Evaluation of an artificial intelligence-facilitated sperm detection tool in azoospermic samples for use in ICSI.

RESEARCH QUESTION: Can artificial intelligence (AI) improve the efficiency and efficacy of sperm searches in azoospermic samples? DESIGN: This two-phase proof-of-concept study began with a training phase using eight azoospermic patients (>10,000 sperm images) to provide a variety of surgically collected samples for sperm morphology and debris variation to train a convolutional neural network to identify spermatozoa. Second, side-by-side testing was undertaken on two cohorts of non-obstructive azoospermia patient samples: an embryologist versus the AI identifying all the spermatozoa in the still images (cohort 1, n = 4), and a side-by-side test with a simulated clinical deployment of the AI model with an intracytoplasmic sperm injection microscope and the embryologist performing a search with and without the aid of the AI (cohort 2, n = 4). RESULTS: In cohort 1, the AI model showed an improvement in the time taken to identify all the spermatozoa per field of view (0.02 ± 0.30  ×  10-5s versus 36.10 ± 1.18s, P < 0.0001) and improved recall (91.95 ± 0.81% versus 86.52 ± 1.34%, P < 0.001) compared with an embryologist. From a total of 2660 spermatozoa to find in all the samples combined, 1937 were found by an embryologist and 1997 were found by the AI in less than 1000th of the time. In cohort 2, the AI-aided embryologist took significantly less time per droplet (98.90 ± 3.19 s versus 168.7 ± 7.84 s, P < 0.0001) and found 1396 spermatozoa, while 1274 were found without AI, although no significant difference was observed. CONCLUSIONS: AI-powered image analysis has the potential for seamless integration into laboratory workflows, to reduce the time to identify and isolate spermatozoa from surgical sperm samples from hours to minutes, thus increasing success rates from these treatments.

Quantitative Polymerase Chain Reaction for the estimation of toxigenic microalgae abundance in shellfish production waters.

Certain species of marine microalgae produce potent biotoxins that pose a risk to human health if contaminated seafood is consumed, particularly filter feeding bivalve shellfish. In regions where this is likely to occur water and seafood produce are regularly monitored for the presence of harmful algal cells and their associated toxins, but the current approach is flawed by a lengthy delay before results are available to local authorities. Quantitative Polymerase Chain Reaction (qPCR) can be used to measure phytoplankton DNA sequences in a shorter timeframe, however it is not currently used in official testing practices. In this study, samples were collected almost weekly over six months from three sites within a known HAB hotspot, St Austell Bay in Cornwall, England. The abundance of algal cells in water was measured using microscopy and qPCR, and lipophilic toxins were quantified in mussel flesh using LC-MS/MS, focusing on the okadaic acid group. An increase in algal cell abundance occurred alongside an increase in the concentration of okadaic acid group toxins in mussel tissue at all three study sites, during September and October 2021. This event corresponded to an increase in the measured levels of Dinophysis accuminata DNA, measured using qPCR. In the following spring, the qPCR detected an increase in D. accuminata DNA levels in water samples, which was not detected by microscopy. Harmful algal species belonging to Alexandrium spp. and Pseudo-nitzschia spp. were also measured using qPCR, finding a similar increase in abundance in Autumn and Spring. The results are discussed with consideration of the potential merits and limitations of the qPCR technique versus conventional microscopy analysis, and its potential future role in phytoplankton surveillance under the Official Controls Regulations pertaining to shellfish.

Inter- and Intra-Observer Agreement Between Embryologists for Cytoplasmic String Assessment in Day 5/6 Human Blastocysts.

