The presence of partial compaction patterns is associated with lower rates of blastocyst formation, sub-optimal morphokinetic parameters and poorer morphologic grade.

BACKGROUND: Compaction is an important marker of embryonic genome activation and marks a critical step in the development to blastocyst. The objective of our study was to determine whether visualization of the embryonic compaction process through time-lapse imaging (TL) can assist in predicting the kinetics of embryo development as well as the likelihood for blastocyst formation, grade, or ploidy. METHODS: This study is a retrospective review of prospectively collected datafrom a single academic institution. Couples included were thosewho underwent preimplantation genetic testing for aneuploidy (PGT-A) following in vitro fertilization between Januaryand December 2020. Embryos were cultured in the Embrysocope. Embryo morphokinetic data was prospectively collected and analyzed.TL videos werelater reviewed in detail for compaction pattern. Embryo compaction patterns (CP) were categorized as follows: 1) full compaction (CP-F), 2) partial compaction with cell extrusion (P-ext), 3) partial compactionwith cell exclusion (P-exc) and 4) partial compactionwith both cell extrusion and exclusion (P-both). Assessment of embryo decompaction and re-compaction was evaluated. The association between CP, morphokinetic parameters,blastocyst formation, grade and ploidy were then analyzed. RESULTS: A total of 349 embryos were studied. Amongst embryos which progressed to morula (n = 281), the distribution of compaction patterns were: CP-F 45.6%, P-ext12.5%, P-exc29.5% and P-both 12.5%. Embryos exhibiting a CP-F were more likely to proceed to blastocyst compared with those that demonstrated partial compaction patterns (p = 0.006). When compared to CP-F, partial compaction patterns were significantly associated with poorer ICM and TE grades (P < 0.001). Of the 281 morula, 59.8% (n = 168) demonstrated at least one episode of decompaction and re-compaction. Of the 249 blastocysts formed, 200 were cryopreserved for future use after undergoing PGT-A evaluation. Of those, 42.5% were diagnosed as euploid, 39.0% as aneuploid, 9.0% as mosaic and 9.5% had no result. When compared to CP-F, partialCPs exhibited a significantly greater percentage of mosaic embryos (3.6% v. 15.6%, p = 0.032). Additionally, we found that a greater percentage of embryos demonstrating CP-F exhibited morphokinetics that fell into optimal ranges for embryo development when compared to those with partial compaction patterns. CONCLUSION: Time-lapse visualization of compaction patterns identified exclusions and/or extrusions as negative indicators of blastocyst formation and blastocyst grade. When compared to full compaction patterns, partial compaction patterns were associated with delayed embryonic development as well as lower rates of optimal kinetic development.

New hyaluronan-based biomatrix for 3-D follicle culture yields functionally competent oocytes.

BACKGROUND: Encapsulation of follicles within a biomatrix is one approach to maintaining 3-D follicle architecture during culture. Hyaluronan is one component of the natural extracellular matrix (ECM) that provides support to cells in vivo. This report describes the application of a novel tyramine-linked hyaluronan for 3-D in vitro follicle culture and the production of developmentally competent metaphase II oocytes. MATERIALS AND METHODS: Enzymatically isolated mouse preantral follicles or follicle clusters (FL-C) from fresh or vitrified ovaries were encapsulated in 3 mg/ml of hyaluronan gel (HA). Follicle growth, antrum formation and meiotic maturation to metaphase II oocytes was monitored. Chromatin staining was used to assess GV oocyte progression towards meiotic competence. Functional competence of in vitro matured (IVM) oocytes was evaluated by in vitro fertilization and ability to develop to blastocyst. Modifying the HA gel by inclusion of laminin (HA-LM), mouse sarcoma extracellular matrix (Matrigel;HA-MG) or placental extracellular matrix (HA-PM) was also tested to see if this might further enhance IVM outcomes. RESULTS: A total of 402 preantral follicles were cultured in HA gel. After hCG trigger, 314 oocyte-cumulus complexes ovulated from the embedded follicles. Meiotic maturation rate to the metaphase II stage was 73% (228/314). After insemination 83% (188/228) of IVM oocytes fertilized with a subsequent blastulation rate of 46% (87/188). A pilot transfer study with 3 recipient mice resulted in the birth of a single pup. HA gel supported individually isolated follicles as well ovarian tissue fragments containing clusters of 6-8 preantral follicles. Meiotic maturation was lower with FL-clusters from vitrified versus fresh ovaries (34% and 55%, respectively; p < 0.007). Modification of the HA gel with ECMs or laminin affected antrum formation and follicle retention. Maturation rates to the metaphase II stage were however not significantly different: 74% for HA gel alone as compared to HA-LM (67%), HA-MG (56%) and HA-PM (58%). CONCLUSION: Hyaluronan gel is an effective and versatile extracellular matrix based biomaterial for 3-D culture of ovarian follicles. This culture model allowed ovulation of functionally competent metaphase II oocytes, capable of fertilization, genomic activation and blastulation. Future testing with human follicles that require longer in vitro culture times should be considered.

