Daniel J. Kaser

Clinical outcomes following selection of human preimplantation embryos with time-lapse monitoring: a systematic review

Sir, We have read the recent article by Kaserand Racowsky (2014) with great interest and we highly welcome this thorough systematic review. We entirely agree with the main conclusions as presented in the abstract. There are currently no high-quality data to firmly support the clinical use of this technology for selection of preimplantation embryos. Prospective studies are needed to clarify the role. We would, however, like to discuss the results from the review upon which Drs Kaser and Racowsky base this conclusion. The authors thoroughly review the time-lapse studies that have presented data on clinical outcome and present the results of their descriptive comparison, including their interpretation of the results from our prospective study (Kirkegaard et al., 2013). Amongst others the authors use the results from our study to argue that there are no differences in timing between the pregnant and the nonpregnant group of all the measured parameters. Our objection is that our study was not powered to test pregnancy as a clinical outcome for all the parameters that the authors review. Accordingly, we specifically desisted from drawing any conclusions on the ability of time-lapse parameters to predict pregnancy in general. This is clearly stated in our paper. Following standard scientific conduct, we did publish timings of all the parameters in the pregnant and non-pregnant group, yet acknowledged that the study was powered only to test the parameters from the targeted logistic regression analysis. We even clarified this in a response to a letter addressing the sample size (Kirkegaard et al., 2014). We believe that the underpowered sample size entails a high risk of falsely concluding that there is no difference. For example, we did not test appearance, abuttal, syngamy, and breakdown of the male and female pronucleus (PN) as predictors of implantation in our logistic regression analysis (as stated in the review), but only PN breakdown. Therefore it is hardly justified to state that we did not find any difference in the above parameters, without acknowledging the lack of power to detect such differences. This is true for several of the conclusions the authors draw from our publication, including the conclusion that we found no difference between implanting and non-implanting embryos in terms of cleavage and blastocyst kinetics in general. We consider it plausible that the majority of the other published studies are far too small to detect any presumed differences in timing with regard to pregnancy. No randomized controlled studies of single embryo transfers have been published so far. We therefore find it very poorly supported, that reliable prediction of blastocyst formation may be the main advantage of TLM, as stated in the review. It is correct that we conclude that TLM may decrease variability (Sundvall et al., 2013). But as the cited study involved manual, in contrast to computer-assisted annotation, it cannot be stated that the reduced variability is a result of the semi-quantification. The statement that TLM may decrease intraand inter-observer variability among embryologists, as a result (our underlining) of computer-assisted annotation of developmental milestones and semi-quantitative process for embryo evaluation, is therefore unsupported. In summary, we entirely agree that larger prospective studies with clinical outcomes are needed to clarify the role of time-lapse. That the existing literature suggests no association with implantation potential is in our opinion so far unjustified due to several factors, most importantly due to lack of power of the studies. This was acknowledged in the original publications, but unfortunately not in the review.

Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance

Abstract BACKGROUND Successful cryopreservation of oocytes and embryos is essential not only to maximize the safety and efficacy of ovarian stimulation cycles in an IVF treatment, but also to enable fertility preservation. Two cryopreservation methods are routinely used: slow-freezing or vitrification. Slow-freezing allows for freezing to occur at a sufficiently slow rate to permit adequate cellular dehydration while minimizing intracellular ice formation. Vitrification allows the solidification of the cell(s) and of the extracellular milieu into a glass-like state without the formation of ice. OBJECTIVE AND RATIONALE The objective of our study was to provide a systematic review and meta-analysis of clinical outcomes following slow-freezing/thawing versus vitrification/warming of oocytes and embryos and to inform the development of World Health Organization guidance on the most effective cryopreservation method. SEARCH METHODS A Medline search was performed from 1966 to 1 August 2016 using the following search terms: (Oocyte(s) [tiab] OR (Pronuclear[tiab] OR Embryo[tiab] OR Blastocyst[tiab]) AND (vitrification[tiab] OR freezing[tiab] OR freeze[tiab]) AND (pregnancy[tiab] OR birth[tiab] OR clinical[tiab]). Queries were limited to those involving humans. RCTs and cohort studies that were published in full-length were considered eligible. Each reference was reviewed for relevance and only primary evidence and relevant articles from the bibliographies of included articles were considered. References were included if they reported cryosurvival rate, clinical pregnancy rate (CPR), live-birth rate (LBR) or delivery rate for slow-frozen or vitrified human oocytes or embryos. A meta-analysis was performed using a random effects model to calculate relative risk ratios (RR) and 95% CI. OUTCOMES One RCT study comparing slow-freezing versus vitrification of oocytes was included. Vitrification was associated with increased ongoing CPR per cycle (RR = 2.81, 95% CI: 1.05–7.51; P = 0.039; 48 and 30 cycles, respectively, per transfer (RR = 1.81, 95% CI 0.71–4.67; P = 0.214; 47 and 19 transfers) and per warmed/thawed oocyte (RR = 1.14, 95% CI: 1.02–1.28; P = 0.018; 260 and 238 oocytes). One RCT comparing vitrification versus fresh oocytes was analysed. In vitrification and fresh cycles, respectively, no evidence for a difference in ongoing CPR per randomized woman (RR = 1.03, 95% CI: 0.87–1.21; P = 0.744, 300 women in each group), per cycle (RR = 1.01, 95% CI: 0.86–1.18; P = 0.934; 267 versus 259 cycles) and per oocyte utilized (RR = 1.02, 95% CI: 0.82–1.26; P = 0.873; 3286 versus 3185 oocytes) was reported. Findings were consistent with relevant cohort studies. Of the seven RCTs on embryo cryopreservation identified, three met the inclusion criteria (638 warming/thawing cycles at cleavage and blastocyst stage), none of which involved pronuclear-stage embryos. A higher CPR per cycle was noted with embryo vitrification compared with slow-freezing, though this was of borderline statistical significance (RR = 1.89, 95% CI: 1.00–3.59; P = 0.051; three RCTs; I2 = 71.9%). LBR per cycle was reported by one RCT performed with cleavage-stage embryos and was higher for vitrification (RR = 2.28; 95% CI: 1.17–4.44; P = 0.016; 216 cycles; one RCT). A secondary analysis was performed focusing on embryo cryosurvival rate. Pooled data from seven RCTs (3615 embryos) revealed a significant improvement in embryo cryosurvival following vitrification as compared with slow-freezing (RR = 1.59, 95% CI: 1.30–1.93; P < 0.001; I2 = 93%). WIDER IMPLICATIONS Data from available RCTs suggest that vitrification/warming is superior to slow-freezing/thawing with regard to clinical outcomes (low quality of the evidence) and cryosurvival rates (moderate quality of the evidence) for oocytes, cleavage-stage embryos and blastocysts. The results were confirmed by cohort studies. The improvements obtained with the introduction of vitrification have several important clinical implications in ART. Based on this evidence, in particular regarding cryosurvival rates, laboratories that continue to use slow-freezing should consider transitioning to the use of vitrification for cryopreservation.

Techniques for contained morcellation in gynecologic surgery

OBJECTIVE To demonstrate 2 step-by-step techniques for contained morcellation of uterine tissue. DESIGN Instructional video showing laparoscopic electromechanical morcellation within an endoscopic pouch, and alternatively, tissue extraction via ultra-minilaparotomy. SETTING Academic medical center. PATIENT(S) Women undergoing laparoscopic myomectomy or hysterectomy. INTERVENTION(S) For contained electromechanical morcellation, the specimen is placed within an endoscopic pouch, the edges of which are exteriorized through a 15-mm cannula. The cannula is repositioned inside the pouch for insufflation. A bladed fixation trocar enters the pouch through an assistant port and is secured by its retention disk and balloon tip. Gas inflow is changed to this assistant port, through which the laparoscope is inserted. A power morcellator is introduced via the 15-mm port site, and morcellation thus proceeds within the containment system. Residual fragments of tissue are collectively retrieved by withdrawing the endoscopic pouch. For tissue extraction via ultra-minilaparotomy, the specimen is placed within a pouch that is drawn up through a flexible, self-retaining retractor seated in a 2 to 3-cm incision. The specimen is cored out sharply with a scalpel. MAIN OUTCOME MEASURE(S) None. RESULT(S) Contained morcellation is technically feasible, efficient (mean additional operative time is approximately 30 minutes), and prevents intraperitoneal dispersion of tissue fragments. Our group has safely performed >100 such procedures and removed specimens weighing nearly 1,500 grams. Potential complications include viscous injury upon insertion of the bladed trocar, and pouch failure. CONCLUSION(S) These techniques allow surgeons to adopt the new standard of contained morcellation and permit removal of extensive pathology with a minimally invasive approach.

