and blastocyst stage (649±42.9AU; ± SEM; p &amp;lt; 0.05). Embryos that did not reach the blastocyst stage presented a significantly different NADH activity profile during embryo development compared to those that did(p &amp;lt; 0.05). Additionally, abnormal embryos also presented significantly decreased NADH activity levels at the 2-cell stage (Normal: 987.1±36.2; abnormal: 726.9±121.7AU; p &amp;lt; 0.05) to the morulae stage (Normal: 1226±31.5; Abnormal:893.3±189AU; p &amp;lt; 0.05). Our study indicates that measuring NADH activity levels during early embryo development present no negative effects in embryo developmental rates, blastocyst formation and implantation potential. Thus, non-invasive measurements of NADH could be applied to determine embryo metabolic activity during embryo development using simple technology and imaging techniques. Limitations, reasons for caution The study was conducted using a mouse model focused in early embryo development and implantation potential. Thus, studies on live birth are required to fully assess safety to further validate potential wider applications. Validation in ageing models is also required to assess potential applications for embryo selection. Wider implications of the findings Non-invasive measurements of metabolic activity could be applied to determine embryo metabolic activity using simple and safe technology. Further applications could link the use of simple non-invasive metabolic imaging with the latest time-lapse technology and artificial intelligence applications. Trial registration number N/A

Higher preconceptional maternal body mass index is associated with faster early preimplantation embryonic development: the Rotterdam periconception cohort

BackgroundOverweight and obesity affect millions of people globally, which has also serious implications for reproduction. For example, treatment outcomes after in vitro fertilisation (IVF) are worse in women with a high body mass index (BMI). However, the impact of maternal BMI on embryo quality is inconclusive. Our main aim is to study associations between preconceptional maternal BMI and morphokinetic parameters of preimplantation embryos and predicted implantation potential. In addition, associations with clinical IVF outcomes are investigated.MethodsFrom a tertiary hospital, 268 women undergoing IVF or IVF with intracytoplasmic sperm injection (ICSI) were included; 143 normal weight, 79 overweight and 46 obese women. The embryos of these women were cultured in the EmbryoScope, a time-lapse incubator. The morphokinetic parameters of preimplantation embryos and predicted implantation potential, assessed by the KIDScore algorithm were longitudinally evaluated as primary and secondary outcomes, respectively. The tertiary outcomes included clinical outcomes, i.e., fertilization, implantation and live birth rate.ResultsAfter adjustment for patient- and treatment-related factors, we demonstrated in 938 embryos that maternal BMI is negatively associated with the moment of pronuclear appearance (βtPNa -0.070 h (95%CI -0.139, -0.001), p = 0.048), pronuclear fading (βtPNf -0.091 h (95%CI -0.180, -0.003), p = 0.043 and the first cell cleavage (βt2 -0.111 h (95%CI -0.205, -0.016), p = 0.022). Maternal BMI was not significantly associated with the KIDScore and tertiary clinical treatment outcomes. In embryos from couples with female or combined factor subfertility, the impact of maternal BMI was even larger (βtPNf -0.170 h (95%CI -0.293, -0.047), p = 0.007; βt2 -0.199 h (95%CI -0.330, -0.067), p = 0.003). Additionally, a detrimental impact of BMI per point increase was observed on the KIDScore (β -0.073 (se 0.028), p = 0.010).ConclusionsHigher maternal BMI is associated with faster early preimplantation development. In couples with female or combined factor subfertility, a higher BMI is associated with a lower implantation potential as predicted by the KIDScore. Likely due to power issues, we did not observe an impact on clinical treatment outcomes. However, an effect of faster preimplantation development on post-implantation development is conceivable, especially since the impact of maternal BMI on pregnancy outcomes has been widely demonstrated.