The impact of endometriosis on embryo morphokinetics: embryos from endometriosis patients exhibit delayed cell cycle milestones and decreased blastulation rates.

PURPOSE: To compare morphokinetic parameters in embryos obtained from women with and without endometriosis. METHODS: We evaluated a total of 3471 embryos resulting from 434 oocyte retrievals performed at a single academic center. One thousand seventy-eight embryos were obtained from women affected by endometriosis and 2393 came from unaffected controls. All embryos were cultured in a time-lapse incubator chamber for up to 6 days. IVF cycle outcomes and morphokinetic parameters collected prospectively were retrospectively reviewed. RESULTS: Morphokinetic data suggest that embryo development is impaired in embryos obtained from women with endometriosis (EE). EE were slower to achieve the 2-8 cell stages compared to control embryos (CE) (p < 0.001); additionally, time to compaction was delayed compared to CE (p = 0.015). The timing of late developmental events, including morulation and blastulation was also delayed in the endometriosis cohort (p < 0.001). In addition to demonstrating delayed cell cycle milestones, EE were less likely than controls to progress to morula, blastocyst, and expanded blastocyst stages (p < 0.001). Furthermore, a smaller proportion of embryos in the endometriosis group fell into optimal kinetic ranges for cc2 (p = 0.003), t5 (p = 0.019), tSB (p < 0.001), and tEB (p = 0.007). There were no significant differences in clinical pregnancy or live birth rates between groups. CONCLUSION: Embryos from endometriosis patients demonstrate impairments in both early and late developmental events, and progress to the morula, blastocyst, and expanded blastocyst stages at lower rates than control embryos. Despite these differences, IVF outcomes are similar for patients with and without endometriosis.

Utilizing a Human-Computer Interaction Approach to Evaluate the Design of Current Pharmacogenomics Clinical Decision Support.

A formal assessment of pharmacogenomics clinical decision support (PGx-CDS) by providers is lacking in the literature. The objective of this study was to evaluate the usability of PGx-CDS tools that have been implemented in a healthcare setting. We enrolled ten prescribing healthcare providers and had them complete a 60-min usability session, which included interacting with two PGx-CDS scenarios using the "Think Aloud" technique, as well as completing the Computer System Usability Questionnaire (CSUQ). Providers reported positive comments, negative comments, and suggestions for the two PGx-CDS during the usability testing. Most provider comments were in favor of the current PGx-CDS design, with the exception of how the genotype and phenotype information is displayed. The mean CSUQ score for the PGx-CDS overall satisfaction was 6.3 ± 0.95, with seven strongly agreeing and one strongly disagreeing for overall satisfaction. The implemented PGx-CDS at our institution was well received by prescribing healthcare providers. The feedback collected from the session will guide future PGx-CDS designs for our healthcare system and provide a framework for other institutions implementing PGx-CDS.

Diverse Approaches to Ovarian Tissue Cryopreservation Have Equivalent Outcomes in Markers of Tissue Viability.

