Sustainability in the IVF laboratory: recommendations of an expert panel.

The healthcare industry is a major contributor to greenhouse gas emissions. Assisted reproductive technology is part of the larger healthcare sector, with its own heavy carbon footprint. The social, economic and environmental costs of this collective carbon footprint are becoming clearer, as is the impact on human reproductive health. Alpha Scientists in Reproductive Medicine and the International IVF Initiative collaborated to seek and formulate practical recommendations for sustainability in IVF laboratories. An international panel of experts, enthusiasts and professionals in reproductive medicine, environmental science, architecture, biorepository and law convened to discuss the topics of importance to sustainability. Recommendations were issued on how to build a culture of sustainability in the workplace, implement green design and building, use life cycle analysis to determine the environmental impact, manage cryostorage more sustainably, and understand and manage laboratory waste with prevention as a primary goal. The panel explored whether the industry supporting IVF is sustainable. An example is provided to illustrate the application of green principles to an IVF laboratory through a certification programme. The UK legislative landscape surrounding sustainability is also discussed and a few recommendations on 'Green Conferencing' are offered.

Laser-assisted zona pellucida thinning does not facilitate hatching and may disrupt the in vitro hatching process: a morphokinetic study in the mouse.

STUDY QUESTION: Does laser-assisted zona thinning of cleavage stage mouse embryos facilitate hatching in vitro? SUMMARY ANSWER: No, unlike laser zona opening, zona thinning does not facilitate embryo hatching. WHAT IS KNOWN ALREADY: Artificial opening of the zona pellucida facilitates hatching of mouse and human embryos. Laser-assisted zona thinning has also been used for the purpose of assisted hatching of human embryos but it has not been properly investigated in an animal model; thinning methods have produced inconsistent clinical results. STUDY DESIGN, SIZE, DURATION: Time-lapse microscopy was used to study the hatching process in the mouse after zona opening and zona thinning; a control group of embryos was not zona-manipulated but exposed to the same laser energy. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eight-cell CB6F1/J mouse embryos were pooled and allocated to three groups (n = 56 per group): A control group of embryos that were exposed to a dose of laser energy focused outside the zona pellucida (zona intact); one experimental group of embryos in which the zona pellucida was opened by complete ablation using the same total number of pulses as the control group; a second experimental group of embryos in which the zona pellucida was thinned to establish a smooth lased area using the same number of pulses as used in the other two groups. The width of the zona opening was 25 µm and width of the thinned area was 35 µm. Development was monitored by time-lapse microscopy. Overall treatment differences for continuous variables were analyzed by analysis of variance and pairwise comparisons using the Student t-test allowing for unequal variances, while for categorical data, a standard chi-squared test was utilized for all pairwise comparisons. MAIN RESULTS AND THE ROLE OF CHANCE: The frequency of complete hatching was 33.9% in the control group, 94.4% after zona opening, and 39.3% after zona thinning (overall group comparison, P < 0.0001). Overall, 60.7% of the zona-thinned embryos did not complete the hatching process and remained trapped within the zona; when they did hatch, they did not necessarily hatch from the zona-thinned area. Hatching in about one-third of the zona-intact embryos began with breaches at multiple sites by small groups of cells. Likewise, 53.6% of zona-thinned embryos had multiple breaches, always involving an area outside the thinned zone. Zona opening decreased multiple breaching and led to blastocyst escape an average of 14 h earlier than zona-thinned embryos and 5.5 h before control embryos (P = 0.0003). LIMITATIONS, REASONS FOR CAUTION: The experiments presented here were limited to in vitro experiments performed in the mouse. Whether human embryos would behave the same way under similar circumstances is unknown. We postulate that zona thinning is not beneficial in human embryos. WIDER IMPLICATIONS OF THE FINDINGS: The experiments demonstrate that zona thinning is not equivalent to zona opening for assisted hatching. The study provides reason for systematic reviews of assisted hatching trials to take the method of assisted hatching into consideration and not combine the results of zona thinning and zona opening procedures. STUDY FUNDING/COMPETING INTERESTS: Institutional funds were used for the study. No competing interests are declared.

The biological basis for defining bi-parental or tri-parental origin of offspring from cytoplasmic and spindle transfer.

The bi-parental genetic state is not a given after assisted reproduction. This is based on a biological definition of parentage that concerns generational inheritance of genetic material. Often three or more individuals may participate in artificial reproduction. Only cytoplasmic and spindle transfer can result in the genetic tri-parental state. All other forms involving three or more assisting persons with no heritable genetic contribution must be considered differently. Can a cytoplasmic donor be a biological parent based on a potential contribution of mitochondrial DNA to the offspring? - only if the mitochondrial DNA sequence can be traced back to the donor, a phenomenon which may not be very common. When considering spindle transfer for avoiding transmission of mitochondrial disease, all offspring is likely to be tri-parental.

Evidence-based medicine and its application in clinical preimplantation embryology.

The randomized clinical trial (RCT) was developed to assess the efficacy of medical interventions by comparing a treatment group with a control group that did not receive treatment or received placebo. It has been evident that this study design is more suited to evaluation of drugs and less to complex interventions such as surgical procedures. The same can be said about assisted reproduction technology, which clearly belongs in the complex category. Yet, there have been no specific guidelines for the design of RCT in clinical embryology. Moreover, the reliability of the published data from arbitrarily designed trials has not been debated. The near absence of allocation concealment and blinding and the difficulties inherent in randomizing oocytes and embryos in addition to patients require further consideration. This work evaluates systematic reviews to assess the efficacy of interventions, including the use of different culture media, culture under reduced oxygen tension, blastocyst culture, co-culture with somatic cells, assisted hatching, preimplantation genetic screening and intracytoplasmic morphologically selected sperm injection. The overall quality of a RCT and the appropriate application of new technologies in clinical embryology may be improved if a specific set of guidelines were to be developed for such investigations.

