Embryo Score Research Articles

The association of embryo quality with the number of days of ovarian stimulation in women undergoing IVF/ICSI-ET cycle- a prospective observational study

Objective: An attempt was made to evaluate the impact of the length of ovarian stimulation on the embryo quality, with recommended strength of gonadotropin doses in women who underwent IVF/ICSI-ET cycles. Setting: Women, who underwent oocyte retrieval and fresh embryo transfer between August 2019 and November 2019, were evaluated. Design: Prospective observational study. Method: Women with primary and secondary infertility were recruited from the OPD. Ovarian stimulation was started with gonadotropins according to our center’s protocol (Antagonist protocol). Oocyte retrieval was performed approximately 36 hours after the hCG administration (trigger) followed by IVF/ICSI. Fertilization analysis was performed approximately 16 to 18 hours postinsemination. After fertilization, embryo scoring was carried out on the day of embryo transfer procedure. The primary outcome was the development of good quality embryos in relation to the days of ovarian stimulation. Statistical Analysis: Data were analyzed by Statistical Package for Social Studies (SPSS) 21.0 software package (IBM Corporation, USA): Chi-square test for categorical variables, nonparametric Kruskal Wallis test, ANOVA test, and Pearson correlation for continuous variables were applied. These tests were applied as appropriate and statistically significant level calculated at P < 0.05. Results: The length of ovarian stimulation has little impact on the quality of embryo and pregnancy outcome. Conclusion: The length of ovarian stimulation has little impact on the quality of embryo and pregnancy outcome. Future studies on large number with proper randomization are invited to be conducted to confirm the finding of present study.

Deep learning can improve day 5 embryo scoring and decision making in an embryology laboratory

Deep learning can improve day 5 embryo scoring and decision making in an embryology laboratory

Embryo selection in developing countries- inner cell mass to blastocyst dimension ratio as a predictor of pregnancy outcome

Is there a correlation between inner cell mass (ICM) to blastocyst dimensions (diameter and area) ratio and pregnancy outcome, which can be used to select embryo for transfer in developing countries? A prospective observational study was performed for a duration of 18 months at a tertiary level IVF centre. 185 subjects who had autologous or donor egg derived blastocyst transfers were included in the study. 385 Blastocysts were photographed at 116 hours post ICSI. ICM to blastocyst dimensions ratio was calculated using Image J software. Morphological scoring of embryos was performed according to Istanbul consensus. Predictive value of ICM to blastocyst diameter and area ratios to implantation and clinical pregnancy rate were studied by ROC curve analysis. Correlation coefficients were drawn between dimensions ratio and morphological scoring. Mean diameter ratio of ICM to blastocyst among embryos that gave clinical pregnancy was 0.33 +/- 0.05 as against 0.30 +/- 0.09 among embryos that did not give clinical pregnancy (p value – 0.015). Mean area ratio of ICM to blastocyst among embryos that gave clinical pregnancy was 0.14 +/- 0.04 as against 0.13 +/- 0.07 (p value – 0.26). Morphological scoring was calculated from grading as per Istanbul consensus and quantified as 1/ICM score x 1/ Trophectoderm score x Expansion score. Mean morphological scoring of embryos that gave clinical pregnancy was 2.45 +/- 1.27 as against 2.04 +/- 1.08 among embryos that did not give clinical pregnancy (p value – 0.03). Correlation coefficient between morphological scoring and mean diameter ratio of ICM to blastocyst was 0.23 (p - 0.003), showing mean diameter ratio is positively correlated with morphological scoring. Correlation coefficient between mean area ratio of ICM to blastocyst to morphological scoring was 0.14 (p - 0.06). Area under curve (AUC) for ICM to blastocyst diameter ratio to predict clinical pregnancy was 0.586 (p-0.04), for area ratio was 0.576 (p-0.04) and for morphological scoring was 0.595 (p-0.04). The predictive ability for clinical pregnancy was highest for existing morphological scoring followed by ICM to blastocyst diameter ratio and least for area ratio. Ratio between ICM to blastocyst dimensions would serve as a simple, cost-effective tool for selecting embryos prior to transfer in developing countries.