To investigate inter- and intra-observer agreement in the assessment of cytoplasmic string (CS) by embryologists on day 5/6 human blastocysts using the EmbryoViewer software. This was a prospective study involving five embryologists working between 2019 and 2020. Inter-observer agreement was calculated using assessments performed on 104 day 5/6 blastocysts regarding the presence, number, and location of CS and CS vesicle activity using timelapse videos. Intra-observer agreement was calculated when the same embryologists repeated the observations after a month's break. Inter- and intra-observer agreement was assessed using Fleiss' kappa coefficient and the intra-class correlation coefficient (ICC). The inter-observer agreement on the presence of CS (kappa: 0.477, 95% CI: 0.301-0.639) and their vesicles (kappa: 0.494, 95% CI: 0.345-0.643) was moderate, while the specific characteristics of CS assessment ranged from fair to moderate (kappa scores between: 0.157 and 0.563). The intra-observer agreement indicated an improvement on the level of agreement (kappa scores between: 0.162 and 0.795) compared to the inter-observer agreement. This study has shown a moderate level of inter- and intra-observer agreement when assessing day 5/6 human blastocysts for the presence of CS and their vesicles. When the specific characteristics of CS assessment occurred (such as the number of CS/vesicles) a slight to moderate level of agreement was seen among the embryologists. Agreement of specific characteristics of CS was not optimal, suggesting the need for further training using specifically designed CS quality assurance programme (QAP) modules, to determine if inter- and intra-observer agreement can be improved.

Detailed analysis of cytoplasmic strings in human blastocysts: new insights.

The aim of this study was to determine if there was an association between the presence of cytoplasmic strings (CS) and their characteristics, with blastocyst quality, development and clinical outcome in human blastocysts. This two-centre cohort study was performed between July 2017 and September 2018 and involved a total of 1152 blastocysts from 225 patients undergoing in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). All embryos were cultured in Embryoscope+ and were assessed for CS using time-lapse images. A single assessor examined all blastocysts and reviewed videos using the EmbyroViewer® Software. Blastocyst quality was assessed on day 5 of embryo development. The number of CS, location and duration of their activity was recorded on days 5/6. A positive association between the presence of CS in human blastocysts with blastocyst quality was identified. Blastocysts with a higher number of CS present, were of higher quality and were in the more advanced stages of development. Top quality blastocysts had CS activity present for longer, as well as having a higher number of vesicles present travelling along the CS. Blastocysts that had CS present, had a significantly higher live birth rate. This study has confirmed that a higher number of CS and vesicles in human blastocysts is associated with top quality blastocysts and is not a negative predictor of development. They had a higher number of CS present that appeared earlier in development and, although ceased activity sooner, had a longer duration of activity. Blastocysts with CS had a significant increase in live birth rate.

The presence of cytoplasmic strings in human blastocysts is associated with the probability of clinical pregnancy with fetal heart.

PURPOSE: Is the presence of cytoplasmic strings (CS) in human blastocysts associated with the probability of clinical pregnancy with fetal heart (CPFH) after transfer. METHODS: This case-control study involved 300 single blastocyst transfers. 150 of these resulted in a CPFH (cases) while 150 did not (controls). All embryos were cultured in Embryoscope+ and AI software (IVY) was used to select the blastocyst with the highest score from the cohort for transfer. An embryologist, blind to the transfer outcome, recorded the CS number, location, and duration of their activity. RESULTS: There was a significant difference in the number of blastocysts that contained CS, with 97.3% of women's blastocysts resulting in +CPFH containing the CS compared to 88.7% of blastocysts in women who did not have a pregnancy (p = 0.007, OR; 4.67, CI 95% 1.5-14.2). CS appeared 2.4 h earlier in embryo development in the +CPFH group compared to their negative counterparts (p = 0.007). There was a significant difference in the average number of CS/blastocyst with a higher number being present in those that achieved a clinical pregnancy (mean: 6.2, SD 2.9) compared to those that did not (mean: 4.6, SD 3.0) (p ≤ 0.0001). There was a significant increase in the number of vesicles seen traveling along the CS with more seen in the blastocysts resulting in a +CPFH (mean: 4.3 SD 2.1) compared to those in the -CPFH group (mean: 3.1, SD 2.1). CONCLUSION: This study has shown that the presence of cytoplasmic strings in human blastocysts is associated with the probability of clinical pregnancy with fetal heart.

Oocyte meiotic spindle morphology is a predictive marker of blastocyst ploidy-a prospective cohort study.

