Blastocyst quality and reproductive and perinatal outcomes: a multinational multicentre observational study.

STUDY QUESTION: Does the transfer of single low-grade blastocysts result in acceptable reproductive and perinatal outcomes compared to the transfer of single good-grade blastocysts? SUMMARY ANSWER: The transfer of single low-grade blastocysts resulted in a reduced live birth rate of around 30% (14% for very low-grade blastocysts) compared to 44% for single good-grade blastocysts, but does not lead to more adverse perinatal outcomes. WHAT IS KNOWN ALREADY: It is known that low-grade blastocysts can result in live births. However, the current studies are limited by relatively small sample sizes and single-centre designs. Furthermore, evidence on perinatal outcomes after transferring low-grade blastocysts is limited. STUDY DESIGN, SIZE, DURATION: We conducted a multi-centre, multi-national retrospective cohort study of 10 018 women undergoing 10 964 single blastocyst transfer cycles between 2009 and 2020 from 14 clinics across Australia, China, and New Zealand. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blastocysts were graded individually based on assessment of the morphology and development of the inner cell mass (ICM) and trophectoderm (TE), and were grouped into three quality categories: good- (AB, AB, or BA), moderate- (BB), and low-grade (grade C for ICM or TE) blastocysts. CC blastocysts were individually grouped as very low-grade blastocysts. Logistic regression with generalized estimating equation was used to analyse the association between blastocyst quality and live birth as well as other reproductive outcomes. Binomial, multinomial logistic, or linear regression was used to investigate the association between blastocyst quality and perinatal outcomes. Odds ratio (OR), adjusted OR (aOR), adjusted regression coefficient, and their 95% CIs are presented. Statistical significance was set at P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: There were 4386 good-grade blastocysts, 3735 moderate-grade blastocysts, and 2843 low-grade blastocysts were included in the analysis, for which the live birth rates were 44.4%, 38.6%, and 30.2%, respectively. Compared to good-grade blastocysts, the live birth rate of low-grade blastocysts was significantly lower (aOR of 0.48 (0.41-0.55)). Very low-grade blastocysts were associated with an even lower live birth rate (aOR 0.30 (0.18-0.52)) and their absolute live birth rate was 13.7%. There were 4132 singleton live births included in the analysis of perinatal outcomes. Compared with good-grade blastocysts, low-grade blastocysts had comparable preterm birth rates (<37 weeks, aOR 1.00 (0.65-1.54)), birthweight Z-scores (adjusted regression coefficient 0.02 (0.09-0.14)), and rates of very low birth weight (<1500 g, aOR 0.84 (0.22-3.25)), low birth weight (1500-2500 g, aOR 0.96 (0.56-1.65)), high birth weight (>4500 g, aOR 0.93 (0.37-2.32)), small for gestational age (aOR 1.63 (0.91-2.93)), and large for gestational age (aOR 1.28 (0.97-1.70)). LIMITATIONS, REASONS FOR CAUTION: Due to the nature of the retrospective design, residual confounding could not be excluded. In addition, the number of events for some perinatal outcomes was small. Between-operator and between-laboratory variations in blastocyst assessment were difficult to control. WIDER IMPLICATIONS OF THE FINDINGS: Patients undergoing IVF should be informed that low-grade blastocysts result in a lower live birth rate, however they do not increase the risk of adverse perinatal outcomes. Further research should focus on the criteria for embryos that should not be transferred and on the follow-up of long-term outcomes of offspring. STUDY FUNDING/COMPETING INTEREST(S): H.Z. is supported by a Monash Research Scholarship. B.W.J.M. is supported by a NHMRC Investigator grant (GNT1176437). R.W. is supported by an NHMRC Emerging Leadership Investigator grant (2009767). B.W.J.M. reports consultancy, travel support, and research funding from Merck. The other authors do not have competing interests to disclose. TRIAL REGISTRATION NUMBER: N/A.