Ovarian tissue cryopreservation (OTC) is an accepted method of fertility preservation. However, OTC is not standardized and many variations exist in the freezing strategy, tissue processing, and surgical approach. In this pilot study, we used a sheep model to compare slow freezing versus vitrification techniques, as well as the feasibility of processing ovarian tissue into a hyaluronan suspension of small ovarian units. Twelve ovaries were harvested from six female ewes. Paired tissues from each animal were assigned to different treatments and underwent freezing, thawing, autotransplantation, and second-look surgery, for a total of 18 surgical procedures and 3 measured time points. Treatments included whole tissue strips versus gel suspension and slow freezing versus vitrification. At each of the time points, tissue viability was measured by immunohistochemical analysis of CD31 and cleaved caspase-3 (CCASP3). CD31 and CCASP3 expression levels were equivalent between slow freezing and vitrification, and between whole ovarian tissue strips and gel suspension of fragmented ovarian tissue, at all time points. These preliminary data using a sheep model suggest that ovarian tissue is robust and likely to be minimally affected by aggressive fragmentation using a hyaluronan suspension. Furthermore, we provide evidence in support of vitrification as a viable option in OTC. Hyaluronan suspension of ovarian cortical fragments is novel and may represent a desirable method for reimplantation of frozen-thawed ovarian tissue in patients where occult malignant cells are a concern.

Randomized study of G-TL and global media for blastocyst culture in the EmbryoScope: morphokinetics, pregnancy, and live births after single-embryo transfer.

OBJECTIVE: To evaluate the efficacy of two different in vitro fertilization culture media for blastocyst development, pregnancy, and live birth rate. Global (GB) medium (used without refreshment) and G-TL medium (designed specifically for culture in time-lapse incubators) were compared. DESIGN: Prospective randomized study of sibling embryo culture in two culture media. SETTING: In vitro fertilization clinic. PATIENT(S): Women undergoing fresh or frozen cycles using autologous or donor oocytes. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The primary endpoints were implantation, pregnancy, and live birth rate (LBR) after single blastocyst transfer. Secondary endpoints included embryo morphokinetics, development of good-quality blastocysts, and euploidy rate. RESULT(S): Kinetic data from 10,768 sibling pronucleate embryos cultured in the EmbryoScope were compared. GB embryos initiated compaction earlier and formed morula sooner than their G-TL counterparts. The mean timing for start of blastulation did not differ. The interval between start of blastulation and time of blastocyst formation was observed to be <12 hours for proportionately more GB compared with G-TL-cultured embryos. Despite a higher rate of observed dysmorphisms in GB embryos, the euploidy rate among biopsied blastocysts did not differ between media. A total of 820 single-embryo transfer cycles were performed. Implantation rates were similar between media, independent of whether the embryo transferred was fresh (GB 58.7% vs. G-TL 61.7%) or frozen (GB 64.1% vs. G-TL 60.5%). Live birth rates were also not different. With GB medium, the LBR for fresh and frozen transfers was 54.2% and 53.1%, respectively, as compared with 51.1% and 50%, respectively, with G-TL. CONCLUSION(S): Uninterrupted culture in a time-lapse incubator without medium refreshment was well supported by both media tested. Differences in morphokinetics did not necessarily dictate the superiority of one media over the other. Both pregnancy and LBR were not significantly influenced by choice of culture medium. The euploidy rate was also independent of culture medium.

Cardiac Monitoring for Thoracic Radiation Therapy: Survey of Practice Patterns in the United States.