OBJECTIVE: To evaluate oocyte meiotic spindle (OMS) morphology at intracytoplasmic sperm injection (ICSI) as a predictor of blastocyst ploidy and whether OMS morphology could aid standard morphology-based blastocyst selection. DESIGN: Prospective cohort study. SETTING: In vitro fertilization clinic. PATIENT(S): Patients undergoing ICSI cycles with an intention to perform preimplantation genetic testing for aneuploidy (PGT-A) from October 2014 to December 2017. INTERVENTION(S): The OMS was visualized with the use of polarized light microscopy at the time of ICSI and the morphology classified as normal, dysmorphic, translucent, not visible, or in telophase. Blastocyst biopsy for PGT-A was performed on embryos with suitable development. MAIN OUTCOME MEASURE(S): The association of OMS morphology with the resulting blastocyst ploidy was evaluated on an "intention-to-treat" (ITT) and an "as-treated analysis" (ATA) basis. RESULT(S): The morphology of 2,056 OMSs were classified. A strong association of OMS morphology with fertilization, cleavage to at least 6 cells on day 3, and good/top-quality blastocyst formation was present. Normal OMS was positively associated with blastocyst euploidy compared with all other OMS types combined, per either ITT or ATA. Even after controlling for female age, blastocyst quality, and developmental stage, the presence of a normal OMS was strongly associated with the probability of blastocyst euploidy. CONCLUSION(S): OMS morphology is a predictive marker of blastocyst ploidy and can potentially aid standard morphology-based blastocyst selection.

Fine-tuning blastocyst selection based on morphology: a multicentre analysis of 2461 single blastocyst transfers.

RESEARCH QUESTION: Which blastocyst morphology parameter is associated with live birth after controlling for female age and endometrial receptivity? DESIGN: Retrospective study including fresh single blastocyst transfers (n = 2461) where the value of serum progesterone on day of human chorionic gonadotrophin trigger (PdHCG) was available. Generalized estimating equation regression models evaluated the independent effects of developmental stage (DevSt), inner cell mass (ICM) and trophectoderm grade on live birth rates while controlling for the confounding effects of female age and PdHCG. RESULTS: DevSt was strongly associated with the probability of live birth (P < 0.0001) independently of female age (odds ratio [OR] 0.89, 95% confidence interval [CI] 0.87-0.91) and PdHCG (OR 0.80, 95% CI 0.74-0.87). For full blastocysts, expanded blastocysts and hatching blastocysts, addition of ICM and trophectoderm grading in the multivariable analysis suggested that besides female age (OR 0.92, 95% CI 0.90-0.94) and PdHCG (OR 0.80, 95% CI 0.73-0.87), only DevSt (P = 0.001) and trophectoderm quality (P = 0.004) were independent predictors of live birth, while the predictive capacity of ICM was no longer significant. The mean probability of live birth was highest for AA blastocysts (35.0%), followed by BA blastocysts (31.2%) and AB blastocysts (27.7%). CONCLUSION: This large study analyses for the first time the independent role of blastocyst morphology in predicting live birth while controlling for female age and PdHCG. Its findings suggest that DevSt and then trophectoderm grade are stronger predictors of live birth over ICM grade when selecting a single blastocyst for transfer.

Time-lapse algorithms and morphological selection of day-5 embryos for transfer: a preclinical validation study.

OBJECTIVE: To determine the agreement between published time-lapse algorithms in selecting the best day-5 embryo for transfer, as well as the agreement between these algorithms and embryologists. DESIGN: Prospective study. SETTING: Private in vitro fertilization center. PATIENT(S): Four hundred and twenty-eight embryos from 100 cycles cultured in the EmbryoScope. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Interalgorithm agreement as assessed by the Fleiss kappa coefficient. RESULT(S): Of seven published algorithms analyzed in this study, only one of the 18 possible pairs showed very good agreement (κ = 0.867); one pair showed good agreement (κ = 0.725), four pairs showed fair agreement (κ = 0.226-0.334), and the remaining 12 pairs showed poor agreement (κ = 0.008-0.149). Even in the best-case scenario, the majority of algorithms showed poor to moderate kappa scores (κ = 0.337-0.722) for the assessment of agreement between the embryo(s) selected as "best" by the algorithms and the embryo that was chosen by the majority (>5) of embryologists, as well as with the embryo that was actually selected in the laboratory on the day of transfer (κ = 0.315-0.802). CONCLUSION(S): The results of this study raise concerns as to whether the tested algorithms are applicable in different clinical settings, emphasizing the need for proper external validation before clinical use.