Past performance of assisted reproduction technologies as a model to predict future progress: a proposed addendum to Moore's law.

The ultimate goal of IVF is to achieve healthy, single, live births following each single-embryo transfer. A timeline for this eventuality has never been defined. National implantation rates from 2003-2010 provided by the Society for Assisted Reproductive Technologies (SART) in the USA were evaluated. Regression analysis was applied to the annual trends. A high correlation was noted showing a linear increase from year to year ranging between 0.3% and 1.5% when maternal age was not higher than 42. This relationship can be retrospectively applied to earlier SART data reports. This incline may be partly technology driven and resembles Moore's law, which describes annual improvements in microchip performance. Based on the assumption that technology will continue to drive progress, the length of time required to reach 100% implantation was calculated. The interval varied between 43 years (AD 2053) for the youngest age group (<35 years old) and 294 years for the 41-42-year age group. The timeframe is shifted for the younger patients to an earlier date of 2027 if a subset of clinics with high implantation regression slopes and low variance is selected. The implications of these findings for infertility treatment and fertility preservation are discussed. Success after IVF has steadily improved. Data from US-based clinics are annually collected by the Society for Assisted Reproductive Technologies (SART; www.sart.org). Through SART, individual clinic's outcomes may be assessed. Although live birth and pregnancy are considered the gold standard of success, the investigators took the approach that those outcomes are often biased due to transfer of multiple embryos. The present analysis was therefore performed on individual embryos, by using the implantation rate to compare national and individual clinic datasets. National implantation rates show a linear increase from year to year ranging between 0.3% and 1.5% for patients aged <43 years. We postulate that this linear trend can be traced back to 1985 even though statistical analysis could only be applied to the implantation data from 2003-2010. We expect that this annual incline is partly technology driven. This is an intriguing effect also seen in the computer industry where there has been a doubling of computer speed and memory for the past 47 years, a phenomenon anticipated by Moore's law. We predict that the annual increase in implantation will also continue as new technologies become available. Based on current trends, the length of time for 100% implantation rates was calculated. Time to achieving 100% implantation varied between 43 years (AD 2053) for the youngest age group (<35 years old) to 294 years for women 41-42 years old. Some clinics may report a perfect success earlier than others. However, implantation does not guarantee birth.

Controlled ovarian hyperstimulation (COH) parameters associated with euploidy rates in donor oocytes.

Although oocyte donors are young and are expected to provide a high rate of euploid oocytes, significant differences of euploidy rates for donor embryos exist between different IVF centers (1). Laboratory conditions can lead to differences of euploidy (2,3,4,5,6,7); but, the role of COH has not been investigated. In this study, we investigated whether euploidy rates in the embryos created from donor oocytes are influenced by controlled ovarian hyperstimulation parameters used during assisted reproduction. Euploidy rates in egg donor cycles undergoing PGT-A (N = 423) were examined retrospectively for associations with donor age, gonadotropin doses (dose per day), the fraction of gonadotropin provided by hMG (F(hMG)), days of stimulation, estradiol per mature oocyte on day of trigger, number of mature oocytes retrieved, number of embryos biopsied, incidence of euploidy and physician of record. Differences in euploidy rates between physicians were examined using analysis of variance. The proportion of euploid embryos per donor cycle was examined for associations with COH parameters using pairwise post-hoc comparisons, adjusting for multiple testing. The set of variables from this analysis was then submitted to a principal component analysis. Linear regression analysis was used to assess the relationships between stimulation parameters and the incidence of euploidy (the dependent variable). Euploidy rates and cycle parameters varied significantly among treating physicians. Euploidy rates (expressed as a fraction of biopsied embryos) were associated (p = 0.01) only with the F(hMG) but not with the number of MII retrieved or other variables. On the other hand, the number of euploid embryos (in contrast to the euploidy rate) was associated with the number of MII produced. Donor euploidy rates are significantly associated with the fraction of total gonadotropin comprising human menopausal gonadotropin (or F(hMG)) during controlled ovarian hyperstimulation but are not associated with other cycle parameters. The study provides the first suggestion that patient stimulation parameters can affect the incidence of euploidy in embryos generated through the use of standard assisted reproductive techniques. The study is limited by its retrospective approach and because the aCGH analysis used is less sensitive than more recent NGS technology. Further, it provides a suggestion that the use of hMG is beneficial for obtaining euploid embryos.

Human Reproductive Cell Cryopreservation, Storage, Handling, and Transport: Risks and Risk Management.

Millions of human oocytes and embryos are stored in thousands of locations across the globe. This inventory continues to grow as cryopreservation becomes more successful and more widely applied. The results of studies assessing pregnancy and neonatal outcomes following frozen embryo transfer (FET) have been encouraging, showing lower incidences of small for gestational age neonates and preterm birth compared with fresh transfers. However, many of these studies have also shown that the odds of large for gestational age births and macrosomia are higher after FET. The origin of these conditions remains unclear. Cryostorage presents many potential risks to the cryopreserved cells/tissues, including loss of viability and contamination, but risks are faced also by care providers-for example, injury and financial liability-and by patients-for example, accidental loss of their reproductive tissues, the burden of embryos they no longer wish to use and failing to meet their contractual obligations. Studies are urgently needed to explore and understand all dimensions of cryostorage to help ART clinics develop effective strategies to manage associated risks. The future of cryostorage, as for many other areas of ART and medicine, is automation.