16. CORRELATION BETWEEN NUMBER OF CHROMOSOMES ALTERATIONS AND TIME-LAPSE EMBRYO SCORE: PRELIMINARY OUTCOMES

Introduction Despite of conventional morphological evaluation is the classical method for embryo selection it clearly has limitations. Recently, time-lapse monitoring allows the embryo quality classification based on morphokinetics. Moreover, Preimplantation Genetic Test for Aneuploidy (PGT-A) is applied to investigate the genetic status of the embryos and the selection of euploid embryos for transfer is supposed to lead to improved clinical outcomes. The association of morphokinetics and PGT-A opened new opportunities to refine blastocyst selection. However, knowledge of the impact of chromosome alterations on embryo morphokinetics need to be increased. The aim of this study was to correlate embryo score from time-lapse imaging with chromosome status of the embryos evaluated by PGT-A. Material & methods This is a preliminary retrospective cohort study of prospectively collected data for 81 ICSI cycles performed at a private assisted reproduction center in Brazil, between April and November 2018. A total of 260 embryos developed into blastocysts suitable for biopsy and were analyzed in this study. Embryos were cultured in the Embryoscope® time-lapse imaging chamber (Vitrolife®) and the image acquisition system was set to capture images from each embryo every 15 minutes during the entire culture interval. Embryos were scored from zero to 10 based on the timings of cell divisions with the start of ICSI until the blastocyst formation and expansion on day 5 or 6 of development. PGT-A was elective, but strongly encouraged for patient's ≥37 years old, implantation failure, recurrent miscarriage or severe male factor cases. Embryos were biopsied at a blastocyst stage and PGT-A was carried out at a global reference laboratory (Igenomix Brasil) through NGS. Results The women age varied from 25 to 42 years (38.5±2.9) and 1 to 10 blastocysts were biopsied per patient (3.2±2.0). Of the 260 biopsied blastocyst, 38.7% were euploid. The embryos scores were slightly lower in aneuploid (5.9±2.1) than euploid (6.4±2.1) blastocysts with a marginally significant statistical difference (p=0.067). The number of altered chromosomes were annotated and inversely correlated to embryo score (Pearson correlation: r=-0.210; p=0.001). Then we classified the number of altered chromosomes as none (euploid), 1-2, 3-6 and more than 6 (chaotic). We only observed a significant difference between the time-lapse embryo score of chaotic (3.9±1.3) and euploid (6.4±2.1; p=0.026) blastocysts. The 1-2 (6.1±2.0) or 3-6 (5.4±2.3) alterations showed similar time-lapse embryo score to euploid embryos (p=1.000 and p=0.161, respectively). The multivariate linear regression model have confirmed the higher number of altered chromosomes lower the time-lapse embryo score (Coef=-0.149; p=0.026) adjusted for confounders as women age and time of culture. Conclusions The findings of our study suggest the number of altered chromosomes affect the embryo morphokinetics, leading to a reducing time-lapse embryo score. Although there seems to be a relationship between the ploidy status and complexity of chromosome alterations with the embryo morphokinetics, time-lapse embryo score is not enough to predict chromosome alterations, and therefore, replace PGT-A. These are preliminary outcomes and further studies are being conducted to investigate the association of chromosome alterations with embryo morphokinetics and relate them with clinical outcomes.

Which factors are correlated with embryo euploidy in PGT-A cycles? - A multivariate logistic regression analysis of 3054 embryos