Mitochondrial DNA Copy Number in Human Blastocyst: A Novel Biomarker for the Prediction of Implantation Potential

The relationship between mitochondrial DNA (mtDNA) copy number and the outcome of embryo transfer is under debate. Our aim was to explore the relationship between mtDNA copy number in human blastocysts and embryonic development to determine whether mtDNA represents a novel biomarker for the prediction of implantation potential. A total of 246 blastocysts were analyzed by next-generation sequencing. There was no correlation between mtDNA copy number and maternal age in all blastocyst groups and euploid blastocyst groups. Additionally, the mtDNA copy number was not significantly higher in aneuploid blastocysts. Subsequently, no relationship was observed between mtDNA copy number and blastocyst quality. The assessment of clinical pregnancy outcome after the transfer of euploid blastocysts to the uterus indicated that the mtDNA copy number was significantly lower in the clinical pregnancy group than in those who failed implantation. The cut-off value of mtDNA copy number was 320.5, which was a highly predictive value. Blastocysts with an increased mtDNA copy number had lower implantation potential, and mtDNA copy number was largely equal in terms of maternal age, chromosome ploidy, and quality of blastocysts.

AMH has no role in predicting oocyte quality in women with advanced age undergoing IVF/ICSI cycles

It has been widely acknowledged that anti-Müllerian hormone (AMH) is a golden marker of ovarian reserve. Declined ovarian reserve (DOR), based on experience from reproductive-aged women, refers to both the quantitative and qualitative reduction in oocytes. This view is challenged by a recent study clearly showing that the quality of oocytes is similar in young women undergoing IVF cycles irrespective of the level of AMH. However, it remains elusive whether AMH indicates oocyte quality in women with advanced age (WAA). The aim of this study was to investigate this issue. In the present study, we retrospectively analysed the data generated from a total of 492 IVF/ICSI cycles (from January 2017 to July 2020), and these IVF/ICSI cycles contributed 292 embryo transfer (ET) cycles (from June 2017 to September 2019, data of day 3 ET were included for analysis) in our reproductive centre. Based on the level of AMH, all patients (= > 37 years old) were divided into 2 groups: the AMH high (H) group and the AMH low (L) group. The parameters of in vitro embryo development and clinical outcomes were compared between the two groups. The results showed that women in the L group experienced severe DOR, as demonstrated by a higher rate of primary diagnosis of DOR, lower antral follicle count (AFC), higher level of basal follicle stimulating hormone (FSH) and cancelation cycles, lower level of E2 production on the day of surge, and fewer oocytes and MII oocytes retrieved. Compared with women in the H group, women in the L group showed slightly reduced top embryo formation rate but a similar normal fertilization rate and blastocyst formation rate. More importantly, we found that the rates of implantation, spontaneous miscarriage and livebirth were similar between the two groups, while the pregnancy rate was significantly reduced in the L group compared with the H group. Further analysis indicated that the higher pregnancy rate of women in the H group may be due to more top embryos transferred per cycle. Due to an extremely low implantation potential for transfer of non-top embryos from WAA (= > 37 years old) in our reproductive centre, we assumed that all the embryos that implanted may result from the transfer of top embryos. Based on this observation, we found that the ratio of embryos that successfully implanted or eventually led to a livebirth to top embryos transferred was similar between the H and the L groups. Furthermore, women with clinical pregnancy or livebirth in the H or L group did not show a higher level of serum AMH but were younger than women with non-pregnancy or non-livebirth. Taken together, this study showed that AMH had a limited role in predicting in vitro embryo developmental potential and had no role in predicting the in vivo embryo developmental potential, suggesting that in WAA, AMH should not be used as a marker of oocyte quality. This study supports the view that the accumulation of top embryos via multiple oocyte retrieval times is a good strategy for the treatment of WAA.

Blastocyst collapse as an embryo marker of low implantation potential: a time-lapse multicentre study.