Should we look for a low-grade threshold for blastocyst transfer? A scoping review.

Embryo quality is a key determinant of the success of IVF. Although the focus has been on selecting the best embryo for transfer, the classification of low-grade blastocysts (LGB) in existing scoring systems has received less attention. This is worrisome; embryo freezing allows optimal use of all created embryos, thus maximizing the cumulative live birth rate, which is arguably the most important outcome for infertile couples. A PubMed search was conducted in August 2020, using '((('poor-quality' OR 'poor quality') OR ('low-grade' OR 'low grade')) AND ('embryo' OR 'blastocyst')) AND ('pregnancy' OR 'live birth')'. This scoping review shows that LGB have similar euploidy and pregnancy success rates after implantation and have no adverse effects on pregnancy or perinatal outcomes. Evidence for pregnancy outcomes is lacking for different grades of LGB, with most studies clustering all LQB as one to compare with optimal blastocysts.

Should we freeze it? Agreement on fate of borderline blastocysts is poor and does not improve with a modified blastocyst grading system.

STUDY QUESTION: What is the inter-observer agreement among embryologists for decision to freeze blastocysts of borderline morphology and can it be improved with a modified grading system? SUMMARY ANSWER: The inter-observer agreement among embryologists deciding whether to freeze blastocysts of marginal morphology was low and was not improved by a modified grading system. WHAT IS KNOWN ALREADY: While previous research on inter-observer variability on the decision of which embryo to transfer from a cohort of blastocysts is good, the impact of grading variability regarding decision to freeze borderline blastocysts has not been investigated. Agreement for inner cell mass (ICM) and trophectoderm (TE) grade is only fair, factors which contribute to the grade that influences decision to freeze. STUDY DESIGN, SIZE, DURATION: This was a prospective study involving 18 embryologists working at four different IVF clinics within a single organisation between January 2019 and July 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: All embryologists currently practicing blastocyst grading at a multi-site organisation were invited to participate. The survey was comprised of blastocyst images in three planes and asked (i) the likelihood of freezing and (ii) whether the blastocyst would be frozen based on visual assessment. Blastocysts varied by quality and were categorised as either top (n = 20), borderline (n = 60) or non-viable/degenerate quality (n = 20). A total of 1800 freeze decisions were assessed. To assess the impact of grading criteria on inter-observer agreement for decision to freeze, the survey was taken once when the embryologists used the Gardner criteria and again 6 months after transitioning to a modified Gardner criterion with four grades for ICM and TE. The fourth grade was introduced with the aim to promote higher levels of agreement for the clinical usability decision when the blastocyst was of marginal quality. MAIN RESULTS AND THE ROLE OF CHANCE: The inter-observer agreement for decision to freeze was near perfect (kappa 1.0) for top and non-viable/degenerate quality blastocysts, and this was not affected by the blastocysts grading criteria used (top quality; P = 0.330 and non-viable/degenerate quality; P = 0.18). In contrast, the cohort of borderline blastocysts received a mixed freeze rate (average 52.7%) during the first survey, indicative of blastocysts that showed uncertain viability and promoting significant disagreement for decision to freeze among the embryologists (kappa 0.304). After transitioning to a modified Gardner criteria with an additional grading tier, the average freeze rate increased (64.8%; P < 0.0001); however, the inter-observer agreement for decision to freeze was unchanged (kappa 0.301). Therefore, significant disagreement for decision to freeze among embryologists is an ongoing issue not resolved by the two grading criteria assessed here. LIMITATIONS, REASONS FOR CAUTION: Blastocyst assessment was performed from time-lapse images in three planes, rather than with a microscope in the laboratory. The inter-observer agreement for decision to freeze may be lower for embryologists working in different clinics with different grading protocols. WIDER IMPLICATIONS OF THE FINDINGS: The decision to freeze a blastocyst with borderline morphology is a common clinical issue that has the potential to arise for any patient during blastocyst culture. Disagreement for decision to freeze these blastocysts, and therefore clinical usability in frozen embryo transfer cycles, affects consistency in patient care due to a potential impact on cumulative live birth rates, as well as financial, emotional and time costs associated with the frozen embryo transfer cycles. We demonstrate significant disagreement for decision to freeze borderline blastocysts among embryologists using the same grading scheme within a large multisite organisation, a phenomenon which was not improved with a modified grading system. Decision-making around borderline embryos is an area requiring further research, especially as studies continue to demonstrate the reduced but modest live birth rates for low quality blastocysts (Grade C). These results provide support for emerging technology for embryo assessment, such as artificial intelligence. STUDY FUNDING/COMPETING INTEREST(S): None declared. TRIAL REGISTRATION NUMBER: Not applicable.

