Intracytoplasmic Sperm Injection Procedure Research Articles

The impact of SARS-CoV-2 mRNA vaccine on intracytoplasmic sperm injection outcomes at a fertility center in Iraq: A prospective cohort study.

Coronavirus disease 2019 (COVID-19) is a major public health problem that requires preventative vaccines. However, there is vaccine hesitancy among women of reproductive age in Iraq. This study aimed to investigate SARS-CoV-2 vaccination effects on intracytoplasmic sperm injection (ICSI) and related fertility parameters. The study population comprised 54 infertile patients undergoing the ICSI procedure at a fertility clinic: vaccinated (n = 17) and non-vaccinated (n = 37). SARS-CoV-2-IgG/mL was assayed in follicular fluid from patients. Fertility parameters were assessed using oocyte and embryo quality and pregnancy outcomes between study groups, with respect to the time interval from vaccination to ova pick up. There were no significant differences between non-vaccinated and vaccinated groups in respect of oocytes quality with regard to the mean number of picked up oocytes (p = 0.564), abnormal oocyte (p = 0.827), oocytes metaphase I and II (p = 0.306; p = 0.165), germinal vesicles (p = 0.076), grade I, II, and III fertilized oocytes (p > 0.05), and for maturation rate (p = 0.13). There were also no significant differences (p > 0.05) in embryo quality parameters with the mean number of grade I, II, and III fertilized oocytes and the fertilization rate, the number of transferred embryo (0.086). There were no significant differences between vaccinated and unvaccinated groups with respect to follicular fluid SARS-CoV-2-IgG (p = 0.854), and pregnancy outcomes (p = 0.550). The COVID-19 mRNA vaccine has no effect on ICSI, fertility parameters, and pregnancy outcome.

Influx of zwitterionic buffer after intracytoplasmic sperm injection (ICSI) membrane piercing alters the transcriptome of human oocytes.

Does piercing oocyte membranes during ICSI allow the influx of surrounding zwitterionic buffer into human oocytes and result in altered developmental competence? Human oocytes directed to IRB-approved research were used to determine the unrestricted influx of surrounding buffer into the oocyte after piercing of membranes via confocal fluorescence microscopy (n = 80 human MII oocytes) and the influence of the select buffer influx of HEPES, MOPS, and bicarbonate buffer on the oocyte transcriptome using ultra-low input RNA sequencing (n = 40 human MII oocytes). Piercing membranes of human MII oocytes during sham-ICSI resulted in the unrestricted influx of surrounding culture buffer into the oocyte that was beyond technician control. Transcriptome analysis revealed statistically significant decreased cytoskeletal transcripts in the pierced buffer cohorts, higher levels of embryo competency transcripts (IGF2 and G6PD) in the bicarbonate buffer cohort, higher levels of stress-induced transcriptional repressor transcripts (MAF1) in the HEPES and MOPS cohorts, and decreased levels of numerous chromosomal maintenance transcripts (SMC3) in the HEPES buffer cohort. The HEPES buffer cohort also revealed higher levels of transcripts suggesting increased oxidative (GPX1) and lysosomal stress (LAMP1). The influence of zwitterionic buffer on intrinsic cellular mechanisms provides numerous concerns for their use in IVF clinical applications. The primary concern is the ICSI procedure, in which the surrounding buffer is allowed influx into the oocytes after membrane piercing.Selecting a physiological bicarbonate buffer may reduce imposed stress on oocytes, resulting in improved embryo development and clinical results because intracellular MOPS, and especially HEPES, may negatively impact intrinsic biological mechanisms, as revealed by transcriptome changes. These findings further support the utilization of bicarbonate buffer as the oocyte-holding medium during ICSI.

Evaluation of Oxidative and Nitrosative Stress Markers Related To Inflammation in The Cumulus Cells and Follicular Fluid of Women Undergoing Intracytoplasmic Sperm Injection: A Prospective Study.

Oxidative/nitrosative stress in the oocyte microenvironment could have an impact on intracytoplasmic sperm injection (ICSI) outcomes. The presence of reactive oxygen species (ROS) and reactive nitrogen species (RNS) can stimulate the secretion of pro-inflammatory cytokines, leading to chronic inflammation and potentially affecting embryo as well as oocyte quality. This study aimed to examine the relationship of lipid peroxidation [measured by the malondialdehyde (MDA) assay] with protein carbonyl [measured by the 2,4 dinitrophenylhydrazine (DNPH) assay] levels in cumulus cells (CCs), as well as nitric oxide (NO), peroxynitrite (ONOO-), and C-reactive protein (CRP) levels in follicular fluid (FF). The potential relationship of these levels with ICSI outcome was also evaluated. In this prospective study, 63 FF samples and their corresponding CCs were collected for ICSI procedures. Spectrophotometry was used to assess levels of DNPH, MDA, NO, and ONOO-. CRP levels were evaluated using an immunoturbidimetric assay. The patients under 37 years with normal ovarian reserve had significantly lower levels of MDA, DNPH, NO, ONOO-, and CRP compared to those over 37 years. Additionally, we observed higher levels of MDA, DNPH, NO, ONOO-, and CRP in the group with an oocyte maturity rate of less than 60%. No significant difference was observed between the DNPH levels and factors such as infertility duration, embryo quality, pregnancy, or the number of retrieved oocytes. A higher level of MDA, NO, ONOO-, and CRP was found to be significantly related to the lower number of retrieved oocytes, longer periods of infertility, poor embryo quality, and negative pregnancy outcomes. Oxidative/nitrosative stress, linking to inflammation in the oocyte microenvironment, can be considered as a potentially useful biomarker for assessing the development and competence of oocytes and embryos and predicting ICSI outcomes.