Is early embryo development as observed by time-lapse microscopy dependent on whether fresh or frozen sperm was used for ICSI? A cohort study.

PURPOSE: The aim of this study was to compare timings of key events of embryo development from those originating from either fresh or cryopreserved ejaculate sperm using time-lapse technology. METHODS: In this retrospective observational cohort study, time-lapse technology was used to monitor 1927 embryos from 234 women undergoing intracytoplasmic sperm injection (ICSI) and utilizing either fresh (n = 172 cycles) or cryopreserved ejaculate sperm (n = 62 cycles) for insemination were included in the study. Key developmental events as described in time-lapse were compared with the use of generalized estimating equations (GEE) to adjust for any auto-correlation between the observations. In addition, multivariable logit regression models were used to account for any known baseline differences between the two groups. RESULTS: There were no differences in conventional embryo development such as number of 8-cell embryos by 72 h (p = 0.359), the number of blastocysts by 120 h (p = 0.417), and the number of top quality blastocysts (p = 0.956) between the two groups compared. There were no statistical differences in the timings of any of the key embryo developmental events (PN_t1, NEBD, cytokinesis, t2, t3, t4, t5, t6, t7, t8, tM, tSB, tEB, tHB, s1, s2, s3, cc2, and cc3) when either fresh or cryopreserved ejaculate sperm was used for ICSI. This was also confirmed with conventional morphological assessment. CONCLUSIONS: This observational cohort study has shown that there are no differences in the morphokinetic parameters of early embryo development when either fresh or frozen ejaculate sperm are used for ICSI insemination.

Inter-observer and intra-observer agreement between embryologists during selection of a single Day 5 embryo for transfer: a multicenter study.

STUDY QUESTION: What is the inter-observer and intra-observer agreement between embryologists when selecting a single Day 5 embryo for transfer? SUMMARY ANSWER: The inter-observer and intra-observer agreement between embryologists when selecting a single Day 5 embryo for transfer was generally good, although not optimal, even among experienced embryologists. WHAT IS KNOWN ALREADY: Previous research on the morphological assessment of early stage (two pronuclei to Day 3) embryos has shown varying levels of inter-observer and intra-observer agreement. However, single blastocyst transfer is now becoming increasingly popular and there are no published data that assess inter-observer and intra-observer agreement when selecting a single embryo for Day 5 transfer. STUDY DESIGN, SIZE, DURATION: This was a prospective study involving 10 embryologists working at five different IVF clinics within a single organization between July 2013 and November 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: The top 10 embryologists were selected based on their yearly Quality Assurance Program scores for blastocyst grading and were asked to morphologically grade all Day 5 embryos and choose a single embryo for transfer in a survey of 100 cases using 2D images. A total of 1000 decisions were therefore assessed. For each case, Day 5 images were shown, followed by a Day 3 and Day 5 image of the same embryo. Subgroup analyses were also performed based on the following characteristics of embryologists: the level of clinical embryology experience in the laboratory; amount of research experience; number of days per week spent grading embryos. The agreement between these embryologists and the one that scored the embryos on the actual day of transfer was also evaluated. Inter-observer and intra-observer variability was assessed using the kappa coefficient to evaluate the extent of agreement. MAIN RESULTS AND THE ROLE OF CHANCE: This study showed that all 10 embryologists agreed on the embryo chosen for transfer in 50 out of 100 cases. In 93 out of 100 cases, at least 6 out of the 10 embryologists agreed. The inter-observer and intra-observer agreement among embryologists when selecting a single Day 5 embryo for transfer was generally good as assessed by the kappa scores (kappa = 0.734, 95% CI: 0.665-0.791 and 0.759, 95% CI: 0.622-0.833, respectively). The subgroup analyses did not substantially alter the inter-observer and intra-observer agreement among embryologists. The agreement when Day 3 images were included alongside Day 5 images of the same embryos resulted in a change of mind at least three times by each embryologist (on average for <10% of cases) and resulted in a small decrease in inter-observer and intra-observer agreement between embryologists (kappa = 0.676, 95% CI: 0.617-0.724 and 0.752, 95% CI: 0.656-808, respectively).The assessment of the inter-observer agreement with regard to morphological grading of Day 5 embryos showed only a fair-to-moderate agreement, which was observed across all subgroup analyses. The highest overall kappa coefficient was seen for the grading of the developmental stage of an embryo (0.513; 95% CI: 0.492-0.538). The findings were similar when the individual embryologists were compared with the embryologist who made the morphological assessments of the available embryos on the actual day of transfer. LIMITATIONS, REASONS FOR CAUTION: All embryologists had already completed their training and were working under one organization with similar policies between the five clinics. Therefore, the inter-observer agreement might not be as high between embryologists working in clinics with different policies or with different levels of training. WIDER IMPLICATIONS OF THE FINDINGS: The generally good, although not optimal uniformity between participating embryologists when selecting a Day 5 embryo for transfer, as well as, the surprisingly low agreement when morphologically grading Day 5 embryos could be improved, potentially resulting in increased pregnancy rates. Future studies need to be directed toward technologies that can help achieve this. STUDY FUNDING/COMPETING INTERESTS: None declared. TRIAL REGISTRATION NUMBER: Not applicable.