Spontaneous blastocyst collapse during in vitro embryo development has been suggested as a novel marker of embryo quality. Therefore, the aim of this multicentre study was to carry out a retrospective multicentre analysis to investigate the correlation between blastocyst collapse and pregnancy outcome. Here, 1297 intracytoplasmic sperm injection (ICSI)/in vitro fertilization (IVF) fresh cycles, with an elective single blastocyst transfer (eSET) were included in this study. Embryos were cultured individually in 6.0% CO2, 5.0% O2, 89.0% N2, using single step medium (GTLTM VitroLife, Sweden) or sequential medium (CookTM, Cook Medical, Australia) and selected for transfer using standard morphological criteria. With the use of time-lapse monitoring (TLM), blastocysts were analyzed by measuring the maximum volume reduction and defined as having collapsed, if there was ≥ 50% volume reduction from the expanded blastocyst and the collapse event. Following embryo replacement, each blastocyst was retrospectively allocated to one of two groups (collapsed or not collapsed). Here, 259 blastocysts collapsed once or more during development (19.9%) and the remaining 1038 either contracted minimally or not collapsed (80.1%). A significantly higher ongoing pregnancy rate (OPR) of 51.9% (95% CI 48.9-59.9%) was observed when blastocysts that had not collapsed were replaced compared with cycles in which collapsed blastocysts were transferred 37.5% (95% CI 31.6-43.4%). This study suggests that human blastocysts that collapse spontaneously during development are less likely to implant and generate a pregnancy compared with embryos that do not. Although this is a retrospective study, the results demonstrated the utility of collapse episodes as new marker of embryo selection following eSET at blastocyst stage.

12. EMBRYOS FROM THE SAME COHORT SHOW HIGH VARIABILITY IN THE MTDNA LEVELS

Introduction Mitochondria play a vital role in cell function. Recently, there has been an increasing research on mtDNA as biomarker of embryo implantation. Reports showed that high levels of mtDNA in blastocyst are associated with low implantation potential. Based on the available evidence, it is unclear whether high levels of mtDNA is a constant feature in some group of women or it depends on each embryo, showing variability among sibling embryos from the same cohort. The aim of this study was to investigate the variability in the levels of mtDNA among sibling euploid embryos from the same cohort. Material and Methods A prospective non-selection study was performed from January 2018 to December 2018. We included 249 blastocysts biopsies from 89 couples who attended our clinic for PGT-A with oocyte donation and with at least two of their biopsied embryos diagnosed as euploid non-mosaic. The aneuploid testing was performed by NGS-Veriseq® (Illumina). The mtDNA analysis was performed once the embryo diagnosis was known using MitoSeek. Sequencing reads mapping to the mtDNA genome were extracted from bam files. The relative measure of mtDNA copy number was calculated by dividing the mtDNA reads by the nuclearDNA and subjected to mathematical correction factor according to the embryo genome. Given that the mtDNA content is dependent upon the number of cell division that preceded the biopsy, the mtDNA content was also corrected by a factor according to the day when the embryos were biopsied. Statistical analysis was performed using SPSSv20.0. Results In order to avoid confusion related to the oocyte quality we included only egg donation cycles, therefore the average maternal age at time of oocyte retrieval was 26± 4 years (range: 19.0–33.0). Overall, the mean average of mtDNA per genome was 0.0010±0.0008 (range: 0.0002-0.0039). We analysed the mtDNA within an embryo cohort. To evaluate the extent of the variability in the mtDNA among embryos from the same cohort we calculate the coefficient of variation, being the average 28.77%. Furthermore, each embryo was classified according to the mtDNA 10th-percentile which belongs. The mean difference in the 10th-percentile between the embryo with the higher and the lower mtDNA within a cohort was 30. This difference was not related with the age of the egg donor neither the male factor (p>0.05). Only 13.5% of patients showed no difference in the mtDNA content within the embryos from the same cohort. Conclusion Our results suggest high variability in the mtDNA content within euploid embryos from the same cohort. MtDNA could be used to predict reproductive potential amongst chromosomally normal embryos however the utility of quantifying mtDNA levels depend on the existence of variability among embryos from the same cohort. This study reports that mtDNA content is not a marker of prognosis amongst patients because it is variable among the embryos from the same cohort, thus mtDNA could be used as an additional marker of embryo competence and a tool for prioritize the euploid embryo to be transferred.