Intuitive Cell Manipulation Microscope System with Haptic Device for Intracytoplasmic Sperm Injection Simplification.

In recent years, the demand for effective intracytoplasmic sperm injection (ICSI) for the treatment of male infertility has increased. The ICSI operation is complicated as it involves delicate organs and requires a high level of skill. Several cell manipulation systems that do not require such skills have been proposed; notably, several automated methods are available for cell rotation. However, these methods are unfeasible for the delicate ICSI medical procedure because of safety issues. Thus, this study proposes a microscopic system that enables intuitive micropipette manipulation using a haptic device that safely and efficiently performs the entire ICSI procedure. The proposed system switches between field-of-view expansion and three-dimensional image presentation to present images according to the operational stage. In addition, the system enables intuitive pipette manipulation using a haptic device. Experiments were conducted on microbeads instead of oocytes. The results confirmed that the time required for the experimental task was improved by 52.6%, and the injection error was improved by 75.3% compared to those observed in the conventional system.

Evaluation of medical equipment of the infertility treatment centers in North western of Libya

Background Cell culture, particularly in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are both of which assisted reproductive technology (ART) approaches that are most frequently utilized to treat infertility around the world. Objective Highlight the differences and similarities in instruments and equipment in ART laboratories that govern their success, limitations, and potential differences between laboratories by identifying the facilities of laboratories to improve outcomes of ICSI and IVF techniques in clinical centers on the western coast of Libya. Method: descriptive study, comparison of five (5) assisted reproduction laboratories instrument and equipment parameters using the same quality-control application in clinical centers on the western coast of Libya.Result: Among the seven clinical centers specializing in the diagnosis and treatment of infertility that were contacted, four infertility treatment centers and the Medical Research Center (MRC) provided a response. The MRC and four clinical centres had good laboratory facilities for ICSI and IVF techniques compared to Jordan ART laboratory facilities.Conclusion: This study highlights the imperative of enhancing laboratory infrastructure to support optimal implementation of ICSI and IVF procedures.

ICSI using testicular spermatozoa after failure of ICSI with ejaculated spermatozoa could be a good choice: A propensity score‐matched cohort study

AbstractBackgroundEjaculated spermatozoa are considered to possess a higher fertilisation potential than testicular spermatozoa. In selected cases, the use of testicular spermatozoa from non‐azoospermic infertile men resulted in a higher implantation and pregnancy rate than the use of ejaculated spermatozoa.ObjectiveThe primary objective was to compare the live birth rate and cumulative live birth rate between couples with failed intracytoplasmic sperm injection procedure using ejaculated spermatozoa who subsequently had an intracytoplasmic sperm injection cycle with testicular spermatozoa and those who subsequently had an intracytoplasmic sperm injection cycle with ejaculated spermatozoa. The secondary objective was to determine the indications for the use of testicular spermatozoa after intracytoplasmic sperm injection failure with ejaculated spermatozoa.Materials and methodsA retrospective study of matched couples using propensity score matching analysis was performed. After an intracytoplasmic sperm injection failure (cycle_1), intracytoplasmic sperm injection with either ejaculated spermatozoa (ejaculated sperm group), or testicular spermatozoa (testicular sperm group), was performed (cycle_2). The matching was on intracytoplasmic sperm injection performed in cycle_1 according to spermatozoa used (testicular or ejaculated) in cycle_2. Logistic regression was used to evaluate the influence of sperm origin on cumulative live birth rate. Univariate analysis on parameters of cycle_1 was used to identify the prognostic factors to propose an intracytoplasmic sperm injection with testicular spermatozoa in case of cycle_1 failure. The study outcomes were live birth rate and cumulative live birth rate.ResultsAmong the 6034 couples available, 63 were selected to constitute the testicular sperm group and 63 were selected by propensity score matching to constitute the ejaculated sperm group. After matching, the DNA fragmentation index was higher in the testicular sperm group (13.43% ± 9.65% vs. 8.93% ± 4.47%, p = 0.013); no significant difference was observed for the fertilisation rate, the number of obtained embryos, blastulation rate and frozen embryo rate. In cycle_2, the live birth rate was higher in the testicular group (22.2% vs. 0.0%, p < 0.001), as was the cumulative live birth rate (25.4% vs. 6.3%, p = 0.065). The prognostic factors identified for the proposal of intracytoplasmic sperm injection procedure with testicular spermatozoa after intracytoplasmic sperm injection failure with ejaculated spermatozoa were: teratozoospermia, cryptozoospermia and high DNA fragmentation index.DiscussionAccording to the present study and current knowledge, the use of testicular spermatozoa after failed intracytoplasmic sperm injection procedure in non‐azoospermic men could be proposed instead of sperm donation in case of high sperm DNA fragmentation index, cryptozoospermia and teratozoospermia. A good oocyte response to ovarian stimulation during the previous assisted reproductive technology attempt will increase the chance of success. Although the main limitation of the current study is its retrospective nature, the use of the propensity score matching to perform causal inference study increases its reliability.ConclusionThe present study supports that the use of testicular spermatozoa outside the classical indication of azoospermia is a good option when the indication is well established. However, before proposing a testicular biopsy, an improvement in sperm characteristics should be considered by treating the causes of sperm alteration.