Tracking quality: can embryology key performance indicators be used to identify clinically relevant shifts in pregnancy rate?

STUDY QUESTION: Can embryology key performance indicators (KPIs) detect shifts in laboratory performance that precede changes in clinical outcomes? SUMMARY ANSWER: Day 5 usable blastocyst rate (D5BUR) is an important embryology KPI that complements total usable blastocyst rate (TBUR) in its ability to rapidly identify adverse embryology outcomes, prior to changes in clinical outcomes. WHAT IS KNOWN ALREADY: The hypothesis that monitoring performance of an IVF laboratory using statistical process controls (SPCs) can act as an early warning signal of shifts in laboratory conditions is a hypothesis that requires validation. The Vienna consensus report recently defined KPIs for monitoring fresh IVF and ICSI cycles, but the effectiveness of using these KPIs for detecting clinically relevant shifts following changes in laboratory processes is unknown. STUDY DESIGN, SIZE, DURATION: A retrospective, multicentre, analysis of KPIs for 1971 fresh IVF and ICSI cycles during three consecutive 5-month periods (P1, P2 and P3) during which the culture medium was changed in the middle period. PARTICIPANTS/MATERIALS, SETTING, METHODS: ICSI fertilisation rate, IVF fertilisation rate, D5BUR, TBUR and clinical pregnancy rate (CPR) were tracked monthly and analysed for SPC using Shewhart control charts. Out-of-control KPIs were identified by warning (2-sigma) and control (3-sigma) limits. The effect of the laboratory culture medium change on embryology KPIs and cumulative CPR was investigated using a one-way ANOVA or Pearson Chi-squared test and logistic regression. MAIN RESULTS AND THE ROLE OF CHANCE: D5BUR decreased from 32 to 25% after the culture medium was changed, and the decrease was detected within 1 week after the change (P < 0.0001). D5BUR subsequently increased after a change back to the original medium. A decrease in CPR (51-36%) after the medium change was also observed but was not detected until 3 months after the shift in D5BUR (P = 0.0005). Overall, the change in culture medium independently influenced D5BUR, CPR and cumulative CPR. Importantly, TBUR (41%) was not affected by the change in culture medium, remaining within control limits for all three culture periods, indicating that the overall blastocyst rate alone may not sufficiently monitor embryology laboratory performance. LIMITATIONS REASONS FOR CAUTION: The statistical KPI monitoring system demonstrated by the current study may be less effective at identifying KPI shifts in smaller clinics with lower cycle volumes. Live-birth rate per cycle started was not included as a clinical KPI. WIDER IMPLICATIONS OF THE FINDINGS: This study demonstrates that statistical KPI monitoring systems have the potential to provide systematic, early detection of adverse outcomes in ART laboratories after planned or unexpected shifts in conditions. STUDY FUNDING/COMPETING INTEREST(S): No external funds were used for the study. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Should extended blastocyst culture include Day 7?