National survey on use of time-lapse imaging systems in IVF laboratories

PurposeSeveral time-lapse imaging (TLI) systems for non-invasive continuous monitoring of developing embryos are currently available. The present study explored the prevalence, means of acquisition, and clinical application of TLI systems in USA in vitro fertilization (IVF) laboratories.MethodsAn online cross-sectional survey of 294 USA IVF laboratory directors was conducted in February and March 2016. Those directing more than one laboratory were asked to complete the survey for their home program and for their smallest laboratory by number of IVF/intracytoplasmic sperm injection (ICSI) cycle starts. Use of TLI was analyzed using logistic regression to calculate odds ratios (OR).ResultsOf 294 directors surveyed, 162 (55%) reported data on 204 laboratories. Thirty-five laboratories (17%) possessed at least one TLI system (median 2, interquartile range 1–4, total range 1–11). The more oocyte retrievals a laboratory performed annually, the more likely the laboratory was to possess a TLI system. Fifteen laboratories (43%) purchased their own systems, while others leased, loaned, or received donated systems. Twenty-five laboratories (71%) reported using TLI for embryo selection; all used TLI always, or usually, in combination with standard morphology evaluation. Twenty laboratories (80%) offered TLI to all patients. Some laboratories charged patients for TLI. Directors with TLI systems were more inclined to believe that TLI has value for embryo selection in clinical IVF.ConclusionsTLI system possession in USA IVF laboratories is low, although positively associated with the number of retrievals performed and with directors’ opinions on the technology’s utility. Over 70% of laboratories with TLI systems use them clinically, and less than half purchased their systems.

Robotic Single-Site Myomectomy: A Step-by-Step Tutorial

Salient details of the interventional Acessa Procedure for radiofrequency ablation of symptomatic uterine fibroids are presented. The procedure is unique: ablation is guided by intra-abdominal laparoscopic ultrasound, does not require laparoscopic suturing or subsequent overnight hospital stay, but does require basic laparoscopic and ultrasound skills. Hemostasis is achieved by coagulation of the handpiece track upon withdrawal of the handpiece from the fibroid and uterus. Fibroids 0.7 to 15 cm in diameter have been treated. The patient is a 31 year-old, nulligravida black female who presented with menorrhagia, abdominal fullness and pelvic pressure. Ultrasound detected a 17-cm uterus with multiple intramural and subserosal fibroids, the two largest being 6 cm and 3 cm in greatest diameters. The patient desired outpatient, surgical, and uterine-conserving therapy. She was discharged 5 hours post-procedure with acetaminophencodeine and nonsteroidal anti-inflammatory drugs. The average patient misses a median of 5 workdays.

Robotic single-site myomectomy: a step-by-step tutorial

OBJECTIVE To provide a step-by-step description of our published technique of single-site robot-assisted laparoscopic myomectomy with the goal of promoting its safe adoption. DESIGN Surgical video tutorial. SETTING University medical center. PATIENT(S) Ten women undergoing single-site robot-assisted laparoscopic myomectomy between November 2014 and March 2015. INTERVENTION(S) A 2.5-cm vertical incision is made within the umbilicus, through which a multilumen single-site port (da Vinci Single-Site; Intuitive Surgical) is seated. An 8.5-mm 0-degree laparoscope is introduced, and the teleoperator (da Vinci Si Surgical Platform; Intuitive Surgical) is docked, allowing subsequent placement of two curved 5-mm instrument cannulae. Two wristed, semirigid needle drivers are loaded onto robotic arms 1 and 2. An 8-mm assistant cannula is also placed through the multilumen single-site port; a flexible 2-mm CO2 laser fiber and all conventional 5-mm laparoscopic instruments are introduced through this cannula as needed. Intramyometrial dilute vasopressin is injected, and fibroid enucleation is performed. The hysterotomy is repaired in layers with unidirectional barbed suture (Stratafix; Ethicon). The teleoperator is undocked. The single-site port is exchanged for a self-retaining wound retractor with gel-sealed cap. An endoscopic pouch is placed in the abdomen, and the specimen is placed within the pouch. The edges of the pouch are exteriorized. Extracorporeal tissue extraction is performed with a scalpel. A running mass closure of the fascia and peritoneum is performed, followed by a subcuticular closure of the skin. MAIN OUTCOME MEASURE(S) Median number and size of fibroids removed, specimen weight, operative time, estimated blood loss, and perioperative complications. RESULT(S) The technique described in our video was successfully employed in our first 10 patients. The median number of fibroids removed was 2.5 (range: 1-8); the median size of the largest myoma was 6 cm (range: 4-8 cm); the median specimen weight was 70 g (range: 26-154 g); the median operating time was 202 minutes (range: 141-254 minutes); the median blood loss was 87.5 mL (range: 10-300 mL). No conversions to multiport robotic or open myomectomy occurred. No major complications occurred, and no patients required blood transfusion. CONCLUSION(S) Robot-assisted laparoscopic single-site myomectomy employing a multilumen port and wristed, semirigid needle drivers is a safe and reproducible technique. Our technique allows surgeons to offer myomectomy and contained, extracorporeal tissue extraction via a single 2.5-cm umbilical incision in select patients with low tumor burden.