#166 : Investigating the Effect of Parental Age on Intracytoplasmic Sperm Injection Reproductive Outcomes in Indonesia: Prognostic Tools and Prediction Approaches to Maximize Human Embryo Implantation

Background and Aims: Reproductive issues impact one in eight Indonesian married couples. Intra Cytoplasmic Sperm Injection (ICSI) is still expensive; hence many couples don’t get evaluated or enroll fertility programs. We decided to study the correlation between age, hormonal status, and ICSI embryo quality because Indonesia’s infertility rate is rising. This is the first investigation into the factors behind ICSI outcomes in Indonesia. Method: In 2022, two fertility centers in Surabaya and Tangerang, Indonesia, performed ICSI on couples. 150 couples receiving ICSI at two research sites comprise the sample. Surgically retrieved or frozen sperm cycles and incomplete medical records were excluded. The Spearman test was used to investigate the correlation between parental age, hormonal status, and ICSI embryo quality on day 1 and day 3. (D1 and D3). Results: 150 ICSI cycles were studied. The hormonal baseline varied from normal to subnormal, while paternal and maternal age ± SDs were 37.03 ± 6.08 and 34.28 ± 5.11. The Spearman test demonstrated a significant negative connection between paternal age and embryo quality D1 (p<0.005, r=-0.363) and D3 (good category: p<0.005, r=-0.172; intermediate category: p<0.005, r=-0.250; poor category: p<0.005, r=-0.221). The Spearman test showed a significant negative connection between maternal age and embryo quality D1 (p<0.005, r=-0.413) and D3 (good category: p<0.005, r=-0.231; intermediate category: p<0.005, r=-0.294; poor category: p<0.005, r=-0.291). Conclusion: This study reveals that married couples have a substantial impact on their hormonal condition, the rate of fertilization, the quality of D1 and D3 embryos, and the number of embryos generated following ICSI. To increase the success rate of ICSI procedures, it is necessary to maximize fertility at a younger age.

Effects of histones related to sperm chromatin on embryo development and ART outcomes.

In the process of spermatogenesis and maturation, histones of the sperm nucleus were gradually replaced by protamine. Abnormal sperm nucleoprotein histotype conversion can make sperm DNA unstable and affect sperm function. The aim of this study is to investigate the impact of high and low proportion of sperm histone positivity in semen sample on embryonic development and assisted reproductive technology results, and to evaluate its diagnostic value in assisted reproduction. Sperm nuclear status was detected with aniline blue staining. Under acidic conditions, aniline blue combines with histones rich in lysine residues to form blue compounds. The groups were divided according to the critical value of sperm histone positive ratio of 30%. Using the intracytoplasmic sperm injection procedure, the fertilization rate and normal fertilization rate in the normal sperm histone positive ratio group were significantly higher than those in the abnormal group, and the difference was statistically significant (P < .001). Using the in vitro fertilization procedure, the effect of sperm histone positive ratio on each index was not statistically different. Overall the study provides some preliminary evidence that abnormal sperm histones may be a factor that affects the fertilization success of intracytoplasmic sperm injection procedures. However, more research is needed to confirm this finding to determine the exact mechanism by which abnormal sperm histones affect fertilization.

Ensembling noisy segmentation masks of blurred sperm images

Background:Sperm tail morphology and motility have been demonstrated to be important factors in determining sperm quality for in vitro fertilization. However, many existing computer-aided sperm analysis systems leave the sperm tail out of the analysis, as detecting a few tail pixels is challenging. Moreover, some publicly available datasets for classifying morphological defects contain images limited only to the sperm head. This study focuses on the segmentation of full sperm, which consists of the head and tail parts, and appear alone and in groups. Methods:We re-purpose the Feature Pyramid Network to ensemble an input image with multiple masks from state-of-the-art segmentation algorithms using a scale-specific cross-attention module. We normalize homogeneous backgrounds for improved training. The low field depth of microscopes blurs the images, easily confusing human raters in discerning minuscule sperm from large backgrounds. We thus propose evaluation protocols for scoring segmentation models trained on imbalanced data and noisy ground truth. Results:The neural ensembling of noisy segmentation masks outperforms all single, state-of-the-art segmentation algorithms in full sperm segmentation. Human raters agree more on the head than tail masks. The algorithms also segment the head better than the tail. Conclusions:The extensive evaluation of state-of-the-art segmentation algorithms shows that full sperm segmentation is challenging. We release the SegSperm dataset of images from Intracytoplasmic Sperm Injection procedures to spur further progress on full sperm segmentation with noisy and imbalanced ground truth. The dataset is publicly available at https://doi.org/10.34808/6wm7-1159.