Introduction Aneuploidy is generally accepted as one of the main causes of IVF/ICSI treatment failures worldwide. For this reason, increasing number of clinics & infertility specialists seek and promote preimplantation genetic testing for aneuploidy (PGT-A) to improve the clinical outcome for indications advanced maternal age (AMA), repeated implantation failure (RIF), recurrent miscarriage (RM), severe male factor infertility. Once an euploid embryo is found, elective single euploid blastocyst transfer (eSEBT) has been shown to improve the overall clinical outcome by providing high implantation rates and lower multiple pregnancy rates. Although It is well known that aneuploidy is strongly correlated with maternal biologic age, the possibility of finding an euploid embryo in a given cycle remains largely unknown and association of aneuploidy with many patient or cycle-related parameters remain controversial. The aim of this study was to evaluate which patient, cycle or laboratory factors have an impact on aneuploidy rate in euploidy for blastocyst stage human embryos. Material & Methods This retrospective study compiled 1362 ICSI cycles of 1136 patients undergoing PGT-A with clinical indications recurrent implantation failure (RIF), recurrent miscarriage (RM) and advanced maternal age (AMA) between January 2016 and December 2018. Trophectoderm(TE) biopsy was performed and next generation sequencing (NGS) was used for comprehensive chromosomal screening (CCS). We checked the possible influence of patient characteristics, treatment history, ovarian stimulation variables, embryo scores and day on aneuploidy. To identify characteristics that may be associated with the euploidy rate multivariate logistic regression analysis was performed. Generalized estimating equations were used after we had taken into account the patient effect. Results The mean age of patients was 38.1±4.7 years. Of these 1136 patients 187(16,5%) have male factor, 256(22,5%) diminished ovarian reserve, 83(7,3%) PCOS, (9,4%) 131(11.5%) endometriosis, 89(7,8%) tubal factor, 277(24,3%) unexplained infertility, and 113(9,9%) male and female factor. Clinical indications of PGT-A these of 1136 patients were RIF in 552(48,5%), RM in 162(14,2%), AMA in 270(23,7%), RIF+AMA in 152(13,4%). Out of 3084 embryos that were biopsied at blastocyst stage, 3054 concluded with valid result (99.02%). After analysis of biopsied materials by NGS, 1327 (43.5%) were found to be euploid and 1727(56,5%) were aneuploid. Severe male factor infertility, duration of ovarian stimulation, total dose of gonadotropins used, number of oocytes retrieved, serum estradiol level on the day of hCG trigger, serum progesterone level on the day of trigger, trophectoderm and inner cell mass scores have been found to be the parameters which significantly affect euploidy in univariate analysis (p Conclusions Due to the limited number of embryos available for embryo biopsy, relative estimation of finding a euploid embryo, which is one of the most frustrating issue for both patients and clinicians, remains a challenge in PGT-A cycles. Our study and results shed light on the nature as well as the magnitude of the patient-, cycle- or laboratory-related parameters that can be used to predict the probability of euploidy before a PGT-A is planned.

PGT-A and RCT proof in AMA and SMF couples

Aneuploidy is the most common genetic abnormality in human embryos. Preimplantation genetic diagnosis for aneuploidy (PGT-A) can increase pregnancy rates in infertile couples requiring assisted reproductive technology (ART). Large datasets from testing preimplantation embryos demonstrate that over half of embryos produced by in vitro fertilization (IVF) are aneuploid. Conventional embryo morphology evaluation as well as morphokinetics can not properly discriminate euploid from aneuploid embryos. Particularly, trisomic embryos that behave very similarly to euploid embryos. For these reasons, PGT-A started to be applied for several indications. In the present talk the results of two randomized trials (RCTs) in advanced maternal age (AMA) and severe male factor (SMF) will be discussed. In both studies, livebirths/ongoing pregnancy rates were compared among patients with blastocyst transfer as a control group and a study group in which day-3 biopsy was performed, and array CGH was applied for 24-chromosome analysis, with the transfer of euploid blastocysts. The final results of the AMA study conducted in women between 38 and 41 years of age included a total of 205 cycles. The PGT-A group exhibited significantly fewer embryo transfers (68.0%, vs. 90.5% for control; p=0.0001), and lower miscarriage rates (2.7%, vs. 39.0% for control; p=0.0007). Delivery rate after the first transfer attempt was significantly higher in the PGT-A group per transfer (52.9% vs. 24.2%; p=0.0002), and per patient (36.0% vs. 21.9%; p=0.0309). No significant differences were observed in the cumulative delivery rates per patient six months after closing the study. However, the mean number of embryo transfers needed per live birth was lower in the PGD-A group compared to control group (1.8 vs. 3.7), as was the time to pregnancy (7.7 vs. 14.9 weeks). The interim analysis of SMF study was performed when 101 cycles were completed and included women We can conclude that the benefits of testing embryos for common chromosomal abnormalities, include: increased ongoing implantation and pregnancy rates per transfer, decreased miscarriage rates per patient, and faster time to pregnancy when compared to conventional embryo scoring by morphology alone. Also, we concluded that PGT-A with the new approaches with throphectoderm biopsy and Next Generation Sequencing (NGS) the cost per treatment can decrease, being like a conventional cycle if we consider the endpoint of a healthy livebirth per cycle.