Mosaic human embryos – our experiences

Introduction Embryonic mosaicism is described as a presence of 2 or more distinct cell lines in embryo. This phenomenon is common and more prevalent in human preimplantation embryos than in later prenatal samples, affecting 15-90% of cleavage stage embryos and 30-40% of blastocyst stage embryos. Mosaic embryos have lower implantation potential than euploid. Material and Methods 98 infertile couples from 4 IVF centres from Czech Republic had undergone PGT for aneuploidy and structural imbalances of chromosomes. Preimplantation genetic testing was performed on NGS platform MiSeq using VeriSeq PGS Kit (Illumina) and analyzed on BlueFuse Multi software (Illumina). Scoring of aneuploidy and mosacism was done according to manufacturer's instructions. Results From August 2016 to December of 2017 the 387 trophectoderm and blastomere samples from 98 couples were tested for aneuploidies and rearrangements of chromosomes. Results were obtained from 95.3% of samples (369/387). No imbalanceswere found in 26.9% of samples (104/387), conditioned embryotransferwas recommended in 7.2% of samples (28/387), because analysed couple´s embryos were mosaic or aneuploid. The mosaicism was detected in 32.8% of samples in the study (127/387). Mosaicism of 1 chromosome was found only in 5.7% of samples (22/387). In 17.1% of samples (66/387) mosaicism more than 1 chromosome was found. Subchromosomal changes of chromosomes in mosaic were detected in 10.1% of samples (39/387). PGT of structural rearrangements was performed in 17.6% of samples (68/387) from 14 couples. 17.6% of samples (12/68) had balanced karyotype. In the study only 2 couples decided to transfer mosaic embryos after genetic counselling and in both cases positive biochemical pregnancies were recorded. Conclusion PGT by NGS was performed for diagnosis of aneuploidies and structural abnormalities in early human embryos. In 28 cases only mosaic embryos could be transferred. Only 2 couples decided to transfer mosaic embryo. For many couples, the decision to transfer mosaic embryo is complicated by fear about the baby's health, because the presence and significance of mosaicism in early human embryos is still poorly understood and interpretation of mosaic result is difficult with respect to which chromosome or chromosomes are involved in mosaic and with respect to accuracy of testing.

Comprehensive mitochondrial DNA analysis and IVF outcome.