Human oocytes image classification method based on deep neural networks

BackgroundThe effectiveness of in vitro fertilization depends on the assessment and selection of oocytes and embryos with the highest developmental potential. One of the tasks in the ICSI (intracytoplasmic sperm injection) procedure is the classification of oocytes according to the stages of their meiotic maturity. Oocytes classification traditionally is done manually during their observation under the light microscope. The paper is part of the bigger task, the development of the system for optimal oocyte and embryos selection. In the hereby work, we present the method for the automatic classification of oocytes based on their images, that employs DNN algorithms.ResultsFor the purpose of oocyte class determination, two structures based on deep neural networks were applied. DeepLabV3Plus was responsible for the analysis of oocyte images in order to extract specific regions of oocyte images. Then extracted components were transferred to the network, inspired by the SqueezeNet architecture, for the purpose of oocyte type classification. The structure of this network was refined by a genetic algorithm in order to improve generalization abilities as well as reduce the network’s FLOPs thus minimizing inference time. As a result, overline{Acc} at the level of 0.964 was obtained at the level of the validation set and 0.957 at the level of the test set. Generated neural networks as well as code that allows running the processing pipe were made publicly available.ConclusionsIn this paper, the complete pipeline was proposed that is able to automatically classify human oocytes into three classes MI, MII, and PI based on the oocytes’ microscopic image.

P-278 What are the effects of ICSI procedure time intervals on in vitro fertilization outcomes?

Abstract Study question Do time intervals between oocyte pick-up (OPU), oocyte denudation (OD), and Intra-cytoplasmic Sperm Injection (ICSI) impact ICSI outcomes? Summary answer ICSI time intervals impact oocyte maturation, blastulation and pregnancy rates. There is no effect on fertilization and cleavage rates. What is known already Cytoplasmic and nuclear oocyte maturation are crucial for the success of in vitro fertilization and embryo development. Cumulus cells enhance oocyte maturation. Therefore, prolonged oocyte culture prior to denudation may induce oocyte apoptosis. There is no consensus for optimal time intervals between different ICSI procedures and their effects on fertilization, embryo and blastocyst, development and pregnancy rates. Study design, size, duration A retrospective analysis of 794 ICSI fresh cycles was performed at the Alyssa Fertility Group Center, between Juin 2021 and December 2022. Participants/materials, setting, methods ICSI cycles were performed on women aged ≤40 years, with at least 3 mature oocytes. Semen partner concentrations greater than 1million/mL were included. Time intervals between OPU, OD, and ICSI were recorded. We evaluate the effects of these timing intervals on oocyte maturation, fertilization, embryo development and pregnancy rates. Statistical analysis was performed using SPSS 22.0. Kolmogorov–Smirnov, Student t, Pearson tests, and ROC curve analysis were used. Level of signification p-value &amp;lt;0.05. Main results and the role of chance The mean women’s age was 33,2 years (SD 4,4). Mean times in hours for OPU/OD, OD/ICSI, and OPU/ ICSI were respectively: 01:28(SD 00:37); 02:35(SD 01:01); 04:03(SD 01:08). Positive correlations were seen between maturation rate and OPU/OD; OPU/ICSI intervals respectively (r = 0,217, p = 0,002; r = 0,193, p = 0,005).There were no significant correlation between ICSI time intervals, fertilization and cleavage rates. Top quality embryos rates were positively associated with OPU/OD time interval (r = 0,136: p = 0,049). The blastulation rate was positively associated with OPU/OD, OD/ICSI, and OPU/ICSI time intervals respectively(r = 0,183: p = 0,008; r = 0,211: p = 0,002; r = 0,279: p &amp;lt; 0,001).Pregnancy rates were positively correlated to OD/ICSI and OPU/ICSI respectively: (r = 0,158: p = 0,022; r = 0,188: p = 0,006). The ROC curves analysis revealed that the cut off value of time intervals between: OPU/OD was 01:10 (AUC = 0,554; IC 95% 0,476-0,632), p = 0,185; OD/ICSI was 02:20 (AUC = 0,600; IC 95% 0,516-0,669), p = 0,023 and for OPU/ICSI: 03:50, (AUC = 0,600; IC 95% 0,524-0,676), p = 0,014. Limitations, reasons for caution Trigger time is an important factor for oocyte maturation that should be noted. This study is limited by its retrospective nature. Potential unmeasured confounding factor cannot be excluded Wider implications of the findings Time intervals between all the ICSI procedures affect reproductive outcomes. According to our findings, excessive oocyte incubation time before denudation should be avoided. ICSI should be performed 2 hours after OD. Time intervals should be adjusted according to laboratory practice to improve ICSI outcomes. Trial registration number Not applicable

P-157 Improved embryo development and clinical outcome using bicarbonate buffer as the oocyte holding medium during Intracytoplasmic sperm injection (ICSI) compared to MOPS buffer

