From Zygote to Blastocyst: Application of Ultrashort Lasers in the Field of Assisted Reproduction and Developmental Biology

Although the use of lasers in medical diagnosis and therapies, as well as in fundamental biomedical research is now almost routine, advanced laser sources and new laser-based methods continue to emerge. Due to the unique ability of ultrashort laser pulses to deposit energy into a microscopic volume in the bulk of a transparent material without disrupting the surrounding tissues, the ultrashort laser-based microsurgery of cells and subcellular components within structurally complex and fragile specimens such as embryos is becoming an important tool in developmental biology and reproductive medicine. In this review, we discuss the mechanisms of ultrashort laser pulse interaction with the matter, advantages of their application for oocyte and preimplantation embryo microsurgery (e.g., for oocyte/blastomere enucleation and embryonic cell fusion), as well as for nonlinear optical microscopy for studying the dynamics of embryonic development and embryo quality assessment. Moreover, we focus on ultrashort laser-based approaches and techniques that are increasingly being applied in the fundamental research and have the potential for successful translation into the IVF (in vitro fertilization) clinics, such as laser-mediated individual embryo labelling and controlled laser-assisted hatching.

Laser-assisted hatching in lower grade cleavage stage embryos improves blastocyst formation: results from a retrospective study

Background Laser-assisted hatching (LAH) has been widely applied to facilitate blastocyst hatching in IVF-ET treatment, however, the effect of LAH on subsequent development and clinical outcomes of the lower grade cleavage stage embryos (LGCE) remains unknown. Our study aimed at evaluating the effect of LAH on blastocyst formation and the clinical pregnancy outcomes of LGCE embryos after transfer. Methods A total of 608 cycles of IVF/ICSI treatment from November 2017 to September 2019 were included in our study as follows: 296 in the LAH group and 312 in the N-LAH group. The total blastocyst rate, usable blastocyst rate, good-grade blastocyst rate and clinical pregnancy rate were statistically compared between the two groups. Results The total blastocyst rate (50.7% vs 40.2%, P < 0.001), usable blastocyst rate (31.0% vs 18.6%, P < 0.001) were significantly higher in the LAH group than those in the N-LAH group. After analysis of generalized estimating equations, LAH was positively correlated with the blastocyst rate (B = 0.201, OR 95% CI = 1.074–1.393, P = 0.002), usable blastocyst rate (B = 0.478, OR 95% CI = 1.331–1.955, P < 0.001). However, the clinical pregnancy rate after blastocyst transfer did not differ between LAH group and N-LAH group (49.4% vs 40.0%, P > 0.05, respectively). Conclusions A higher proportion of total blastocysts and usable blastocysts can be obtained by LAH in LGCE, which may be beneficial to the outcome of the IVF/ICSI-ET cycle.

P–771 I hear, I forget. I see and I remember. I do and I understand. An Insight into the need for training for add on techniques

Study question Do embryologists need additional training or certifications before using add on techniques in the lab ? Summary answer Out of 173 respondents majority feel add on techniques require training and/or certifications, the mode of training varies for different add ons. What is known already Cochrane reviews have suggested that minimal evidence exists for the use of add on treatments in ART, the data on the prevalence of add ons in IVF is unclear but the presence of technologies in ART laboratories world over suggests a increasing trend of adoption of unproven techniques. No data exists suggesting the role of embryologists in performing the Add on techniques and how their training or lack of, can impact patient safety. The most common method of training comes from the manufacturers and there is a lack of structured trainings for add on treatments worldover. Study design, size, duration An internet based survey was designed keeping in mind commonly available laboratory add ons. It comprised of 9 sections and a total of 18 multiple choice questions. Answer choices ranged from a simple yes or no to more complex choices suggesting the type of training and the potential benefits of training. Participants/materials, setting, methods The Survey includes results from 173 embryologists from india with varying degree of experience. Add ons included in the survey were Sperm DNA Fragmentation test, IMSI, PICSI, Microfluidics, MACS, Advanced culture media, Oocyte vitrification, Assisted hatching, Time Lapse imaging, spindle view and Electronic Witnessing. The most common practice suggestions were tabulated and identified. Main results and the role of chance The survey reports huge need for training for different add on treatments (SDF –91.4%, IMSI - 81.2%, PICSI- 66.5%, Microfluidics- 55.9%, MACS –55.3%, MicroTese- 86.9%, using advanced culture medias{Calcium ionophore- 73.4%, Hyaluronan rich media- 52.1%, growth cytokine rich media–48.5%, Theophylline for sperm motility–50.9%},oocyte Vitrification 85.5%, Assisted Hatching 75.4%, Time-lapse and Electronic witnessing 77.3%, Polarised microscopy for spindle assessment 73.5%). The Most preferred mode of training for more invasive procedures was Hands on training, followed by On the job training and validation followed by workshops(SDF- 62.6%, IMSI- 61%,PICSI–56.1%, MACS and Microfluidics 38.8%,microTese- 50.6%,Oocyte freezing 85.5%, assisted hatching 67.8%,Time-lapse and electronic witnessing 77.3%, Spindle view 73.5% ). The most preferred mode of training for non invasive procedures was Workshops and Observerships, followed by CME’s, followed by product Leaflets(44.4%,42.1%,13.5% respectively). The most common answer for the disadvantages of not being trained was unable to use the technology to its fullest potential(88.5%), whereas the most common answer for the benefit of being trained was better outcomes with said technologies(76.6%). Limitations, reasons for caution This study includes responses from embryologists who have varied levels of experience, while the need for training can be established based on these results, a junior level embryologist might answer the survey differently as compared to a senior or a lab director. Wider implications of the findings: This is a first of its kind large survey, suggesting the need for training and validation from the perspective of the embryologist. This data can be used in formulating guidelines for future trainings and can help regulators in deciding on the most preferred mode of training. Trial registration number -