STUDY QUESTIONDo mitochondrial DNA (mtDNA) copy number and heteroplasmy in human embryos affect the ongoing pregnancy rate?SUMMARY ANSWEROur study suggests that mtDNA copy number above a specific threshold is associated with the ongoing pregnancy rate.WHAT IS KNOWN ALREADYMitochondria play a vital role in cell function. Recently, there has been increasing research on mtDNA as a biomarker of embryo implantation. Although reports showed that high levels of mtDNA in the blastocyst are associated with low implantation potential, other publications were unable to confirm this. Confounding factors may influence the mtDNA copy number in euploid embryos. On the other hand it has been speculated that both mtDNA heteroplasmy and copy number contribute to mitochondrial function. Next generation sequencing (NGS) allows us to study in depth mtDNA heteroplasmy and copy number simultaneously.STUDY DESIGN, SIZE, DURATIONA prospective non-selection study was performed. We included 159 blastocyst biopsies from 142 couples who attended our clinic for preimplantation genetic testing for aneuploidies (PGT-A), from January 2017 to December 2017. All embryos were biopsied on Day 5 or Day 6. The aneuploid testing was performed by NGS. All blastocysts were diagnosed as euploid non-mosaic and were transferred. The mtDNA analysis was performed once the embryo diagnosis was known.PARTICIPANTS/MATERIALS, SETTING, METHODSSequencing reads mapping to the mtDNA genome were extracted from indexed bam files to identify copy number and heteroplasmy. The relative measure of mtDNA copy number was calculated by dividing the mtDNA reads by the nuclear DNA value to normalize for technical variants and the number of cells collected at the biopsy. All the results were subjected to a mathematical correction factor according to the embryo genome. Heteroplasmy was assigned by MitoSeek.MAIN RESULTS AND THE ROLE OF CHANCEThe mean average copy number and SD of mtDNA per genome was 0.0016 ± 0.0012. Regarding heteroplasmy, 40 embryos were heteroplasmy carriers (26.32%). MtDNA variants were detected in coding and non-coding regions and the highest number of variants in an embryo was eight. With respect to IVF outcome for mtDNA copy number analysis, we set a threshold of 0.003 for the following analysis. The vast majority of the embryos were below the threshold (142/159, 89.31%) and 17 embryos were classified as having higher mtDNA levels. We showed a reduction in ongoing pregnancy rate associated with elevated mtDNA copy number (42.96% versus 17.65%, P < 0.05). This result was independent of maternal age and day of the biopsy: these factors were included as confounding factors because mtDNA copy number was negatively correlated with female age (25 –30 y: 0.0017 ± 0.0011, 30 –35 y: 0.0012 ± 0.0007, 35 –40 y: 0.0016 ± 0.0009, over 40 y: 0.0024 + 0.0017, P < 0.05). Embryos biopsied on Day 5 were more likely to have higher quantities of mtDNA compared with those biopsied on Day 6 (0.0017 versus 0.0009, P < 0.001). According to IVF outcome and heteroplasmy, a lower ongoing pregnancy rate was reported for embryos that carried more than two variants. However, this did not reach statistical significance when we compared embryos with a number of variants lower or higher than two (39.15 versus 20.0, P = 0.188). Finally, a clear positive association between the mtDNA variants and copy number was reported when we compare embryos with or without heteroplasmy (0.0013 ± 0.0009 versus 0.0025 ± 0.0014, P < 0.001) and among different numbers of variants (0:0.0013 ± 0.0009, 1–2:0.0023 ± 0.0012, >2:0.0043 ± 0.0014, P < 0.05).LIMITATIONS, REASONS FOR CAUTIONA limitation may be the size of the sample and the high-throughput sequencing technology that might not have detected heteroplasmy levels below 2% which requires high sequence depth A clinical randomized trial comparing the clinical outcome after the transfer of embryos selected according to mtDNA levels or only by morphological evaluation will be necessary. More research into the impact of mtDNA heteroplasmy and copy number on IVF outcome is needed.WIDER IMPLICATIONS OF THE FINDINGSOur results demonstrate that embryos with elevated mtDNA copy number have a lower chance of producing an ongoing pregnancy. MtDNA copy number is higher in older women and is dependent upon the number of cell divisions that preceded biopsy. Moreover, our data suggest that mitochondrial activity could be a balance between functional capacity and relative mtDNA copy number.STUDY FUNDING/COMPETING INTEREST(S)There are no conflicts of interest or sources of funding to declare.Trial registration numberNot applicable.

The association between quality of supernumerary embryos in a cohort and implantation potential of the transferred blastocyst.