Is oocyte meiotic spindle morphology associated with embryo ploidy? A prospective cohort study.

OBJECTIVE: To explore whether an association exists between oocyte meiotic spindle morphology visualized by polarized light microscopy at the time of intracytoplasmic sperm injection and the ploidy of the resulting embryo. DESIGN: Prospective cohort study. SETTING: Private IVF clinic. PATIENT(S): Patients undergoing preimplantation genetic screening/diagnosis (n = 113 patients). INTERVENTION(S): Oocyte meiotic spindles were assessed by polarized light microscopy and classified at the time of intracytoplasmic sperm injection as normal, dysmorphic, translucent, telophase, or no visible spindle. Single blastomere biopsy was performed on day 3 of culture for analysis by array comparative genomic hybridization. MAIN OUTCOME MEASURE(S): Spindle morphology and embryo ploidy association was evaluated by regression methods accounting for non-independence of data. RESULT(S): The frequency of euploidy in embryos derived from oocytes with normal spindle morphology was significantly higher than all other spindle classifications combined (odds ratio [OR] 1.93, 95% confidence interval [CI] 1.33-2.79). Oocytes with translucent (OR 0.25, 95% CI 0.13-0.46) and no visible spindle morphology (OR 0.35, 95% CI 0.19-0.63) were significantly less likely to result in euploid embryos when compared with oocytes with normal spindle morphology. There was no significant difference between normal and dysmorphic spindle morphology (OR 0.73, 95% CI 0.49-1.08), whereas no telophase spindles resulted in euploid embryos (n = 11). Assessment of spindle morphology was found to be independently associated with embryo euploidy after controlling for embryo quality (OR 1.73, 95% CI 1.16-2.60). CONCLUSION(S): Oocyte spindle morphology is associated with the resulting embryo's ploidy. Oocytes with normal spindle morphology are significantly more likely to produce euploid embryos compared with oocytes with meiotic spindles that are translucent or not visible.

Morphokinetic parameters using time-lapse technology and day 5 embryo quality: a prospective cohort study.

