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.

Live birth resulting from a conjoined oocyte confirmed as euploid using array CGH: a case report.

The significance of conjoined oocytes in the clinical IVF laboratory setting has been of question due to the extremely limited data available. The most reliable criterion for true binovularity is the inclusion of two oocytes within a common zona pellucida or their fusion in the zonal region. This is a relatively rare event and owing to the limited number of embryo transfers performed and information on their outcomes, it is highly probable that these oocytes would be discarded without attempts at fertilization and subsequent embryo culture. To our knowledge, this is the first reported pregnancy resulting from a conjoined oocyte. Our experience involved a blastocyst transfer of a genetically screened embryo, performed after removal of the germinal vesicle from the conjoined oocyte/embryo on day 3. A clinical pregnancy with a gestational sac and fetal heartbeat was achieved and a healthy baby girl was delivered via Caesarean section at 37 weeks' gestation.

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.

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.

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.

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.

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.

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 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.

Is measurement of progesterone level prior to FSH stimulation useful in GnRH-antagonist cycles?

AIM: To reduce patient inconvenience during in vitro fertilization (IVF) cycles, some protocols delay intensive monitoring until mid-follicular stimulation. Others assess hormone levels prior to follicle-stimulating hormone (FSH) administration, not commencing stimulation until baseline progesterone (P4) levels (< 5 nmol/l) are achieved. Higher P4 levels (> 4.8 nmol/L) on the day of FSH trigger have been implicated in poorer pregnancy rates. This study evaluates the association of P4 levels at day 1-2 in gonadotrophin-releasing hormone (GnRH)-antagonist cycles with pre-trigger P4 levels and clinical pregnancy rates (CPRs). METHOD: All fresh GnRH-antagonist IVF cycles between June 2011 and June 2012, in which pre-FSH P4 levels were not routinely performed (group 1), were retrieved from the IVF Australia database and compared with controls (group 2). RESULTS: There were 163 cycles in each group. P4 levels on the day of trigger were significantly higher in group 1 (3.75 vs. 2.77, p < 0.05). The incidence of pre-trigger P4 levels >4.8 nmol/l was significantly higher in group 1 (30 vs. 16, p < 0.05). The number of oocytes retrieved was higher in group 1 (11.1 vs. 9, p < 0.05), however fertilization rates were significantly lower in that group (53.6% vs. 61.2%, p < 0.05); CPRs were similar between the two groups (27.8% vs. 31.8%, p = ns). Overall, pregnancy rates were lower in cycles with pre-trigger P4 level of > 4.8 nmol/L compared with those with lower levels (15% vs. 32.5%, p < 0.05). CONCLUSION: We found that measurement of P4 level at early follicular phase was associated with significantly lower pre-trigger levels. However, this did not translate into a difference in CPR between the monitored and unmonitored groups. We have confirmed that elevation in pre-trigger P4 level is associated with halving of the CPR, indicating that the most important P4 measurements are those in the late follicular/pre-trigger phase.

Meiotic spindle normality predicts live birth in patients with recurrent in vitro fertilization failure.

OBJECTIVE: To investigate the impact of meiotic spindle normality on live birth rates in women with recurrent IVF failure. DESIGN: A retrospective comparative study. SETTING: A large private sector IVF service in Sydney, Australia. PATIENT(S): Five hundred five intracytoplasmic sperm injection (ICSI)/ET cycles of patients who fulfilled the criteria for recurrent IVF failure, three or more previous fresh or frozen ETs with no ongoing pregnancy. INTERVENTION(S): Polarized light microscopy was used at the time of ICSI. MAIN OUTCOME MEASURE(S): Clinical pregnancy rates (PR) and live birth rates (LBR) were compared depending on the morphology of the meiotic spindle of the oocyte(s) from which the embryo(s) were transferred. RESULT(S): Women receiving embryos where at least one was derived from a normally spindled oocyte had significantly increased clinical PR and LBR when compared with those who had only embryos derived from abnormally spindled oocytes (clinical PR: 31% vs. 7%; odds ratio [OR], 6.45; 95% confidence interval [CI] 3.65-11.40) and (LBR: 24% vs. 4%; OR, 7.24; 95% CI 3.62-14.49). Comparison between the abnormal spindle groups showed significantly higher clinical PR and LBR from the group of patients receiving embryos where at least one was derived from an oocyte with no visible spindle compared with the group receiving embryos from dysmorphic spindles only (clinical PR: 9% vs. 6%; OR, 0.58; 95% CI 0.22-1.57 and LBR: 8% vs. 1%; OR, 0.16; 95% CI 0.03-0.77). CONCLUSION(S): Normally spindled oocytes, as determined by polarized light microscopy, are associated with significantly higher clinical PRs in patients with recurrent IVF failure.

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.

Twice-frozen embryos are no detriment to pregnancy success: a retrospective comparative study.

OBJECTIVE: To compare the pregnancy rates (PR) and live birth rates in once- versus twice-frozen ET treatment cycles in the same cohort of women. DESIGN: A retrospective study. SETTING: Fertility clinics, IVF Australia, New South Wales. PATIENT(S): The study population was all women who underwent thawing of twice-frozen embryos between January 2003 and May 2009. INTERVENTION(S): Twice-frozen, twice-thawed embryos. MAIN OUTCOME MEASURE(S): Pregnancy and live birth rate. RESULT(S): There were 44 women who had 52 twice-frozen ET treatment cycles. The mean age of the women was 32 ± 4.4 years and the mean number of embryos transferred was 1.1 in both the once-frozen and twice-frozen ET treatment cycles. Twice-frozen embryos had a lower post-thaw survival rate compared with the once-frozen embryos. There was no significant difference in the clinical PR or live birth rate per ET between twice-frozen and once-frozen ETs. CONCLUSION(S): Twice-frozen-thawed embryos have a lower post- thaw survival rate but equivalent pregnancy and live birth rates to once-frozen embryos. Further studies are necessary to confirm our findings and to assess long-term safety outcomes.

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.