Despite studies focused on the association between embryo morphology and implantation potential, it is unknown how the collective quality of the supernumerary embryos in a cohort is associated with the implantation rate (IR) of the transferred embryo. This study tested the hypothesis that a relationship exists between the quality of the supernumerary cohort and IR. A retrospective cohort study of first fresh autologous IVF cycles from 05/2012 to 09/2016, with ≥ 3 blastocysts, resulting in a single blastocyst transfer (n = 819) was performed. Cohorts were grouped in two ways: by mean priority score (PS; 1 being best) of supernumerary embryos and by percent supernumerary embryos with low implantation potential. The relationship between cohort quality and IR was assessed using logistic regression. As mean cohort PS increased, IR of the transferred embryo decreased (test for linear trend, p = 0.05). When ≥ 75% of the supernumerary cohort was predicted to have low implantation potential, IR of the transferred embryo was significantly lower compared to when < 75% of the cohort was predicted to have low implantation potential (OR 0.71; 95% CI (0.53-0.94)). All associations were attenuated when adjusting for PS of the transferred embryo. Our findings suggest that quality of supernumerary embryos is associated with IR of the transferred embryo, among patients with ≥ 3 blastocysts available on day 5. As cohort quality declines and the proportion of low implantation potential embryos increases, the IR of the transferred embryo declines. These associations are attenuated when controlling for quality of the transferred embryo, suggesting that the relationship between embryo cohort quality and implantation is not independent of the transferred embryo quality.

Genetic diseases and aneuploidies can be detected with a single blastocyst biopsy: a successful clinical approach.

Can simultaneous detection of aneuploidies and genetic diseases or chromosomal aberrations in blastocysts reduce the chance of transferring embryos with low implantation potential, guaranteeing good clinical outcomes? The screening for chromosomal aneuploidies revealed that 50.6% of blastocysts diagnosed free of genetic disease or balanced, were aneuploid, therefore avoiding the transfer of blastocysts potentially resulting in implantation failures, miscarriages, or in some cases, in health affected live births. PGD is applied in patients at risk of transmitting genetically inheritable diseases to their offspring. It has been demonstrated that aneuploidies can involve chromosomes other than those investigated with PGD, affecting embryo implantation competence. Performing the biopsy at blastocyst level produces higher clinical outcomes allowing a more accurate diagnosis, compared to blastomere biopsy. This consecutive case series study was performed from October 2011 to May 2016. Clinical and biological outcomes from 1122 blastocysts obtained in 304 PGD cycles for monogenic diseases (N = 163) or chromosomal rearrangements (N = 141) were analyzed. When the blastocyst resulted transferable after the PGD analysis or chromosomal rearrangement analysis, its ploidy status by mean of preimplantation genetic screening (PGS) was also detected using the same biopsy sample. Mean female age was 35.4 ± 4.2 years old. All biopsies were performed at blastocyst stage and analyzed by Whole Genome Amplification (WGA) followed by PCR for monogenic diseases, and by array-comparative genotype hybridization (array-CGH) for all cycles. All mature oocytes retrieved were injected and cultured individually until the blastocyst stage at 37°C, 6% CO2, 5% O2. When the blastocyst was formed, it was biopsied and vitrified, awaiting the genetic results. The frozen-thawed embryo transfer was performed in a subsequent cycle. In some cases, when the blastocyst was obtained within the morning of Day 5 of culture, it had been maintained in culture and transferred on Day 6, after receiving the genetic report. A total of 2809 (2718 fresh and 91 frozen-thawed) mature oocytes were injected with a fertilization rate of 75.5% (N = 2120), leading to the development of 2102 embryos. A further 24 frozen embryos, previously vitrified without any genetic testing, were successfully warmed for genetic screening. A total of 2126 embryos were cultured with a blastocyst formation rate of 52.8% (N = 1122); all of them were biopsied from Day 4 to Day 7 of culture. After the genetic analysis, 309 (27.5%) blastocysts resulted transferable, both for monogenic disease or translocation and for their ploidy status, 42 were diploid/aneuploid mosaic, 55 were no result and 716 were not transferable, due to genetic disease or chromosomal rearrangement and/or for their ploidy status. Of note, 316 (50.6% of transferable blastocysts after PGD and 28.2% of total number of biopsied blastocysts) of the blastocysts resulted healthy for the genetic disease or chromosomal rearrangement were aneuploid. Out of 304 PGD/PGS cycles performed, 28.6% (N = 87) resulted in no-transferable blastocysts after only PGD analysis; this percentage increased to 39.8% (N = 121) when also PGS was carried out (Mc Nemar test P < 0.001). A total of 202 embryo-transfers were performed, 53 fresh and 149 cryopreserved, in which 218 healthy or carrier euploid blastocysts were transferred. Clinical pregnancy, implantation and miscarriage rates were 49.0, 47.7 and 9.9%, respectively. To date, 66 deliveries occurred with 70 healthy babies born and 13 pregnancies are still ongoing. Finally, 91 euploid healthy blastocysts are still cryopreserved waiting to be transferred. A higher than expected cycle cancellation rate could be found due to the double genetic analysis performed. For this reason, particular care should be taken in drafting and explaining informed consent, in order to avoid patient drop out. When the biopsy has to be performed in order to prevent the transmission of an inheritable disease, it should be mandatory to analyze also the genetic status of the blastocyst, avoiding useless embryo-transfers in this particular category of patients. In our study, 316 aneuploid healthy blastocysts could have been transferred without performing PGS, leading to implantation failures, miscarriages, or in some cases, to live births affected by different syndromes. No specific funding was obtained for this study. None of the authors have any competing interests to declare. Not applicable.