OBJECTIVE: The American Society of Clinical Oncology (ASCO) 2017 guidelines on cardiac monitoring during cancer treatments identified patients receiving thoracic radiation (TRT) ≥30 Gy (heart in field) at increased risk for developing radiation-induced heart disease (RIHD). ASCO encouraged clinicians to actively screen and monitor for baseline modifiable cardiac risk factors and therapy-induced cardiotoxicity in this high-risk population. Coronary artery calcium (CAC) is an independent risk factor for adverse cardiac events that can be mitigated with preventative medical therapy. It is unclear whether radiation oncologists (ROs) are aware of ASCO guidelines or the implications of CAC observed on computed tomographic scans. We report on practice patterns, perceptions, and experiences of cardiac monitoring for patients receiving definitive TRT, excluding breast patients. MATERIALS AND METHODS: A 28-question survey was emailed to United States ROs 3 times from September 2018 to January 2019. RESULTS: There were 162 respondents from 42 states, 51% in academic practice. Most ROs (81%) were not aware of the ASCO guidelines. Only 24% agreed with the guidelines, only 27% believed symptomatic RIHD could manifest within 2 years of TRT, and 69% thought there was a lack of strong evidence for type and timing of cardiac monitoring tests. If CAC was evident on computed tomographic scans, 40% took no further action to inform the patient or referring doctor. CONCLUSIONS: This survey highlights a critical gap in knowledge about cardiac monitoring and potentially life-saving opportunities for preventive cardiac medical management. Future studies focusing on timing and detection of RIHD may elucidate the utility of cardiac monitoring for TRT patients.

Nationwide Trends in Heart-Sparing Techniques Utilized in Radiation Therapy for Breast Cancer.

PURPOSE: Radiation dose to the heart correlates with cardiac-related deaths and may partially diminish the benefit of radiation for breast cancer. This study assessed the current nationwide trends in heart-sparing techniques for breast cancer radiation. METHODS AND MATERIALS: In November 2017, an institutional review board-approved survey was sent to radiation oncologists in the United States. Questions assessed demographics and the type and frequency of heart-sparing techniques. Data were analyzed using descriptive statistics and χ2 tests. RESULTS: In total, 530 responses (13%) were obtained. Most physicians had practiced >15 years (46%), with most in a private setting (59%). Eighty-three percent of physicians offered prone positioning and/or deep inspiration breath hold (DIBH). This was more common in academic practice (P < .01). Seventy-three percent of physicians used heart-sparing techniques for more than three-fourths of left-sided patients. The most commonly used technique was DIBH, and 43% of physicians used the technique more than three-fourths of the time. Commonly used DIBH systems were Varian RPM (54%) and Vision RT/Align RT (31%). No increase in DIBH use was observed with regional nodal irradiation, and coverage of internal mammary chain nodes varied. Patient tolerance (78%) and cardiac-to-chest wall distance (72%) were the most common determinants of DIBH in left-sided patients. Twenty-three percent of physicians used DIBH for right-sided patients, with lung (64%) and heart sparing (46%) as the most common reasons for use. Lack of facilities was the most common reason not to use DIBH (61%). CONCLUSIONS: Most respondents offer heart-sparing techniques for breast cancer radiation; this is more common in academic centers. DIBH is the most common technique across all practice settings. DIBH is much less commonly used in right-sided patients but is still used by >20% of practitioners, with lung and heart sparing cited as reasons for use. More data are needed to determine if and when this technique should be used in right-sided cases.

Blastomere cleavage plane orientation and the tetrahedral formation are associated with increased probability of a good-quality blastocyst for cryopreservation or transfer: a time-lapse study.

OBJECTIVE: To determine whether blastomere spatial arrangement in early human embryos is reflective of embryonic potential. DESIGN: Retrospective analysis of prospectively collected data. SETTING: Single academic center. PATIENT(S): Patients undergoing a single blastocyst transfer. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Developmental kinetics, blastocyst quality, embryo dysmorphisms, and live birth rate. RESULT(S): A total of 716 embryos were examined in detail for cleavage plane orientation, blastomere arrangement, and morphokinetic behavior. Tetrahedral (TET) and nontetrahedral embryos (nTET) differed significantly in developmental kinetics. The frequency of dysmorphisms, multinucleation, and irregular chaotic division was higher in nTET embryos. Only 44% of nTET versus 62.9% of TET embryos were scored as top-quality blastocysts. After adjusting for age, our data indicated that having TET embryos significantly increased the odds of having a blastocyst for cryopreservation/transfer (odds ratio, 3.58; confidence interval, 2.42-5.28) when compared with nTET. A total of 164 fresh single ETs were performed with blastocyst-stage embryos. The implantation rate for TET- and nTET-derived blastocysts were similar (64.7% and 62%, respectively). The live birth rate was 55% in both groups. A meridonal first division was noted in 85% of the fresh SET blastocysts. CONCLUSION(S): Cleavage plane orientation during the first three divisions appeared to dictate final blastomere spatial arrangement. The TET formation at the four-cell stage was predictive for embryos most likely to develop into good-quality blastocysts for cryopreservation/transfer. Morphokinetic markers of embryo potential were significantly different between TET and nTET embryos.