Impact of sperm DNA fragmentation on ICSI outcome and incidence of apoptosis of human pre-implantation embryos obtained from in vitro matured MII oocytes

BackgroundSperm DNA integrity and oocyte quality significantly affect embryo development and survival. The current study evaluated embryo development and quality, as well as the expression level of apoptosis-related genes and microRNAs in embryo derived from in vitro matured MII oocytes according to sperm DNA fragmentation (SDF) level. MethodsThe semen and immature oocytes were collected from 50 ICSI cycles with any recognizable female factor infertility. After ovarian stimulation, germinal vesicle stage (GV) oocytes were collected and incubated in in vitro maturation (IVM) medium for 24 h. Next, reactive oxygen species (ROS) level of media culture was determined. Using by sperm chromatin dispersion (SCD) test, the SDF levels of processed semen were assessed and categorized into SDF ≤ 30% and SDF>30%. Seventy two hours after intracytoplasmic injection, the embryo development and quality score were recorded in the groups I (GV-MII + SDF≤ 30%) and II (GV-MII + SDF> 30%). Also, the apoptosis incidence of embryos at morula stage was evaluated at molecular and cellular levels by quantitative real time PCR and TUNEL staining, respectively. ResultsCleavage rate did not differ between two groups. The quality score of embryos obtained from IVM matured oocytes and high level of SDF was significantly lower than that of low level of SDF (P < 0.05). The embryos from group II had a significant reduction of the expression of BCL-2 compared to group I (P < 0.05). Also, they showed an increase in relative transcription of pro-apoptotic microRNAs; miR 15a and miR 16-1 versus group I (P < 0.05). A rise of TUNEL positive blastomers of embryo was observed at group II versus group I, but it did not reach to significantly level. ConclusionThe IVM oocytes, probably, did not suffice to recover the high level of paternal genomic damage and inhibition of apoptosis pathway beginning.

Association between Vascular Endothelial Growth Factor and Clinical Outcomes of IVF-ET/ICSI.

To investigate the association between the level of vascular endothelial growth factor (VEGF) and the quality of early embryos as well as clinical outcome after embryo transplantation in embryo culture medium of patients treated with IVF-ET/ICSI, and explore the association between VEGF 936 C/T gene polymorphisms and IVF-ET/ICSI clinical outcomes. Case-control study. The Reproductive Medical Center, Yantaishan Hospital, Yantai, Shandong Province, China, from January to December 2016. Ninety-eight patients undergoing IVF-ET/ICSI treatment were divided into pregnant group (n=58) and nonpregnant group (n=40), based on clinical outcomes. Average age, infertility duration, body mass index, number of previous grafts, basic follicle-stimulating hormone (FSH) levels, luteinizing hormone (LH) levels, chorionic gonadotropin (HCG) intimal thickness, estrogen (E2) levels, progesterone (P) levels, and other relevant indicators were compared between the two groups. The vascular endothelial growth factor (VEGF) levels in the embryo culture medium were detected by enzyme-linked immunosorbent assay (ELISA). Meanwhile, VEGF 936 C/T genotype and allele frequency (%) were compared between the two groups. There was no significant difference in average age, infertility duration, number of previous grafts, basic folliclestimulating hormone (FSH) levels, luteinizing hormone (LH) levels, chorionic gonadotropin (HCG) intimal thickness, estrogen (E2) level, progesterone (P) level, number of retrieved oocytes, embryo number, quality embryo number, quality embryo rate, and cumulative embryo score (CES) of transplanted embryo between the two groups (P=0.601, 0.180, 0.741, 0.119, 0.693, 0.282, 0.064, 0.505, 0.712, 0.822, 0.663, 0.150, 0.178, 0.204 and 0.263, respectively). VEGF level in embryo culture medium was lower in pregnant group than non-pregnant group, with statistically significant difference (p<0.001). VEGF level in the culture medium of grade I embryos was significantly lower than that of grade II embryos (p<0.001). The frequencies of CC genotype and C allele in VEGF 936 in the non-pregnant group were higher than those in the pregnant group (p=0.003, and 0.005, respectively). VEGF level in the embryo culture medium can be used as one reference indicator for predicting embryo quality and pregnancy outcome. The VEGF 936 C/T gene polymorphism may be related to IVF-ET/ICSI treatment outcome, and C allele may be a susceptibility gene for IVF-ET/ICSI failure.