Extended culture to the blastocyst stage is widely practised, improving embryo selection and promoting single embryo transfer. Selection of useable blastocysts typically occurs on Days 5 and 6 of embryo culture. Embryos not suitable for transfer, biopsy or cryopreservation after Day 6 are routinely discarded. Some embryos develop at a slower rate, however, forming blastocysts on Day 7 of culture. Day 7 blastocysts can be viable, they can be of top morphological grade, euploid and result in a healthy live birth. Since ending culture on Day 6 is current practice in most clinics, viable Day 7 blastocysts may be prematurely discarded. Although Day 7 blastocysts make up only 5% of useable blastocysts, those which are suitable for cryopreservation or biopsy are clinically significant. Overall, culturing embryos an additional day increases the number of useable embryos per IVF cycle and provides further opportunity for pregnancy for patients, especially those who have only a few or low-quality blastocysts.

Oocyte mitochondrial deletions and heteroplasmy in a bovine model of ageing and ovarian stimulation.

STUDY HYPOTHESIS: Maternal ageing and ovarian stimulation result in the accumulation of mitochondrial DNA (mtDNA) deletions and heteroplasmy in individual oocytes from a novel bovine model for human assisted reproductive technology (ART). STUDY FINDING: The levels of mtDNA deletions detected in oocytes increased with ovarian ageing. Low levels of mtDNA heteroplasmy were apparent across oocytes and no relationship was identified with respect to ovarian ageing or ovarian stimulation. WHAT IS KNOWN ALREADY: Oocyte quality decreases with ovarian ageing and it is postulated that the mtDNA may have a role in this decline. The impact of ovarian stimulation on oocyte quality is poorly understood. Human studies investigating these effects are often limited by the use of low quality oocytes and embryos, variation in age and ovarian stimulation regimens within the patients studied, as well as genetic and environmental variability. Further, no study has investigated mtDNA heteroplasmy in individual oocytes using next-generation sequencing (NGS), and little is known about whether the oocyte accumulates heteroplasmic mtDNA mutations following ageing or ovarian stimulation. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: A novel bovine model for the effect of stimulation and age in human ART was undertaken using cows generated by somatic cell nuclear transfer (SCNT) from one founder, to produce a homogeneous population with reduced genetic and environmental variability. Oocytes and somatic tissues were collected from young (3 years of age; n = 4 females) and old (10 years of age; n = 5 females) cow clones following multiple natural ovarian cycles, as well as oocytes following multiple mild (FSH only) and standard (based on human a long GnRH agonist protocol) ovarian stimulation cycles. In addition, oocytes were recovered in a natural cycle from naturally conceived cows aged 4-13.5 years (n = 10) to provide a heterogeneous cohort for mtDNA deletion studies. The presence or absence of mtDNA deletions were investigated using long-range PCR in individual oocytes (n = 62). To determine the detection threshold for mtDNA heteroplasmy levels in individual oocytes, a novel NGS methodology was validated; artificial mixtures of the Bos taurus and Bos indicus mitochondrial genome were generated at 1, 2, 5, 15 and 50% ratios to experimentally mimic different levels of heteroplasmy. This NGS methodology was then employed to determine mtDNA heteroplasmy levels in single oocytes (n = 24). Oocyte mtDNA deletion and heteroplasmy data were analysed by binary logistic regression with respect to the effects of ovarian ageing and ovarian stimulation regimens. MAIN RESULTS AND THE ROLE OF CHANCE: Ovarian ageing, but not ovarian stimulation, increased the number of oocytes exhibiting mtDNA deletions (P = 0.04). A minimum mtDNA heteroplasmy level of 2% was validated as a sensitive (97-100%) threshold for variant detection in individual oocytes using NGS. Few mtDNA heteroplasmies were detected across the individual oocytes, with only 15 oocyte-specific variants confined to two of the 24 oocytes studied. There was no relationship (P > 0.05) evident between ovarian ageing or ovarian stimulation and the presence of mtDNA heteroplasmies. LIMITATIONS, REASON FOR CAUTION: The low number of oocytes collected from the natural ovarian cycles limited the analysis. Fertilization and developmental potential of the oocytes was not assessed as the oocytes were destroyed for mtDNA deletion and heteroplasmy analysis. WIDER IMPLICATIONS OF THE FINDINGS: If the findings of this model apply to the human, this study suggests that the incidence of mtDNA deletions increases with age, but not with degree of ovarian stimulation, while the frequency of mtDNA heteroplasmies may be low regardless of ovarian ageing or level of ovarian stimulation. STUDY FUNDING AND COMPETING INTERESTS: Funding was provided by Fertility Associates, the Nurture Foundation for Reproductive Research, the Fertility Society of Australia, and the Auckland Medical Research Foundation. J.C.P. is a shareholder of Fertility Associates and M.P.G. received a fellowship from Fertility Associates. The other authors of this manuscript declare no conflict of interest that could be perceived as prejudicing the impartiality of the reported research.