PURPOSE: The aims of this prospective study were to evaluate whether time-lapse parameters can aid in the prediction of day 5 embryo quality and also to assess their discriminatory capacity. METHODS: In this prospective cohort study, we used time-lapse technology to record specific timings of key events for 380 day 5 blastocysts (originating from 108 patients). Generalized estimating equation regression models were used to evaluate the capacity of these markers to identify a top-quality blastocyst. Multivariable regression models were also constructed, aiming to identify the model with the highest capacity to predict a top-quality blastocyst. The discriminatory capacity of single predictors or composite models was assessed with the use of receiver operating characteristic (ROC) analyses. RESULTS: Eight significant predictive parameters of a top-quality blastocyst were identified: s3, t6, t7, t8, tM, tSB, tB and tEB. A ROC analysis of the identified parameters found s3 (area under the curve--AUC 0.585, 95 % CI 0.534-0.635) to have the best individual discriminatory capacity to predict a top-quality blastocyst prior to embryo compaction. The parameter tEB (AUC 0.727, 95 % CI 0.675-0.775) was the best predictor regardless of embryo stage. A model containing s3, t8 and tEB showed a slightly increased discriminatory capacity for top-quality blastocyst prediction (AUC 0.748, 95 % CI 0.697-0.794). CONCLUSIONS: The identified morphokinetic parameters and their cutoffs, albeit of limited clinical value, add to the increasing knowledge concerning the potential predictive markers of a top-quality blastocyst. Additional evidence is necessary before validated time-lapse parameters can be used for embryo selection in IVF laboratories.

The relationship between sperm head retardance using polarized light microscopy and clinical outcomes.

In human sperm head, birefringence can be seen under polarized light resulting from highly ordered structures within the acrosome and nucleus. Selecting sperm with partial head birefringence improves success of clinical pregnancies in patients with severe male factor infertility. The aim of this study was to establish a range of retardance in sperm heads using polarized light microscopy to select an optimum sperm for intracytoplasmic sperm injection (ICSI). Sperm heads of 63 couples undergoing ICSI in women aged 38 years or younger were imaged at the time of ICSI and later analysed for retardance blinded to embryo and cycle outcomes. Sperm head retardance was similar irrespective of whether fertilization occurred. Quality of embryos on day 3 and day 5 were higher when sperm were selected with head retardance ranging from 0.56 nm or greater to 0.91 nm or less. Selection of sperm with head retardance ranging from 0.56 nm or greater to 0.91 nm or less was associated with higher clinical pregnancy rates (OR 3.74 95% CI 1.43 to 9.77). Optimum sperm for selection at the time of ICSI was with head retardance within the range 0.56 nm or greater to 0.91 nm or less.

An analysis of the impact of embryo transfer difficulty on live birth rates, using a standardised grading system.

BACKGROUND: Embryo transfer (ET) is a vital part of the IVF process. While some studies have supported the hypothesis that ET difficulty alters success rate, others suggest the contrary. This large population study aimed to test this hypothesis, using a standardised ET grading system. METHODS: ET data for fresh and frozen IVF/ICSI cycles between 2005 and 2010, retrieved from the IVFAustralia database, were grouped by degree of difficulty (easy, medium and difficult). Live birth rates, as the primary outcome, were compared between the groups. Biochemical pregnancy, Clinical Pregnancy Rate (CPR) and other pregnancy outcomes were assessed. The influence of blood ± mucus contamination on the transfer catheter tip and the use of intra-transfer ultrasound (US) were also analysed. RESULTS: Of 6484 transfers, 5976 (92.2%) were considered easy, 374 (5.7%) medium and 134 (2.1%) difficult. Basic patient characteristics were similar between the cohorts. There were statistically significant differences in the live delivery rates (25.3% vs 19.5%, p < 0.05), CPR (30.7% vs 24.6%, p < 0.05), and + ve ßhCG rate (35.9% vs 28%, p < 0.05) between the easy and medium/difficult groups. There was no significant difference in the rates of ectopic pregnancy, stillbirth and miscarriage between the groups. The presence of blood on the catheter did not affect pregnancy outcome adversely (p = ns). CONCLUSION: This study, which we believe to be the largest report using a standardised system for grading the difficulty of ET, demonstrates that difficult ETs are associated with lower live birth rates but not higher rates of ectopic or miscarriage. While the presence of blood may be an indicative of a relatively more traumatic and difficult transfer, it does not necessarily lead to a lower birth rate.