Abstract Study question Does bicarbonate buffer used as the oocyte holding medium during ICSI result in improved embryo development and clinical outcome compared to the zwitterionic buffer MOPS? Summary answer Pre-equilibrated bicarbonate buffer is acceptable for ICSI use in atmospheric conditions, and results in superior sibling-oocyte cohort study outcomes compared to ICSI with MOPS buffer. What is known already Maintaining proper pH is of the utmost importance during IVF micromanipulation procedures. However, currently used zwitterionic buffers, HEPES and MOPS, may significantly influence intrinsic biological mechanisms outside pH maintenance, impacting oocyte competence, embryo development, and clinical outcome. Zwitterionic buffer influence is of concern during ICSI because piercing the oolemma allows the influx of the surrounding buffer into the oocyte (Mendola unpublished data, 2022). Bicarbonate is the physiological buffer for pH maintenance in all mammals providing numerous benefits for cellular development. Study design, size, duration An iSTAT handheld blood analyzer was used to analyze the pH shift of pre-equilibrated bicarbonate buffer dishes to determine the acceptablity for use during ICSI in atmospheric conditions. A clinical sibling-oocyte cohort study consisting of 115 patients and 1,997 oocytes (1,016 bicarbonate/981 MOPS) was analyzed to compare the embryo development and clinical outcomes of bicarbonate buffer vs. MOPS as the oocyte-holding medium during ICSI. Morphokinetic time points for all PGT-A tested embryos were also compared. Participants/materials, setting, methods We analyzed the pH shift of pre-equilibrated bicarbonate buffer in atmospheric conditions using 50x9mm culture dishes (Falcon, cat#351006) comparing light and high viscosity paraffin oil overlay, OvOil and Heavy OvOil (Vitrolife), after 5-minute, 10-minute, and 15-minute intervals. We analyzed all data points in triplicate. We analyzed the sibling-oocyte cohort study comparing fertilization rate, abnormal fertilization rate, useable blastocyst rate, mosaicism rate, euploidy/aneuploidy rate, morphokinetic time points, pregnancy rate, and ongoing pregnancy rate. Main results and the role of chance Pre-equilibrated (6.5%CO2/5.0%O2/7.301pH) bicarbonate buffer in 50 × 9mm ICSI culture dishes, with 5.0mL of OvOil overlay, maintained proper pH (7.301/7.328/7.354pH) for the time required to complete the ICSI procedure in atmospheric conditions (0min/5min/10min). The use of the high viscosity paraffin oil overlay, Heavy OvOil, reduced pH drift of the bicarbonate buffer ICSI dishes (7.301/7.323/7.342pH). In this clinical sibling-oocyte cohort study, ICSI was performed in pre-equilibrated bicarbonate buffer culture drops (6µL) in the 50x9mm dishes with 5.0mL Heavy OvOil overlay. Utilization of bicarbonate buffer as the oocyte-holding medium during ICSI is superior to MOPS buffer. Bicarbonate buffer used with ICSI resulted in higher fertilization rates (85.4% vs. 78.8%, P&amp;lt;0.0001), higher useable blastocyst rate per ICSI oocyte (50.6% vs. 46.2%, P&amp;lt;0.05), lower abnormal fertilization rate (4.5% vs. 6.7%, P&amp;lt;0.05), and a lower whole chromosome mosaicism rate (4.2% vs. 8.2%, P&amp;lt;0.03) compared to the MOPS buffer. Pregnancy rates were higher in the bicarbonate group compared to MOPS but not statistically significant. Morphokinetic analysis revealed a slight delay in all MOPS developmental time points compared to bicarbonate, but only statistically significant in the time to compaction (P&amp;lt;0.03). Limitations, reasons for caution During this study, different genetic testing companies, with differing mosaicism thresholds and decisions on included ploidy results, were used for PGT-A analysis of patient samples. Wider implications of the findings We determined that pre-equilibrated bicarbonate buffer can be used for ICSI in atmospheric conditions, therefore avoiding the potential detrimental effect of zwitterionic buffer after influx into the oocyte from the ICSI procedure. Bicarbonate buffer used during ICSI was determined to be superior to MOPS buffer. Trial registration number Not applicable

P-270 The timing of intracytoplasmic sperm injection does not impact live birth rate and neonatal outcomes