P–770 Two in One - Monozygotic splitting and associated perinatal outcomes after oocyte freezing; an exploratory analysis of the UK national database from 1990 to 2016

Study question Is oocyte freezing a risk factor for monozygotic splitting? Summary answer There is a trend towards a higher monozygotic splitting incidence among frozen oocytes, but this did not reach statistical significance. What is known already Laboratory techniques which involve embryo manipulation such as ICSI, assisted hatching, embryo biopsy for pre-implantation genetic testing and extended culture to the blastocyst stage appear to increase the risk of monozygotic splitting. Whilst there is some data that embryo freezing does not appear to increase the risk of monozygotic splitting, there is no comparable analysis on whether oocyte freezing increases the risk of monozygotic splitting. Study design, size, duration This was a retrospective cohort study analysing 988 015 ART (assisted reproductive technique) cycles from the HFEA anonymised database from 1990 to 2016. As frozen oocytes require ICSI, only fresh oocytes with ICSI were taken for comparison and frozen embryo transfers were excluded. Only single embryo transfers were included.[CM1] [MM2] We also noted ages of the female partner at the time of treatment, stage of embryo transfer, and whether pre-implantation genetic testing had been performed. Participants/materials, setting, methods There were 84 085 ICSI cycles with single embryo transfers using fresh oocytes and 596 using frozen oocytes. Monozygotic splitting was defined as the presence of two foetal hearts [CM1] [MM2] on ultrasound. Live birth (LB)was defined as either a singleton or a twin LB resulting from a monozygotically split embryo. Preterm birth (PTB) was defined as birth prior to 37 weeks gestation and early PTB as birth prior to 32 weeks gestation. Main results and the role of chance The frozen oocyte group had fewer women in the under–35 age group (frozen oocytes 16.6% vs fresh oocytes 53.6%, p &lt; 0.0001) and a higher proportion of blastocyst transfers ( frozen oocytes 55.1% vs fresh oocytes 48.8%, p = 0.002) There were only 10 PGT cycles amongst monozygotically split embryos from fresh oocytes in our analysis, and none in the frozen oocyte group. Hence, this was not included as a confounder. There was a non-significant trend toward a higher incidence of monozygotic splitting amongst frozen oocytes (4/596, 2.3%, all monozygotic twins) than amongst fresh oocytes (378/27 019, 1.4%, 372 monozygotic twins and 6 monozygotic triplets); OR 1.688, 95% CI 0.623 to 4.574 and aOR 1.506, 95% CI 0.531 to 4.274 (maternal age and stage of embryo transfer adjusted as confounders). Of the 378 monozygotically split embryos from fresh oocytes, 308 (81.5%) had a LB: of which 47 (15.3%) were singletons and the rest were twins; 241 (78.2%) were PTB and 56 (18.2%) were early PTB. Of the four monozygotic twins from frozen oocytes, all reached a LB; one was a singleton term LB (Birthweight 3–3.5kg) whilst three were twin preterm LBs at 35–36 weeks, with no early PTBs and twin median birthweight 2–2.5 kg. Limitations, reasons for caution Albeit a large national database, this cohort study was restricted due to absence of data on potential confounders such as age at oocyte freezing, method of cryopreservation and length of storage.[CM1] Data was also lacking on amnionicity, obstetric risks including pre-eclampsia, twin-to-twin-transfusion syndrome, intrapartum and late effects. Wider implications of the findings: With rapid rise in egg freezing, our findings would help reassure women that eggs on ice does not predispose to significant risk of two-in-one monozygotic splitting. However, the marginal trend (from 1.4% in fresh to 2.3% in frozen oocytes), does indicate that this subject merits further research. Trial registration number Not applicable. A database based retrospective study