Does meiotic spindle normality predict improved blastocyst development, implantation and live birth rates?

OBJECTIVE: To investigate the relationship between spindle normality and: 1) blastocyst formation; 2) implantation rates; and 3) live birth rates. DESIGN: Prospective observational study. SETTING: A private fertility clinic. PATIENT(S): One hundred patients ≤40 years old undergoing intracytoplasmic sperm injection (ICSI). INTERVENTION(S): Meiotic spindles were imaged before ICSI with the use of Oosight microscopy. With the use of specific criteria, spindle normality was independently assessed by two researchers. Blastocysts were chosen for transfer by standard light microscopic morphologic criteria by researchers who were blinded to the spindle assessment. MAIN OUTCOME MEASURE(S): 1) Blastocyst formation; 2) implantation; and 3) live birth rates. RESULT(S): A total of 808/920 oocytes were metaphase II. Of those, 711 (88%) had a visible spindle: 205 (29%) were normal spindles (NS) and 506 (71%) abnormal spindles (AS). Fertilization rates were significantly higher in NS oocytes. Although NS and AS oocytes both formed morphologically good-quality blastocysts, implantation and live birth rates were higher from NS oocytes. All ongoing pregnancies resulted from NS oocytes. CONCLUSION(S): Spindle assessment with polarized light microscopy provides an early predictor of the pregnancy potential of that oocyte. AS oocytes can form good-quality blastocysts, but these appear to have little chance of live birth. Therefore, spindle assessment should improve the selection of the best embryo for single embryo transfer.

Time course of meiotic spindle development in MII oocytes.

The aim of this study was to examine changes in meiotic spindle morphology over time to potentially optimize timing for ICSI. Using polarized light microscopy, images of MII oocytes were captured after retrieval of oocytes in stimulated cycles at six time intervals in culture: 36-36.5 h, 36.5-37.0 h, 38-38.5 h, 39-39.5 h, 40-40.5 h and 40.5-41 h post hCG. Captured images were analysed for spindle presence and their retardance. Results showed that spindles were detected in 58% (45/78) of oocytes at 36-36.5 h. This percentage rose to a peak (96% vs. 58%, p < 0.001) at 39-39.5 h and stabilized between 39-40.5 h post trigger then significantly declined at 40.5-41 h post hCG (96% vs. 77%, p < 0.001). Average spindle retardance increased from 36-36.5 h (1.8 ± 0.7 nm) until it peaked at 39-40.5 h (3.8 ± 0.8 nm, p < 0.0001) and then declined significantly after 40.5-41 h (3.2 ± 0.9 nm, p = 0.0001). These results show that the meiotic spindle appearance is time dependent with the majority of oocytes having detectable spindles and highest retardance between 39-40.5 h post hCG under currently used stimulation protocol after which they start to disaggregate. 39-40.5 h post hCG may be the optimal time for ICSI.

Are there non-invasive markers in human oocytes that can predict pregnancy outcome?

Predictive criteria for selection of the best embryo for single embryo transfer remain elusive. This study aimed to determine if non-invasive markers in human oocytes, detectable using polarized light microscopy, can predict pregnancy outcome. Twenty-two pregnancy-producing oocytes from 19 patients had their morphological features compared with 30 oocytes from 19 age-matched patients whose transfer did not result in a pregnancy. Both pregnant and non-pregnant patients had similar numbers of oocytes collected (average: 11.9 +/- 2.8 versus 11.3 +/- 2.9) and similar fertilization rates (70.1% versus 69.6%). All embryos transferred were 4-cell cleavage-stage on day 2 with <10% fragmentation. Meiotic spindles were examined at 39-40 h following human chorionic gonadotrophin administration for spindle normality, length, density and angle from first polar body. There was a significant difference in spindle normality in oocytes in the pregnant patients compared with oocytes in the non-pregnant patients (100% versus 33%, P < 0.001). Spindle density was significantly higher in those oocytes resulting in pregnancy (3.0 +/- 1.23 nm versus 2.5 +/- 0.7 nm, P = 0.02). These oocyte markers may provide a useful non-invasive tool in the selection of the embryo most likely to produce a pregnancy.