Abstract Study question Does the timing of intracytoplasmic sperm injection (ICSI) relative to ovulation trigger impact live birth rates and neonatal outcomes? Summary answer ICSI timing relative to ovulation trigger impacts neither live birth rates nor neonatal outcomes. What is known already When conducting ICSI, the timing of the insemination relative to ovulation trigger is difficult to standardise in clinical practice, especially in busy ART laboratories. Flexibility when scheduling ICSI procedures would therefore be beneficial from a laboratory management perspective. However, the impact of this timing on live birth rates and neonatal outcomes remains inconclusive in the literature. Study design, size, duration This retrospective study included a total of 1547 consecutive single autologous embryo transfer cycles (maternal age 35.2 ± 4.8 years) performed at Fertility North between January 2017 and August 2021. ICSI was performed immediately after denudation without timing restrictions relative to ovulation trigger. A total of 422 (27.3%) transfers resulted in a live birth, including 415 singletons, 5 twin deliveries, and 2 lost to follow-up. Only singleton babies were included for neonatal outcome analysis. Participants/materials, setting, methods ICSI timings relative to ovulation induction trigger (mean ± SD 42.5 ± 1.4 hours, range 37.0-46.5 hours) were extracted from the electronic witnessing database. Cycles were grouped according to ICSI timing quartiles (groups Q1-4: &amp;lt;41.7, 41.7-42.7, 42.8-43.4 and 43.5+ hours). Logistic/linear regressions were used to assess impacts of ICSI timing on the subsequent live birth and birthweight outcomes in the singleton babies. Mixed effect model was used to account for clustering effect from repeat cycles by same patients. Main results and the role of chance Q1 group had significantly lower live birth rate (22.7%) but higher maternal age (37.3 ± 5.2 years) in comparison to Q2 (28.5% and 34.5 ± 4.6, P &amp;lt; 0.05 respectively), Q3 (30.2% and 34.8 ± 4.5, P &amp;lt; 0.05 respectively) and Q4 groups (27.7% and 34.4 ± 4.4, P &amp;lt; 0.05 respectively). However, following multivariate logistic regression adjusting for potential confounders including maternal age, body mass index, aetiology, sperm origin (ejaculated or surgical), sperm type (donor/partner), embryo stage at transfer (cleavage or blastocyst), and fresh/frozen transfer; observed differences in live birth rates became no longer significant (Q2 vs Q1, aOR = 0.982, 0.692-1.394, P = 0.919; Q3 vs Q1, aOR = 1.058, 0.884-1.266, P = 0.538; Q4 vs Q1, aOR = 0.969, 0.859-1.094, P = 0.615; Q3 vs Q2, aOR = 1.153, 0.830-1.600, P = 0.396; Q4 vs Q2, aOR = 0.963, 0.818-1.134, P = 0.649; Q4 vs Q3, aOR = 0.836, 0.601-1.164, P = 0.289). Gestational age and birthweight of singletons arising from 4 groups were also comparable (Q1 38.1 ± 2.2 weeks and 3209.1 ± 581.4 grams, Q2 38.2 ± 1.6 weeks and 3159.9 ± 508.2 grams, Q3 38.7 ± 1.8 weeks and 3304.7 ± 546.9 grams, and Q4 38.4 ± 2.0 weeks and 3297.5 ± 526.9 grams; P &amp;gt; 0.05 respectively). Multivariate linear regression further confirmed the absence of significant impacts (standardised coefficient = 0.001, P = 0.980) by ICSI timing on the birthweight of resulting newborns, taking into account gestational age and baby sex plus all above mentioned 7 potential confounders. Limitations, reasons for caution Our study is limited by its retrospective nature, where it is impossible to control all known and unknown confounding factors. The varied denudation duration as a result of different egg numbers was unable to be analysed, although ICSI was aimed to be performed immediately after completion of denudation. Wider implications of the findings Our findings indicate a wide time window in human oocytes to initiate fertilization via ICSI. This data offers reassurance in applying flexible ICSI timing without impacting live birth rates and neonatal outcomes. This is clinically valuable by providing evidence-based guidance to daily practice and patient counselling. Trial registration number not applicable

Assessment of cell-free DNA and apoptosis in an oocyte microenvironment: promising biomarkers to predict intracytoplasmic sperm injection outcomes.

Cell-free DNA (cf-DNA) is defined as DNA fragments that are released into the body fluids from apoptosis or necrosis cells, including follicular fluid (FF), which can affect the microenvironment of the oocyte associated with infertility. We aimed to investigate a relationship between apoptosis of cumulus cells (CCs) and cf-DNA levels in FF and clinical outcomes of women undergoing intracytoplasmic sperm injection (ICSI). Therefore, 82 FF samples were collected, and the corresponding CCs were isolated for ICSI procedures. FF cf-DNA concentration was quantified using ALU-quantitative polymerase chain reaction (PCR), and CCs DNA fragmentation index (DFI) was evaluated by the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) method. We found that cf-DNA and DFI levels were significantly higher in FF and CCs samples related to the age of women ≥37 years compared with the age of women < 37 years. Moreover, in older and younger women, FF cf-DNA and CCs DFI levels were significantly lower when the anti-Müllerian hormone (AMH) level was > 1.1 ng/ml compared with when AMH ≤ 1.1 ng/ml. In addition, patients with a low number of retrieved oocytes ≤ 6 had significantly higher levels of CCs DFI and FF cf-DNA than women with a higher number of retrieved oocytes > 6. Additionally, we observed that higher levels of cf-DNA and DFI were associated with poor oocyte maturity and poor embryo quality. Finally, cf-DNA and DFI levels were significantly lower in pregnant women than in non-pregnant ones. We conclude that DFI and cf-DNA levels in the oocyte microenvironment could have potential use in evaluating oocyte and embryo developmental competence.

The Eff ect of Testosterone Gel for Patients with Poor Ovarian Response Prior to IVF Cycles: Is It really Eff ective?