Construction and validation of a prediction model to minimize twin rates at preserved high live birth rates after IVF.

Elective single-embryo transfer (eSET) at blastocyst stage is widely used to reduce the frequency of multiple pregnancies after IVF. There are, however, concerns about increased risks for the offspring with prolonged embryo culture. Is it possible to select embryos for transfer at the early cleavage stage and still achieve low twin rates at preserved high live birth rates? A prediction model (PM) was developed to optimize eSET based on variables known 2 days after oocyte retrieval (fresh day 2 embryo transfers; double-embryo transfers 1999-2002 (n=2846) and SET 1999-2003 (n=945); n total=3791). Seventy-five variables were analysed for association with pregnancy chance and twin risk and combined for PM construction. This PM was validated in 2004-2016 including frozen-thawed transfers (FET), to compare cumulative live birth rate (CLBR) and twin rate before (1999-2002 fresh embryo transfers plus FET from the same oocyte retrievals until the end of 2007, n=3495) and after (2004-2011 fresh embryo transfers plus FET from the same oocyte retrievals until the end of 2016, n=11195) implementing the model. The PM was constructed from four independent variables: female age, embryo score, ovarian sensitivity and treatment history. The calibration, i.e. the fit of observed versus predicted results, was excellent both at construction and at validation. Without compromising CLBR, twin rate was reduced from 25.2% to 3.8%, accompanied by profound improvements in perinatal outcome. The results provide the first successful construction, validation and impact analysis of a day 2 transfer PM to reduce multiple pregnancies.

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.

Relationship between blastocoel cell-free DNA and day-5 blastocyst morphology.

Cell-free DNA (cfDNA) which is present in the blastocoel cavity of embryos is believed to result from physiological apoptosis during development. This study assessed cfDNA content and caspase-3 protease activity in day-5 IVF blastocysts to determine if there was a correlation with embryo morphology. Day-5 IVF blastocysts were scored according to the Gardner and Schoolcraft system (modified to generate a numerical value) and cfDNA was collected following laser-induced blastocoel collapsing prior to cryopreservation in 25μL of media. cfDNA was quantified via fluorospectrometry and apoptotic activity was assessed via a caspase-3 protease assay using a fluorescent peptide substrate. Data were compared by linear regression. A total of 32 embryos were evaluated. There was a significant (p < 0.01) and positive correlation (cfDNA = 104.753 + (11.281 × score); R2 = 0.200) between embryo score and cfDNA content. A significant (p < 0.05) and positive correlation (cfDNA = 115.9 + (0.05 × caspase-3); R2= 0.128) was observed between caspase-3 activity and cfDNA levels. There was no significant relationship between caspase-3 activity and embryo morphology score. This study provides further evidence that cfDNA is present in blastocoel fluid, can be quantified, and positively correlates with embryonic morphology. There is also evidence that at least a portion of the cfDNA present is from intracellular contents of embryonic cells that underwent apoptosis. Additional studies are warranted to determine other physiological sources of the cfDNA in blastocyst fluid and to determine the relationship with cfDNA content, embryo morphology, and chromosomal ploidy status plus implantation potential.

Predictive Value Of Glucose And β-Hcg Concentration In The Embryo Culture Medium Of Patients Undergoing Intracytoplasmic Sperm Injection

Objective: The aim of this study was to evaluate 3 methods of embryo selection in predicting the embryos with the best method of achieving pregnancy in single embryo transfer cycles, namely embryo morphology, glucose concentration and β-HCG concentration in the embryo culture medium.Patients and Methods: A total of 66 embryos in 66 ICSI cycles were studied: 33 pregnant and 33 non-pregnant cycles. Each embryo was scored using the Veeck's scoring method and the spent culture medium of each embryo was analyzed for its content of glucose and β-HCG using previously validated methods.Results: The results showed that the Veeck's embryo score, the measurement of glucose levels and the measurement of β-HCG levels in the spent culture medium are all reliable and practical methods to be used for this purpose. The Veeck's embryo score had the best predictability followed by β-HCG levels, followed by glucose levels in the culture medium. Combining the 3 methods was superior to each methods studied alone as well as any combination of 2 methods.Conclusion: The measurement of glucose and β-HCG in the spent culture medium of individual embryos is an effective and practical method for embryo selection in patients treated with ICSI. Combining both methods with the Veeck's embryo score gives the best predictability. However, these findings should be confirmed by a prospective randomized study, preferably in single embryo transfer cycles.