Nuclear and mitochondrial DNA in blastocoele fluid and embryo culture medium: evidence and potential clinical use.

The ability to screen embryos for aneuploidy or inherited disorders in a minimally invasive manner may represent a major advancement for the future of embryo viability assessment. Recent studies have demonstrated that both blastocoele fluid and embryo culture medium contain genetic material, which can be isolated and subjected to downstream genetic analysis. The blastocoele fluid may represent an alternative source of nuclear DNA for aneuploidy testing, although the degree to which the isolated genetic material is solely representative of the developing embryo is currently unclear. In addition to nuclear DNA, mitochondrial DNA (mtDNA) can be detected in the embryo culture medium. Currently, the origin of this nuclear and mtDNA has not been fully evaluated and there are several potential sources of contamination that may contribute to the genetic material detected in the culture medium. There is however evidence that the mtDNA content of the culture medium is related to embryo fragmentation levels and its presence is predictive of blastulation, indicating that embryo development may influence the levels of genetic material detected. If the levels of genetic material are strongly related to aspects of embryo quality, then this may be a novel biomarker of embryo viability. If the genetic material does have an embryo origin, the mechanisms by which DNA may be released into the blastocoele fluid and embryo culture medium are unknown, although apoptosis may play a role. While the presence of this genetic material is an exciting discovery, the DNA in the blastocoele fluid and embryo culture medium appears to be of low yield and integrity, which makes it challenging to study. Further research aimed at assessing the methodologies used for both isolating and analysing this genetic material, as well as tracing its origin, are needed in order to evaluate its potential for clinical use. Should such methodologies prove to be routinely successful and the DNA recovered demonstrated to be embryonic in origin, then they may be used in a minimally invasive and less technical methodology for genetic analysis and embryo viability assessment than those currently available.

Maternal age and ovarian stimulation independently affect oocyte mtDNA copy number and cumulus cell gene expression in bovine clones.