Background: Classically, poor women respond with old maternal age and little ovarian standby. Though, some females unpredictably have little reply to measured ovarian stimulation. Aim of the Study: To evaluate the eff ectiveness of transdermal testosterone gel (TTG) before controlled ovarian stimulation (COS) in poor responders undergoing intracytoplasmic sperm injection (ICSI). Methods: An interventional prospective randomized control trial that included 60 females who were defi nite as unfortunate responders undergoing an intracytoplasmic sperm injection (ICSI) procedure. The population was divided into two groups. Approximately 30 patients received 10 mg TTG that was applied daily for 21 days in the cycle preceding COS for ICSI Transdermal testosterone gel (TTG) pretreatment group, while the other 30 patients are the control group. All patients were assessed for their AFC again and to begin in-vitro futilization (IVF) treatment with the beginning of the menstrual cycle. Serum testosterone was measured again in the study group. The primary outcome is the number of retrieved oocytes while the secondary outcome is the number and quality of embryos transferred, chemical gestation rate. Results: No important diff erences in sociodemographic features between both groups compared. Also there was an increase in total number of oocytes in the study group but again, this diff erence was statically non-signifi cant. Although the total dose of gonadotrophin (GT) and the duration of stimulation days were less in study group but this was statically non-signifi cant. No diff erences experiential regarding number and grading of embryos, chemical pregnancy rate nor clinical pregnancy rate. Conclusion: Pretreatment of transdermal testosterone at a dose of 10 mg/day for 21 days not upsurge the number of oocyte retrieved nor the number of embryos or pregnancy rate to a signifi cant level in poor responders undergoing ICSI

GVBD rate is an independent predictor for pregnancy in ICSI patients with surplus immature oocytes.

It was reported that there were still up to 30% immature retrieved oocyte at germinal vesicle (GV) or metaphase I (MI) stage. Whether the spontaneous maturity competency of immature oocytes associated to the clinical outcome of in vitro fertilization (IVF) cycles remains unclear and unexplored. This study aimed to investigate how the oocyte developmental parameters in in vitro maturation (IVM) affect clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles. This retrospective cohort study included couples undergoing ICSI in a university-affiliated hospital. Surplus immature oocytes during ICSI were collected and cultured in vitro. The numbers of germinal vesicle (GV) oocytes undergoing GV breakdown (GVBD) and polar body 1 extrusion within 24 h culture were recorded. The main outcome measurements were demographic baselines and oocyte developmental parameters in IVM associated with pregnancy outcomes. A total of 191 couples were included with an overall GVBD rate of 63.7% (327/513) and oocyte maturation rate of 46.8% (240/513). 53.4% (102/191) of them had embryos transferred freshly, which originated from metaphase II oocytes that matured spontaneously in vivo, and 60.8% (62/102) got pregnant. Among factors with a P-value < 0.2 in univariate logistic regression analyses of pregnancy correlation, GVBD rate (OR 3.220, 95% CI 1.060-9.782, P=0.039) and progesterone level on human chorionic gonadotropin (HCG) day (OR 0.231, 95% CI 0.056-0.949, P=0.042) remained significant in the multivariate model. The area under the curve (AUC) of the predictive nomogram was 0.729 (95% CI 0.632-0.826) with an acceptable calibration. Moreover, decision curve analyses illustrated the superior overall net benefit of models that included the GVBD rate in clinical decisions within a wide range of threshold probabilities. In conclusion, GVBD rate and progesterone level on HCG day may be associated with pregnancy outcomes in infertile couples during the regular ICSI procedure. An elevated GVBD rate within 24 h may greatly increase the likelihood of pregnancy in infertile couples during ICSI. This preliminary study may optimize clinical pregnancy prediction, which provides support in decision-making in clinical practice.

How Could One Sperm and One Oocyte Proceed To Molar Pregnancy? Prevalence of Molar Pregnancy during ICSI Procedure in Over 25,000 Fresh Embryo Transfers: A Retrospective Cross-Sectional Study.

Gestational trophoblastic disease (GTD) is a heterogeneous group of diseases characterized by excessive proliferating trophoblastic tissue. The prevalence of GTD has a varied geographical distribution. However, its frequency following intracytoplasmic sperm injection (ICSI) cycles has not yet been reported. This study aimed to estimate GTD frequency and prevalence after ICSI cycles. This retrospective cross-sectional study included all patients diagnosed with GTD subsequent to ICSI and segmental embryo transfer procedure during 2011-2019 at Royan Institute. GTD diagnosis was established for patients who met all three criteria: beta-human chorionic gonadotropin (β-hCG) levels greater than 100,000 mIU/mL, vesicular ultrasonographic pattern, and presence of pathologic features of hydatidiform mole. Although we assessed the GTD frequency in all ICSI cycles, GTD cases were only observed following fresh embryo transfer ICSI procedures. We evaluated 25,667 fresh embryo transfer ICSI procedures out of 41,540 ICSI cycles. This study identified a total of 10 GTDs confirmed by all criteria which were mentioned previously. Of these 10 GTDs, nine cases had hydatidiform mole, and one had gestational trophoblastic neoplasia. The frequency of GTD was calculated 10 cases in 41,540 (0.240 per 1000) ICSI procedures and 10 in 25,667 (0.389 per 1000) fresh embryo transfers following ICSI cycles. Also, we detected 10 GTD cases in 8,196 (1.220 per 1000) clinical pregnancies. We discuss that the possibility of GTD after ICSI procedure is not as low as expected. Thus, the previous theses are insufficient to explain all aspects of molar pregnancy, and more research is required.