Diagnostic échographique de grossesse arrêtée précoce : étude de la qualité des clichés

ObjectiveUltrasound examination plays a central role in case of suspected non-viable pregnancy. A wrong diagnosis might have major consequence in terms of inadequate care, especially in cases of false positive non-viable pregnancy diagnosis. Ultrasound criterions are today well defined. Our objective was to evaluate the feasibility and reproducibility of a novel image-quoting method of first-trimester non-viable pregnancy. MethodsThirty images of non-viable pregnancy were twice evaluated with blinded proofreading. Two quotations were evaluated: the first for the images of gestational sacs without embryo (gestational sac score), the second for the images with embryo (embryo score). ResultsThe ICC (interclass correlation coefficient) was>0.75 for inter- and intra-observer reproducibility both for the quotations of the gestational sac and for the embryo with a low variability. Reproducibility of quoting crown rump length measurements <5mm was low at first proofreading but after adjustment of the quoting modalities, ICC was also>0.75. ConclusionThe inter- and intra-observer reproducibility of our quoting methods is high with a low variability. They might be a useful tool in current practice in the future.

Morphology vs morphokinetics: a retrospective comparison of inter-observer and intra-observer agreement between embryologists on blastocysts with known implantation outcome.

ObjectiveOur primary aim was to compare the morphology and morphokinetics on inter- and intra-observer agreement for blastocyst with known implantation outcome. Our secondary aim was to validate the morphokinetic parameters' ability to predict pregnancy using a previous published selection algorithm, and to compare this to standard morphology assessments.MethodsTwo embryologists made independent blinded annotations on two occasions using time-lapse images and morphology evaluations using the Gardner Schoolcraft criteria of 99 blastocysts with known implantation outcome. Inter- and intra-observer agreement was calculated and compared using the two methods. The embryos were grouped based on their morphological score, and on their morphokinetic class using a previous published selection algorithm. The implantation rates for each group was calculated and compared.ResultsThere was moderate agreement for morphology, with agreement on the same embryo score in 55 of 99 cases. The highest agreement rate was found for expansion grade, followed by trophectoderm and inner cell mass. Correlation with pregnancy was inconclusive. For morphokinetics, almost perfect agreement was found for early and late embryo development events, and strong agreement for day-2 and day-3 events. When applying the selection algorithm, the embryo distributions were uneven, and correlation to pregnancy was inconclusive.ConclusionsTime-lapse annotation is consistent and accurate, but our external validation of a previously published selection algorithm was unsuccessful.

Improving In Vitro Somatic Embryos Production of Medjool and Khalas Date Palm Cultivars via Modification of Ammonium and Potassium Nitrate

Date palm is one of the most economically powerful fruit tree grown in Egypt. Although Egypt tops the list of date's product in the world. Egyptian date palm industry suffers from many constraints. Low-quality varieties and limited conventional propagation methods are the main obstacles. Extension of date palm plantations in various areas of Egypt and replanting trees to compensate loss due to diseases or human factors are reduced expected to lack adequate planting materials. The knowledge of the interplay between some fundamental medium parameters allows not only the optimization of the micropropagation system but also gives models to investigate and rationalize the process of induction and development of somatic embryogenesis itself. In vitro growth and development of produced embryos and plantlets has been improved by using of full MS salt strength. MS medium modification of some nutrients concentrations, i.e. NH4NO3(1237.5 mg/l) which recorded (13.20 and 9.60 embryos/explant for Medjool and Khalas respectively) at the end of three subculture. KNO3 which gave a higher score of mature somatic embryos (14.40 and 10.00 embryo/culture for Medjool and Khalas respectively) was obtained with 1425 mg/l KNO3 after three subcultures as compared to those obtained from any other treatment combination. The morphological response of somatic embryos production is controlled by some internal factors that fall under the influence of the genetic make-up of the plant and specialized for each genotype that is responsible for the production of somatic embryos (Medjool and Khalas cultivars). The genetic make-up is a decisive factor during somatic embryos production. There are differences between the two cultivars named, Medjool and Khalas. The produced somatic embryos at germination stage were transferred from the two experiments namely ammonium nitrate and potassium nitrate to multiplication and rooting stages for more in vitro growth for 12 weeks at three re-cultures. Individual shoots were cultured on modified MS basal medium in addition to IBA (1.0 mg/l), sucrose (30 g/l) and solidified with phyto-agar (8.0 g/l) for more in vitro growth and development. In vitro plantlets were transferred to acclimatization stage in plastic pots 5*18 cm diameter containing peatmoss, perlite and washed sand at equal volume. In vitro date palm plantlets produced from rooting stage grow well in the greenhouse during acclimatization stage without morphological abnormality. The growth and development of Medjool cultivar were better than Khalas during in vitro culture and ex vitro acclimatization.