STUDY QUESTION: Does maternal ageing and ovarian stimulation alter mitochondrial DNA (mtDNA) copy number and gene expression of oocytes and cumulus cells from a novel bovine model for human IVF? SUMMARY ANSWER: Oocytes collected from females with identical nuclear genetics show decreased mtDNA copy number and increased expression of an endoplasmic reticulum (ER) stress gene with repect to ovarian stimulation, whilst differences in the expression of genes involved in mitochondrial function, antioxidant protection and apoptosis were evident in relation to maternal ageing and the degree of ovarian stimulation in cumulus cells. WHAT IS KNOWN ALREADY: Oocyte quality declines with advancing maternal age; however, the underlying mechanism, as well as the effects of ovarian stimulation are poorly understood. Human studies investigating these effects are often limited by differences in age and ovarian stimulation regimens within a patient cohort, as well as genetic and environmental variability. STUDY DESIGN, SIZE, DURATION: A novel bovine cross-sectional maternal age model for human IVF was undertaken. Follicles were aspirated from young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian clones following multiple unstimulated, mild and standard ovarian stimulation cycles. These bovine cloned females were generated by the process of somatic cell nuclear transfer (SCNT) from the same founder and represent a homogeneous population with reduced genetic and environmental variability. Maternal age and ovarian stimulation effects were investigated in relation to mtDNA copy number, and the expression of 19 genes involved in mitochondrial function, antioxidant protection, oocyte-cumulus cell signalling and follicle development in both oocytes and cumulus cells. MATERIALS, SETTING, METHODS: Young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian bovine clones were maintained as one herd. Stimulation cycles were based on the long GnRH agonist down-regulation regimen used in human fertility clinics. Follicle growth rates, numbers and diameters were monitored by ultrasonography and aspirated when the lead follicles were >14 mm in diameter. Follicle characteristics were analysed using a mixed model procedure. Quantitative PCR (qPCR) was used to determine mtDNA copy number and reverse transcriptase-qPCR (RT-qPCR) was used to measure gene expression in oocytes and cumulus cells. MAIN RESULTS AND THE ROLE OF CHANCE: Method of ovarian stimulation (P = 0.04), but not maternal age (P > 0.1), was associated with a lower mtDNA copy number in oocytes. Neither factor affected mtDNA copy number in cumulus cells. In oocytes, maternal age had no effect on gene expression; however, ovarian stimulation in older females increased the expression of GRP78 (P = 0.02), a gene involved in ER stress. In cumulus cells, increasing maternal age was associated with the higher expression of genes involved in mitochondrial maintenance (TXN2 P = 0.008 and TFAM P = 0.03), whereas ovarian stimulation decreased the expression of genes involved in mitochondrial oxidative stress and apoptosis (TXN2 P = 0.002, PRDX3 P = 0.03 and BAX P = 0.03). LIMITATIONS, REASON FOR CAUTION: The low number of oocyte and cumulus cell samples collected from the unstimulated cycles limited the analysis. Fertilization and developmental potential of the oocytes was not assessed because these were used for mtDNA and gene expression quantification. WIDER IMPLICATIONS OF THE FINDINGS: Delineation of the independent effects of maternal age and ovarian stimulation regimen on mtDNA copy number gene expression in oocytes and cumulus cells was enabled by the removal of genetic and environmental variability in this bovine model for human IVF. Therefore, these extend upon previous knowledge and findings provide relevant insights that are applicable for improving human ovarian stimulation regimens. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Fertility Associates and the University of Auckland. J.C.P. is a shareholder of Fertility Associates and M.P.G. received a fellowship from Fertility Associates. The other authors of this manuscript declare no conflict of interest that could be perceived as prejudicing the impartiality of the reported research.

Assessing embryo quality by combining non-invasive markers: early time-lapse parameters reflect gene expression in associated cumulus cells.