Concurrent Radiation and Immunotherapy: Survey of Practice Patterns in the United States.

OBJECTIVE: The objective of this study was to report on US radiation oncologists' (ROs) practice patterns and perceptions of concurrent radiation (RT) and immunotherapy (IT) (CRI). METHODS: A 22-question survey was emailed to radiation oncologists in February 2018. CRI was defined as RT completed within 1 week before initial IT infusion through 4 weeks after final IT infusion. RESULTS: Of the 323 respondents from 45 states, 88% had experience treating a patient with CRI, including 51% private and 48% academic physicians. The most common reason for not offering CRI was concerns of increased toxicity (50%). Although 84% to 94% of respondents did not change RT dose, more ROs decreased dose when treating central structures (chest/abdomen/pelvis) versus noncentral structures (brain/head and neck/extremities): 13% to 15% versus 4% to 8%, P<0.001. The majority (58% to 80%) of respondents would not delay RT from last IT infusion. Moderate and significant actual toxicities were rare (medical intervention 6%, hospitalization/death <1%). 97.5% of ROs did not routinely prescribed prophylactic steroids for CRI. More ROs believed CRI with SBRT/SRS versus palliative RT had better local control (35% vs. 25%, P<0.05) and higher rates of abscopal responses (41% vs. 25%, P<0.01). CONCLUSIONS: Despite concerns for toxicity, ROs with CRI experience reported minimal toxicities. Most ROs do not alter RT dose, use prophylactic steroids, or delay starting RT from last IT infusion. Uncertainty remains about improved local control outcomes and abscopal responses from CRI, with a perception that concurrent SBRT offers better outcomes than palliative RT. These survey results may help guide ROs until more definitive data are available.

Azoospermia and embryo morphokinetics: testicular sperm-derived embryos exhibit delays in early cell cycle events and increased arrest prior to compaction.

PURPOSE: Sperm play an essential role in embryonic genome activation and embryonic progression to blastocyst. In the present work, we focus on development of embryos created as a result of ICSI with testicular or epididymal sperm from azoospermic males and compare this to outcomes from normospermic males. The objective of this study was to determine if sperm origin influences clinical outcomes, the kinetics of embryo development, or the incidence of cleavage anomalies and multinucleation. METHODS: A total of 93 consecutive intracytoplasmic sperm injection cycles (ICSI) performed for 83 couples were included in this study. Observations were made on 594 fertilized oocytes cultured in the EmbryoScope using time-lapse microscopy (TLM). Epididymal sperm (n = 29) cycles or surgically retrieved sperm from the testis (TESE; n = 37 cycles) of men with either obstructive (OA) or non-obstructive azoospermia (NOA) were used to inject oocytes. A further 27 ICSI cycles were performed using ejaculated sperm from normospermic males, designated as our control sperm (CS) group. Kinetic data and cycle outcomes were retrospectively analyzed. RESULTS: The clinical pregnancy rate was not different between the three groups (TESE 51.4%, PESA 57.7%, and CS 59.3%). A non-significant decrease was observed in both implantation (30.9%) and live birth rate (43%) with TESE as compared to PESA (35.3%, 58%, respectively) and CS groups (45.1%, 56%, respectively). Failure to compact was significantly higher amongst TESE-NOA embryos (35.2%; P < 0.001) as compared to TESE-OA (4%), PESA (9%), and CS (3.8%) embryos. The two points at which TESE-derived embryos (both NOA and OA) behaved most differently from PESA and CS embryos was at cc2 (t3-t2; time to initiation of the second cell cycle) and tSB (time to start of blastulation). A significantly lower percentage of TESE embryos exhibited kinetics typically ascribed to high quality embryos with the greatest developmental potential. Finally, the incidence of direct uneven cleavage (DUC) was observed to be significantly higher after ICSI with sperm retrieved from azoospermic males. CONCLUSIONS: TLM allowed a more in depth comparison of paternal influence on embryo morphokinetics and helped to identify specific differences in cell cycle kinetics. TESE-NOA embryos exhibited a higher incidence of compaction failure.