PGS analysis of over 33,000 blastocysts using high resolution Next Generation Sequencing (HRNGS) of over 33,000 blastocysts using high resolution Next Generation Sequencing (HRNGS)

hr-NGS detects mosaicism in trophectoderm (TE) biopsies when 10%-90% of cells are abnormal. Mosaic TE biopsies may come from embryos that result from euploid, aneuploid or mosaic pregnancies. Mosaic biopsies have higher risk of miscarrying, lower implantation potential but some may go to term. Mosaic embryos should be classified in between euploid and aneuploid in their priority for transfer. This study aims to determine the rate of euploidy, mosaicism and other abnormalities according to maternal age in a large cohort of embryos for purpose of patient counseling.

Miscarriage rate comparison of PGS cycles performed with array comparative genome hybridization (aCGH) and next generation sequencing (NGS)

hr-NGS detects mosaicism in about 20% of trophectoderm biopsies while aCGH in about 4%. Mosaic embryos are at a higher risk of miscarriage, have lower implantation potential; however some may result in a live birth. The objective of this study is to determine if the rate of miscarriage is reduced in PGS cycles by transferring non-mosaic embryos selected by NGS compared to cycles performed by aCGH.

Morphokinetic Characteristics and Developmental Potential of In Vitro Cultured Embryos from Natural Cycles in Patients with Poor Ovarian Response.

Background. Patients with poor ovarian response to ovarian hyperstimulation represent an interesting group for studying the impact of embryo cleavage irregularities on clinical outcome since all embryos, regardless of their quality, are usually transferred to the uterus. The aim of our study was to follow the morphokinetics of fertilized oocytes from natural cycles in poor responders. Methods. Zygotes from 53 cycles were cultured in vitro for 3 days. The morphokinetics of their development and transfer outcomes were retrospectively analyzed for the normally and irregularly cleaved embryos. Results. Of all embryos, 30.2% had single and 20.8% multiple cleavage irregularities with the following prevalence: developmental arrest 30.2%, direct cleavage to more than two cells 24.5%, chaotic cleavage 13.2%, and reverse cleavage 11.3%. These embryos had longer pronuclear phases, first cytokinesis, second embryo cell cycles, and less synchronized divisions. The transfer of normally developing embryos resulted in an implantation rate of 30.8% and a delivery rate of 23.1%, but irregularly cleaved embryos did not implant. Conclusions. The use of time-lapse microscopy in poor responder patients identified embryos with cleavage abnormalities that are related with no or extremely low implantation potential. Gained information about embryo quality is important for counselling patients about their expectations.