STUDY QUESTION: Are there associations between early time-lapse parameters, expression of candidate embryo viability genes in cumulus cells and embryo quality on Day 5? SUMMARY ANSWER: Early time-lapse parameters correlate to the expression levels of candidate embryo viability genes in cumulus cells but a combined analysis including both time-lapse and candidate gene expression did not identify significant predictors of embryo quality on Day 5. WHAT IS KNOWN ALREADY: Recent evidence suggests that early time-lapse parameters are predictive of blastocyst development. Similarly, a number of candidate genes in cumulus cells have been identified as potential markers of embryo viability. Relationships between time-lapse parameters and candidate gene expression in cumulus cells have not been investigated, and a combined analysis of these markers has not been attempted in relation to embryo quality. STUDY DESIGN, SIZE, DURATION: A total of 78 embryos obtained by ICSI from 22 patients were studied by time-lapse and measurement of cumulus cell gene expression of known markers of embryo viability. Time-lapse and cumulus cell gene expression data were assessed in relation to embryo quality on Day 5. PARTICIPANTS/MATERIALS, SETTING, METHODS: All women, aged 32-40 years, underwent ICSI treatment for male infertility. Embryos with annotatable time to pronuclear breakdown (tPNB), division to two cells (t2C), three cells (t3C), four cells (t4C) and five cells (t5C) were included in the study. Expression levels of 27 candidate genes for embryo viability were measured in 78 associated cumulus cell masses using quantitative real-time PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Cumulus cell expression of 11 candidate genes involved in energy metabolism (ATPase, H+ transporting, lysosomal 70 kDa, V1 subunit A (ATP6V1A), NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1, 7.5 kDa (NDUFA1), lactate dehydrogenase A (LDHA), phosphofructokinase platelet (PFKP) and solute carrier family 2 member 4 (SLC2A4), mitochondrial biogenesis (DNA directed RNA polymerase, mitochondrial (POLRMT) and transcription factor A, mitochondrial (TFAM), signalling (prostaglandin-endoperoxide synthase 2), steroidogenesis (cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) and cell stress (heat shock 70 kDa protein 5 (HSPA5) and peroxiredoxin 3 (PRDX3)) correlated to time-lapse parameters of the developing embryo, largely for t3C onwards (all P < 0.05). Expression of ATP synthase, H+ transporting, mitochondrial Fo complex, subunit E (ATP51), HSPA5, PFKP, PRDX3 and versican (VCAN) and the parameter t4C were also related to embryo quality on Day 5 (all P < 0.05). Ordinal logistic regression, where gene expression and time-lapse parameters were combined, did not identify any significant predictors of embryo quality on Day 5. LIMITATIONS AND REASON FOR CAUTION: Data are from a preliminary study, limited by a small sample size and using more than one ovarian stimulation protocol. A possible limitation is that each follicle was treated as an independent observation, although a considerable fraction of embryos were from the same patient. WIDER IMPLICATIONS OF THE FINDINGS: Results presented in this study suggest that some of the variation of time-lapse parameters may be related to cumulus cell gene expression and thus the ovarian microenvironment in which the oocyte developed. Although the current study did not identify significant predictors of embryo quality on Day 5, investigation in a larger cohort may determine whether cumulus cell gene expression and time-lapse parameters can be combined to predict embryo quality. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Fertility Associates Ltd, the Auckland Medical Research Foundation and the University of Auckland. J.C.P. has a 0.5% shareholding in Fertility Associates. All other authors of this manuscript have nothing to declare and no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Dropout rate and cumulative birth outcomes in couples undergoing in vitro fertilization within a funded and actively managed system of care in New Zealand.

OBJECTIVE: To determine the dropout rate between the first and second in vitro fertilization (IVF) cycles in a controlled population derived from a funded and actively managed system of care in New Zealand, including the reason for dropout and associated cumulative live birth rate. DESIGN: Retrospective cohort. SETTING: Multicenter IVF practice. PATIENT(S): Couples qualifying for publicly funded IVF treatment under New Zealand's Clinical Priority Assessment Criteria. Couples (n = 974) started treatment between July 2011 and June 2013, used their own gametes, and were eligible for up to 2 IVF packages of funded care (including the transfer of surplus embryos). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): IVF dropout rate, reason for dropout, and cumulative live birth rate. RESULT(S): A low IVF dropout rate between the first and second IVF cycle was reported within this controlled IVF population, with 10% of couples discontinuing treatment for reasons related to stress. The cumulative live birth rate in this "low dropout" population was 59% at the end of treatment, ranging from 72% (≤30 years) to 42% (38-39 years) according to female age. Most patients who discontinued for stress had a good prognosis, and a third of patients still had embryos in cryostorage. Only 30% of those who discontinued used the funded counseling services. CONCLUSION(S): A low dropout rate (10%) can be achieved within an actively managed IVF population. This was lower than previously reported, suggesting that prognosis, cost, and treatment management are the significant causes of dropout within the general IVF population. Couples with many embryos also require psychological support because of treatment fatigue or repeated transfers.

Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified.