Embryo biopsy for aneuploidy detection in the general infertility population.

Since report of the first live birth following preimplantation genetic screening (PGS) in 1995, the procedure and available technologies for aneuploidy detection have rapidly evolved. Through these efforts, the biology of meiotic and mitotic segregation errors has been partially elucidated. A process that began with polar body biopsy and four-color fluorescence in situ hybridization to detect copy number in a limited number of chromosomes is now hardly recognizable: current molecular methods permit high-density screening of the entire human genome for copy number variants, structural rearrangements, microdeletions, and polyploids to a resolution of 35 kilobases in less than 48 hours. Indeed, with the advent of real-time quantitative analyses of ploidy status that allow same-day trophectoderm biopsy with fresh transfer of a euploid blastocyst, the future is bright for PGS. Questions remain about how best to safely offer this technology to patients, and which patients, if any, will benefit from routine biopsy. Herein, we will review the limited available evidence for application of PGS in the general infertility population as an adjunct method to optimize live birth rates.

Should We Eliminate Fresh Embryo Transfer from ART

Controlled ovarian hyperstimulation (COH) affects the transcriptional program of the mid-secretory endometrium, rendering it less receptive to embryo implantation. It is postulated that the supraphysiologic hormonal milieu at the time of fresh embryo transfer (ET) may impair trophoblast invasion and placental angiogenesis. One approach that circumvents this problem is to uncouple COH from ET by cryopreserving all embryos with subsequent replacement in a natural or programmed cycle. There is emerging evidence that pregnancy rates and obstetric and neonatal outcomes are all improved following delayed cryopreserved embryo transfer (CET). Before such an elective cryopreservation program is adopted, though, the existing observational evidence must be validated by adequately powered randomized controlled trials.

Developmental potential of embryos from intracytoplasmic sperm injection cycles containing fragmented oocytes

OBJECTIVE To determine the incidence of fragmented oocytes in intracytoplasmic sperm injection (ICSI) cycles, describe the developmental potential of their sibling oocytes, and define clinical outcomes from affected cycles. DESIGN Case-control study. SETTING Academic medical center. PATIENT(S) All ICSI cycles from January 2006 to December 2010 (n = 2,844) were reviewed for the presence of fragmented oocytes at cumulus stripping or fertilization check (n = 93). Sibling oocytes and corresponding embryos from index cycles were compared with matched control cycles without fragmented oocytes. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Cycle characteristics, embryo quality, and pregnancy rates per retrieval. RESULT(S) The incidence of ICSI cycles containing at least one fragmented oocyte was 3.3% (93/2,844). Twelve patients were represented more than once in these 93 index cycles. Only the first cycles (n = 81) were included, of which 28 contained fragmented oocytes at cumulus stripping, 48 at fertilization check, and five at both. Compared with matched controls, index cycles had fewer good-quality embryos available for transfer (18.8% vs. 32.1%) and significantly lower rates of implantation (20.3% vs. 32.7%), clinical pregnancy (33.3% vs. 58.0%), and ongoing delivery (29.6% vs. 49.4%). The cumulative ongoing delivered rate was also significantly lower for index cycles (32.1% vs. 55.6%), with no difference in the percentage of cycles with cryopreserved embryos remaining at study conclusion (13.5% vs. 23.5%). CONCLUSION(S) Cohorts containing fragmented oocytes have decreased developmental potential. The biologic mechanisms underlying this occurrence merit further investigation, and patient counseling should reflect the possible decreased success rates associated with this aberrant developmental pattern.