Comparison of clinical outcomes between conventional in vitro fertilization and intracytoplasmic sperm injection in poor responders with only single oocyte retrieved

ObjectiveTo compare the clinical outcomes between conventional insemination (IVF) and intracytoplasmic sperm injection (ICSI) in poor responders with only a single oocyte retrieved. Materials and methodsThis is a retrospective case–control study. Couples who were treated with assisted reproductive technology (ART) with a single oocyte retrieved in Mackay Memorial Hospital from 1996 to 2016 were recruited. All data were categorized into three groups, according to their fertilization method and semen quality: group A, conventional insemination with non-male factor (IVF-NMF, n = 115), group B, ICSI with male factor (ICSI-MF, n = 30), and group C, ICSI with non-male factor (ICSI-NMF, n = 49). ResultsNo statistically significant difference was observed between IVF and ICSI groups in pregnancy outcomes, including the chemical or clinical pregnancy rate, miscarriage rate, and live birth rate. Similar fertilization rates per oocyte obtained were observed in IVF and ICSI patients, but significantly lower per mature oocyte in the ICSI group (IVF: 91.5%, ICSI-MF: 75.0%, ICSI-NMF: 77.8%). Although there is no statistical significance, the lower live birth rate is observed in group C than others (A:11.5%, B:25%, C:5%, p = 0.187). ConclusionIn this study, pregnancy outcomes of conventional in vitro fertilization and ICSI in poor responders with only a single oocyte retrieved were similar. However, the fertilization rate of matured oocytes in ICSI groups is significantly lower than that in the IVF group, indicating that ICSI procedures might cause oocyte damage. Therefore, the choice of fertilization method should be based on semen quality. A randomized controlled trial should be performed to confirm our findings.

Obesity and fertility: a literature review of the association between parental obesity at the time of conception and embryo quality

Obesity [body mass index (BMI) ≥30 kg/m2] has been shown to be a risk factor in reducing the rate of successful in vitro fertilization (IVF) outcomes. In this systematized literature review, we investigate whether embryo quality specifically is affected by single-parent obesity (maternal or paternal) and obesity in both parents at the time of conception. We systematically searched for studies published over the last 10 years that graded embryo quality in conventional IVF and intracytoplasmic sperm injection procedures. Studies that did not use measured weight and height for BMI calculation were excluded. Ten eligible articles were found across 13 public databases. Comparing the results of 1845 couples, both female and male obesity independently caused faster preimplantation embryo development (P &lt;0.05). The fertilization rate was not affected by either female, male, or combined parental obesity (P&gt;0.05). Day 3 embryo quality does not seem to be affected by either female or male obesity alone. The 2 studies that investigated the effect of combined parental obesity on embryo quality reached contradictory findings. Male, but not female, &gt;7% BMI reduction following bariatric surgery significantly increased the number of top-quality embryos compared with their results before surgery (P&lt;0.05). We conclude that embryos of couples with single-parent obesity are not of lower quality compared with those of normal-weight parents. Combined parental obesity may affect embryo quality. BMI reduction could lead toward a successful outcome in obese infertile couples, regardless of whether they are undergoing IVF or not.

Intracytoplasmic sperm injection cycle success in patients under 35 years old with diminished ovarian reserve plus severe male factor.

We aimed to evaluate intracytoplasmic sperm injection (ICSI) outcomes in young patients with diminished ovarian reserve (DOR) plus severe male factor (SMF) compared with age-matched controls with DOR. A total of 189 infertile women under 35 years with DOR undergoing ICSI procedures were included retrospectively. Participants whose partners' sperm analysis was normal considered as the DOR group (n=154) and whose partners' had SMF considered as the DOR + SMF group (n=35). The two groups were compared regarding cycle characteristics and pregnancy outcomes. Demographic features except infertility duration were similar between two groups. The duration of infertility was significantly longer in the DOR + SMF group compared to the DOR group (p=0.02). Ovarian stimulation characteristics, oocyte retrieval parameters, fertilization rate, quality of embryos, embryo cancellation rate, and development up to blastocyst stage were found similar between two groups. Implantation, clinical pregnancy, abortion, and live birth rate, multiple pregnancy rate per cycle were distributed homogenously between the DOR and DOR + SMF groups. Regarding perinatal and neonatal outcomes of groups, fetal height and weight were significantly lower in DOR + SMF group than in DOR group (p=0.001 and 0.01, respectively). Gestational week at delivery was lower in the DOR + SMF group compared to the DOR group (p < 0.0001). Fetal anthropometric measures were lower regarding to preterm delivery in the DOR + SMF group than the DOR group. Large sample-sized studies should be performed to explain the decreased gestational week at the time of delivery in the DOR + SMF group.