OBJECTIVE: To characterize nuclear and mitochondrial DNA (mtDNA) in spent culture media from normally developing blastocysts to determine whether it could be used for noninvasive genetic assessment. DESIGN: Prospective embryo cohort study. SETTING: Academic center and private in vitro fertilization (IVF) clinic. PATIENT(S): Seventy patients undergoing intracytoplasmic sperm injection (ICSI) and 227 blastocysts. INTERVENTION(S): Culture media assessment, artificial blastocoele fluid collapse and DNA analysis using digital polymerase chain reaction (dPCR), long-range PCR, quantitative PCR (qPCR), and DNA fingerprinting. MAIN OUTCOME MEASURE(S): Presence of nuclear and mtDNA in three different commercial culture media from Vitrolife and Irvine Scientific, spent embryo media assessment at the cleavage and blastocyst stages of development, and analysis of the internal media controls for each patient that had been exposed to identical conditions as embryo media but did not come into contact with embryos. RESULT(S): Higher levels of nuclear and mtDNA were observed in the culture media that had been exposed to embryos compared with the internal media controls. Nuclear DNA (∼4 copies) and mtDNA (∼600 copies) could be detected in spent media, and the levels increased at the blastocyst stage. No increase in DNA was detected after artificial blastocoele fluid collapse. Mixed sex chromosome DNA was detected. This originated from contamination in the culture media and from maternal (cumulus) cells. Due to the limited amount of template, the presence of embryonic nuclear DNA could not be confirmed by DNA fingerprinting analysis. CONCLUSION(S): Currently DNA from culture media cannot be used for genetic assessment because embryo-associated structures release DNA into the culture medium and the DNA is of mixed origin.

Manipulation of objects with and without the use of the index finger: implications for digital amputations.

STUDY DESIGN: Clinical Measurement. PURPOSE: To evaluate changes in temporal and amplitude movement accuracy with tasks requiring fine motor manipulation with and without the use of the index finger (WIF). PARTICIPANTS: Twenty right-handed participants (10 males, 10 females, aged 24-47 years) were recruited. METHODS: Three objects, ranging in weight and size, that required the use of 2 or 3 fingers were selected for this study. Motor performance was quantified during manipulation of a pen, cork, and wine glass using a computerized visual guided tracking task. The miniBird (Ascension Technology, Burlington, VT, USA) miniature motion tracking sensor was attached to each object to measure and record the 3D linear and angular motion. RESULTS: Task performance and temporal accuracy of the pen task in the normal condition was more accurate (P=.033). During the WIF condition there was significantly more motion performing the wine task (P<.001). CONCLUSIONS: The protocol directly measures the ability of the hand to coordinate movement in response to a visual tracking target. Both temporal accuracy and amplitude consistency can be objectively evaluated. The current study evaluates the ability of the hand to manipulate 3 objects used in fine motor manipulation, using motion analysis and visual tracking. LEVEL OF EVIDENCE: 3b.

Objective evaluation of fine motor manipulation-a new clinical tool.

A new performance-based tool has been developed to accurately and precisely evaluate finger/hand function during manipulation of any object, independent of geometric and surface properties. The objectives of this study were to show test-retest reliability and evaluate criterion validity. Twenty healthy, right-handed participants were recruited. Three objects ranging in weight and size, requiring two or three fingers, were instrumented with a motion sensor that tracked 3D linear/angular motion. A computerized visual-guided tracking task was used to quantify motor performance during object manipulation. Two testing periods, one week apart were performed to evaluate test-retest reliability. Criterion validity was assessed by comparing performance with this tool to performance on commonly used clinical dexterity tests. Global performance, temporal accuracy, and amplitude consistency during manipulation of the objects compared with the reference waveform were highly reliable on the two testing occasions. Low-moderate correlations between the clinical dexterity tests and the task protocol indicate that different aspects of hand function were measured. The task protocol directly measures the ability of the hand to coordinate movement in response to a visual tracking target. Providing effective and objective ways to evaluate manual dexterity and hand function is a critical part of evidence-based practice.