Does ovarian stimulation regimen affect IVF outcome? a two-centre, real-world retrospective study using predominantly cleavage-stage, single embryo transfer

In this study, ovarian stimulation using highly purified human menopausal gonadotrophin (HP-HMG) and recombinant FSH (rFSH) for IVF were compared in two large assisted reproduction technique centres in Sweden. A total of 5902 women underwent 9631 oocyte retrievals leading to 8818 embryo transfers (7720 on day 2): single embryo transfers (74.2%); birth rate per embryo transfer (27.7%); multiple birth rate (5.0%); incidence of severe ovarian hyperstimulation syndrome (0.71%). Compared with ovarian stimulation with rFSH, women who received HP-HMG were older, had higher dosages of gonadotrophins administered, fewer oocytes retrieved and more embryos transferred. After multivariate analysis controlling for age and generalized estimating equation model, no differences were found in delivery outcomes per embryo transfers between HP-HMG and rFSH, independent of gonadotrophin releasing hormone analogue (GnRH) used. Logit curves for live birth rate suggested differences for various subgroups, most prominently for women with high oocyte yield or when high total doses were used. Differences were not significant, perhaps owing to skewed distributions of the FSH compounds versus age and other covariates. These ‘real-life patients’ had no differences in live birth rate between HP-HMG and rFSH overall or in subgroups of age, embryo score, ovarian sensitivity or use of GnRH analogue regimen.

Influential effect of age on oocyte morphometry, fertilization rate and embryo development following IVF in mice

Abstract Changes of oocyte quality and decreasing of ovarian follicle reserve in advanced age are the major challenges in infertility treatments. One factor associated with aging is the thickness of zona pellucida, which has adverse relation with embryo score. Although, there is no correlation between perivitelline space and granulation with embryo quality, an inversely correlation is observed between these two factors with subsequent embryo quality. The aim was to investigate the influence of age on the oocyte quality, fertilization rate and embryo development following in vitro fertilization setting in mice. NMRI mice (N = 21) were categorized into 3 groups regarding to their ages (groups I–III; 25, 30, 35 weeks old, respectively). Standard in vitro fertilization protocol was conducted for each group. After collection of the oocytes, three points of zona pellucida and perivitelline space diameters were measured in each group and mean values were calculated. Also, the number of oocytes, fertilization rate, the number of cleavage embryos and blastocyst formation were compared among the groups. All the changes were insignificancy age related, as the mean value of zona pellucida and perivitelline space diameters as well as the number of oocytes, rate of fertilization and 2 cells embryos were higher in group I compared to other groups. Also, there was significant difference between some evaluated parameters, such as the number of generated 4 cells cleavage embryos and blastocysts as well as oocytes degeneration rates. The advanced maternal age influenced negatively on the oocyte morphometry and cleavage and blastocyst embryo formation in animal model.