Do age and extended culture affect the architecture of the zona pellucida of human oocytes and embryos?

Advanced female age and extended in vitro culture have both been implicated in zona pellucida (ZP) hardening and thickening. This study aimed to determine the influence of (i) the woman's age and (ii) prolonged in vitro culture of embryos on ZP thickness and density using non-invasive polarized light (LC-PolScope) microscopy. ZP thickness and density (measured as retardance) were determined in oocytes, embryos and blastocysts in women undergoing intracytoplasmic sperm injection (ICSI) in two age groups (older, > 38 years; younger, < or = 38 years). A total of 193 oocytes from 29 patients were studied. The younger group contained 100 oocytes and the older group 93 oocytes. The ZP was significantly thicker in metaphase II oocytes in the older group compared with the younger group (mean +/- SD: 24.1 +/- 2.5 microm vs 23.1 +/- 3.3 microm; p = 0.01) but ZP density was equal (2.8 +/- 0.7 nm). By day 2 of culture, embryos from the two groups had similar ZP thickness (22.2 +/- 2.2 microm vs 21.7 +/- 1.6 microm; p = 0.28) and density (2.9 +/- 0.7 nm vs 2.8 +/- 0.8 nm; p = 0.57). For the embryos cultured to blastocyst (older: n = 20; younger: n = 18) ZP thickness was similar in the two groups (19.2 +/- 2.7 microm vs 19.1 +/- 5.0 microm; p = 0.8) but thinner than on day 2. The older group had significantly denser ZP than the younger group (4.2 +/- 0.5 nm vs 3.3 +/- 1.0 nm, p < 0.01). Blastocysts from both groups had significantly denser ZP than their corresponding day 2 embryos (older: 4.2 +/- 0.5 nm vs 2.9 +/- 0.7 nm, p < 0.001; younger: 3.3 +/- 1.0 nm vs 2.8 +/- 0.8 nm, p = 0.013). It is concluded that there is little relationship between ZP thickness and its density as measured by polarized light microscopy. While ZP thickness decreases with extended embryo culturing, the density of the ZP increases. ZP density increases in both age groups with extended culture and, interestingly, more in embryos from older compared with younger women.

Equivalency of culture media for human in vitro fertilization formulated to have the same pH under an atmosphere containing 5% or 6% carbon dioxide.

Culture media for human in vitro fertilization and early embryonic development were modified to maintain similar pH values under an atmosphere containing 5% or 6% carbon dioxide by adjusting the concentration of sodium bicarbonate. Similar results were obtained with both media groups in a mouse embryo assay and in human in vitro parameters of fertilization and early development and clinical outcomes.

Improvement in early human embryo development using new formulation sequential stage-specific culture media.

OBJECTIVE: To determine whether altering selected components of sequential culture media can improve early development variables of human embryos. DESIGN: Prospective, randomized, sibling oocyte split trial. SETTING: Private ART center. PATIENT(S): Two hundred eight undergoing treatment with in vitro fertilization or microinjection. INTERVENTION(S): Oocytes from each patient were randomly allocated to fertilization and cleavage media of a control and a trial culture medium formulation. MAIN OUTCOME MEASURE(S): Rates of fertilization, cleavage, and uncontrolled division; average embryo morphology score; blastomeres per embryo; embryo score parameter (number of blastomeres x embryo morphology grade); and embryo utilization. The trial media resulted in a higher fertilization rate, higher cleavage rate, lower rate of uncontrolled division, higher number of blastomeres per embryo, higher average embryo morphology score, a higher embryo score parameter, and higher embryo utilization rate compared to the control media. All differences were statistically significant. CONCLUSION(S): Improved sequential stage-specific culture media can reduce the occurrence of severe human embryo fragmentation and improve developmental variables in early IVF- and ICSI-generated embryos.