Clinical significance of intercellular contact at the four-cell stage of human embryos, and the use of abnormal cleavage patterns to identify embryos with low implantation potential: a time-lapse study

To investigate the clinical significance of intercellular contact point (ICCP) in four-cell stage human embryos and the effectiveness of morphology and abnormal cleavage patterns in identifying embryos with low implantation potential. Retrospective cohort study. Private IVF center. A total of 223 consecutive IVF and intracytoplasmic sperm injection treatment cycles, with all resulting embryos cultured in the Embryoscope, and a subset of 207 cycles analyzed for ICCP number where good-quality four-cell embryos were available on day 2 (n = 373 IVF and n = 392 intracytoplasmic sperm injection embryos). None. Morphologic score on day 3, embryo morphokinetic parameters, incidence of abnormal biological events, and known implantation results. Of 765 good-quality four-cell embryos, 89 (11.6%) failed to achieve six ICCPs; 166 of 765 (21.7%) initially had fewer than six ICCPs but were able to establish six ICCPs before subsequent division. Embryos with fewer than six ICCPs at the end of four-cell stage had a lower implantation rate (5.0% vs. 38.5%), with lower embryology performance in both conventional and morphokinetic assessments, compared with embryos achieving six ICCPs by the end of four-cell stage. Deselecting embryos with poor morphology, direct cleavage, reverse cleavage, and fewer than six ICCPs at the four-cell stage led to a significantly improved implantation rate (33.6% vs. 22.4%). Embryos with fewer than six ICCPs at the end of the four-cell stage show compromised subsequent development and reduced implantation potential. Deselection of embryos with poor morphology and abnormal cleavage revealed via time-lapse imaging could provide the basis of a qualitative algorithm for embryo selection.

Selection of embryos for transfer in IVF: ranking embryos based on their implantation potential using morphological scoring

The selection of embryos based on morphology is still the core of daily laboratory practice in IVF/intracytoplasmic sperm injection. At present, the selection of embryos is primarily based on experience and local protocols. Since an evidence-based ranking strategy for embryos on day 3 is currently lacking, this work constructed a multivariable prediction model to rank embryos according to their implantation potential. A total of 6021 fresh embryo transfers between January 2004 and July 2009 were included, eight potential predictive factors were evaluated and a prediction model was developed using multivariable logistic regression. The model was externally validated with data from couples treated between August 2009 and September 2011 in the same clinic. Five factors were included in the final prediction model: early cleavage, number of blastomeres on days 2 and 3 and morphological score and presence of morula on day 3. With validation, the model showed moderate discriminative capacity (c-statistic 0.70) and calibrated well and was able to distinguish embryos with high ongoing implantation potential from embryos with moderate or low ongoing implantation potential. The model can be used by embryologists as an objective tool to rank embryos according to implantation potential, thereby aiding the selection of embryos for transfer.

A critical review of bi-dimensional and three-dimensional ultrasound techniques to monitor follicle growth: do they help improving IVF outcome?

BackgroundThis review focuses on the possibility of improving the outcome of human IVF by studying the follicles where oocytes grow by ultrasound techniques. A comprehensive analysis of bi-dimensional (2D) and three-dimensional (3D) ultrasound (US) assessment of the follicle size and volume is presented.MethodsPublished reports from the year 1999 to 2014 analyzing the relationship between oocyte competence, IVF outcome and ultrasound assessment of the follicle size and volume have been critically analyzed.ResultsUS assessment of growing follicles has been performed mainly by 2D-US, and while overall very useful, it has been found to be of limited usefulness in predicting oocyte competence, recognize which follicles will release a mature metaphase II oocytes and decide the ideal time to trigger ovulation. In fact, a quite wide follicle size range (16–22 mm) has been reported to be associated with mature oocytes with good competence toward fertilization and embryo development. It has been also shown that smaller follicles sometimes contain mature, fertilizable oocytes. However, embryos derived from smaller follicles have probably a lower implantation potential, while follicles larger than 22 mm often contain post-mature eggs.ConclusionsThe study of follicular size by 2D-US is of limited usefulness in helping in the identification of follicles containing the best oocytes and in choosing the best moment to trigger ovulation. Possibly the value of US in this area will be improved by large prospective studies in which automated 3D-US will be used.