Prospective study of automated versus manual annotation of early time-lapse markers in the human preimplantation embryo

STUDY QUESTION How does automated time-lapse annotation (Eeva™) compare to manual annotation of the same video images performed by embryologists certified in measuring durations of the 2-cell (P2; time to the 3-cell minus time to the 2-cell, or t3-t2) and 3-cell (P3; time to 4-cell minus time to the 3-cell, or t4-t3) stages? SUMMARY ANSWER Manual annotation was superior to the automated annotation provided by Eeva™ version 2.2, because manual annotation assigned a rating to a higher proportion of embryos and yielded a greater sensitivity for blastocyst prediction than automated annotation. WHAT IS KNOWN ALREADY While use of the Eeva™ test has been shown to improve an embryologists ability to predict blastocyst formation compared to Day 3 morphology alone, the accuracy of the automated image analysis employed by the Eeva™ system has never been compared to manual annotation of the same time-lapse markers by a trained embryologist. STUDY DESIGN, SIZE, DURATION We conducted a prospective cohort study of embryos (n = 1477) cultured in the Eeva™ system (n = 8 microscopes) at our institution from August 2014 to February 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS Embryos were assigned a blastocyst prediction rating of High (H), Medium (M), Low (L), or Not Rated (NR) by Eeva™ version 2.2 according to P2 and P3. An embryologist from a team of 10, then manually annotated each embryo and if the automated and manual ratings differed, a second embryologist independently annotated the embryo. If both embryologists disagreed with the automated Eeva™ rating, then the rating was classified as discordant. If the second embryologist agreed with the automated Eeva™ score, the rating was not considered discordant. Spearmans correlation (ρ), weighted kappa statistics and the intra-class correlation (ICC) coefficients with 95% confidence intervals (CI) between Eeva™ and manual annotation were calculated, as were the proportions of discordant embryos, and the sensitivity, specificity, positive predictive value (PPV) and NPV of each method for blastocyst prediction. MAIN RESULTS AND THE ROLE OF CHANCE The distribution of H, M and L ratings differed by annotation method (P < 0.0001). The correlation between Eeva™ and manual annotation was higher for P2 (ρ = 0.75; ICC = 0.82; 95% CI 0.82-0.83) than for P3 (ρ = 0.39; ICC = 0.20; 95% CI 0.16-0.26). Eeva™ was more likely than an embryologist to rate an embryo as NR (11.1% vs. 3.0%, P < 0.0001). Discordance occurred in 30.0% (443/1477) of all embryos and was not associated with factors such as Day 3 cell number, fragmentation, symmetry or presence of abnormal cleavage. Rather, discordance was associated with direct cleavage (P2 ≤ 5 h) and short P3 (≤0.25 h), and also factors intrinsic to the Eeva™ system, such as the automated rating (proportion of discordant embryos by rating: H: 9.3%; M: 18.1%; L: 41.3%; NR: 31.4%; P < 0.0001), microwell location (peripheral: 31.2%; central: 23.8%; P = 0.02) and Eeva™ microscope (n = 8; range 22.9-42.6%; P < 0.0001). Manual annotation upgraded 82.6% of all discordant embryos from a lower to a higher rating, and improved the sensitivity for predicting blastocyst formation. LIMITATIONS, REASONS FOR CAUTION One team of embryologists performed the manual annotations; however, the study staff was trained and certified by the company sponsor. Only two time-lapse markers were evaluated, so the results are not generalizable to other parameters; likewise, the results are not generalizable to future versions of Eeva™ or other automated image analysis systems. WIDER IMPLICATIONS OF THE FINDINGS Based on the proportion of discordance and the improved performance of manual annotation, clinics using the Eeva™ system should consider manual annotation of P2 and P3 to confirm the automated ratings generated by Eeva™. STUDY FUNDING/COMPETING INTEREST(S) These data were acquired in a study funded by Progyny, Inc. There are no competing interests. TRIAL REGISTRATION NUMBER N/A.