Delayed blastocyst development dose not influence the outcome of vitrified warmed transfer cycles

During embryonic culture to the blastocyst stage, there are embryos that are delayed in their speed of development with a low implantation rate. Our objective of this study was to compare the clinical outcome according to the day of embryo freezing (day 5 or 6 following fertilization in vitro) when vitrification was postponed until expanded blastocysts developed. Cohort observational study We retrospectively investigated the vitrified-warmed embryo transfer cycles between Jan 2014 and Dec 2015 in Fertility center of CHA Gangnam medical center . Clinical outcome data consecutive of 1,056 vitrified-warming cycles were analyzed. The mean age of the patient was 35.8 ± 4.4 years. According to the time of the vitrification, embryos were divided into day 5 (1,788 embryos of 772 cycles) and day 6 (594 embryos of 284 cycles) group. Blastocysts were assigned to one of three groups scores according to score of embryos. We compared the clinical outcomes of vitrified-warmed blastocyst stage derived from day 5 and day 6. The mean number of blastocysts transferred per cycle, vitrified and transferred embryo score was not different (1.8 ± 0.5 vs. 1.8 ± 0.3, 9.5±0.8 vs. 9.5±1.0, 9.7±0.4 vs. 9.8±0.7, respectively). The survival rates were comparable between the day 5 and day 6 groups (90.15% vs. 88.68%). The clinical pregnancy and implantation rate (56.08 vs. 41.54%, 40.54% vs.29.46%, P<0.05, respectively) of the day 5 were significantly higher than day 6. The abortion rate was lower in day5 as compared to day 6 (13.16% vs 16.10%) but no significant difference. The pregnancy, implantation and abortion rate of high grade blastocyst vitrified on day 5 and day6 was not different (59.08 vs. 51.34%, 48.32% vs.42.76%, 11.45% vs. 13.45%, respectively). Vitrified-warmed blastocyst stage derived from day 5 was significantly higher evidence for clinical pregnancy rate and implantation rate. But lower evidence for abortion rate were frozen either on day 5 or day 6 but no significant difference. But good quality blastocysts that were vitrified on day 6 had similar developmental potential and pregnancy outcomes compared to those of on day5. Thus, the quality of the blastocysts were an important factor that affected the pregnancy outcomes of the vitrified-warmed transfer cycles.

Early miscarriage rate in lean polycystic ovary syndrome women after euploid embryo transfer – a matched-pair study

The early miscarriage rate is reported to be higher in patients with polycystic ovary syndrome (PCOS) compared with non-PCOS patients. However, whether PCOS is an independent risk factor for early miscarriage is still controversial; to what extent embryonic aneuploidy accounts for miscarriages of PCOS is still unknown. In this 1:3 matched-pair study, 67 lean PCOS patients and 201 controls matched for age, body mass index (BMI) and embryo scores undergoing a single euploid blastocyst transfer in vitrified-warmed cycles were analysed. Clinical pregnancy, early miscarriage and live birth rates were compared. Logistic regression analysis was performed to further evaluate the factors associated with early miscarriage and live birth. Clinical pregnancy rates were 50.7% in PCOS and 55.2% in control groups. Early miscarriage rate was significantly (P = 0.029) increased in the PCOS group compared with controls; non-PCOS patients had a significantly higher live birth rate than PCOS patients, P < 0.001. Further regression analyses showed that PCOS was significantly associated with a higher risk of early miscarriage and decreased chance of live birth. In conclusion, PCOS in women undergoing pre-implantation genetic diagnosis may, independently from BMI and karyotype, increase the risk of miscarriage.

Correlation analysis of human embryo LeY glycan antigen expression and embryo quality.

This study assessed the feasibility of using LeY glycan secretion level in human embryos as a method of judging embryo quality. Embryo culture media from patients receiving in vitro fertilization-embryo transfer was collected, and quality scores of embryos were recorded. Secretions of LeY in the culture media in different development stages (from 4-cell to 10-cell), embryos in the same development stage of the same patients (8-cell/I) and embryos in the same development stage of different patients (8-cell/I) were examined by dot-blot. Embryos were divided into a hypersecretion group and hyposecretion group, based on their LeY secretion level. The embryo quality was evaluated by clinical observations, the number which developed to D3 cell stage and the number of successful embryo transplantations. LeY secretion increased as embryos developed from 4-cell to 10-cell (P<0.05); secretion of LeY of 8/I is not identical; development speed of embryos with different secretion level of LeY was also different. The number of embryos which developed to 6-cell or higher was 82.2% in the LeY hypersecretion group but only 60% in the hyposecretion group. The rate of successful transplantation was significantly higher in the hypersecretion group (71.1 vs. 40%). In conclusion, LeY glycan secretion level in human embryos is closely related to embryo quality. LeY may become a useful measure to evaluate embryo quality in the future.