Deep Inspiration Breath Hold: Techniques and Advantages for Cardiac Sparing During Breast Cancer Irradiation.

Historically, heart dose from left-sided breast radiotherapy has been associated with a risk of cardiac injury. Data suggests that there is not a threshold for the deleterious effects from radiation on the heart. Over the past several years, advances in radiation delivery techniques have reduced cardiac morbidity due to treatment. Deep inspiration breath hold (DIBH) is a technique that takes advantage of a more favorable position of the heart during inspiration to minimize heart doses over a course of radiation therapy. In the accompanying review article, we outline several methods used to deliver treatment with DIBH, quantify the benefits of DIBH treatment, discuss considerations for patient selection, and identify challenges associated with DIBH techniques.

Are cleavage anomalies, multinucleation, or specific cell cycle kinetics observed with time-lapse imaging predictive of embryo developmental capacity or ploidy?

OBJECTIVE: To determine whether cleavage anomalies, multinucleation, and specific cellular kinetic parameters available from time-lapse imaging are predictive of developmental capacity or blastocyst chromosomal status. DESIGN: Retrospective analysis of prospectively collected data. SETTING: Single academic center. PATIENT(S): A total of 1,478 zygotes from patients with blastocysts biopsied for preimplantation genetic screening were cultured in the EmbryoScope. INTERVENTION(S): Trophectoderm biopsy. MAIN OUTCOME MEASURE(S): Embryo dysmorphisms, developmental kinetics, and euploidy. RESULT(S): Of the 767 biopsied blastocysts, 41.6% (95% confidence interval [CI], 38%-45%) were diagnosed as euploid. Individual dysmorphisms such as multinucleation, reverse cleavage, irregular chaotic division, or direct uneven cleavage were not associated with aneuploidy. Direct uneven cleavage and irregular chaotic division embryos did, however, exhibit lower developmental potential. The presence of two or more dysmorphisms was associated with an overall lower euploidy rate, 27.6% (95% CI 19%-39%). Early embryo kinetics were predictive of blastocyst development but not ploidy status. In contrast, chromosomal status correlated significantly with start time of blastulation (tSB), expansion (tEB), and the tEB-tSB interval. A lower euploidy rate, 36.6% (95% CI 33%-42%) was observed with tSB ≥ 96.2 hours, compared with 48.2% with tSB < 96.2 (95% CI 42%-54%). A drop in euploidy rate to 30% (95% CI 25%-37%) was observed in blastocysts with delayed expansion (tEB > 116). The proportion of euploid blastocysts was increased with tEB-tSB intervals of ≤13 hours. A logistic regression model to enhance the probability of selecting a euploid blastocyst was constructed. CONCLUSION(S): Morphokinetics may aid in selection of euploid embryos from a cohort of day 5/6 blastocysts.

Beta-arrestin 1 regulation of reward-motivated behaviors and glutamatergic function.

The two highly homologous non-visual arrestins, beta-arrestin 1 and 2, are ubiquitously expressed in the central nervous system, yet knowledge of their disparate roles is limited. While beta-arrestin 2 (ßarr2) has been implicated in several aspects of reward-related learning and behavior, very little is known about the behavioral function of beta-arrestin 1 (ßarr1). Using mice lacking ßarr1, we focused on the role of this scaffolding and signal transduction protein in reward-motivated behaviors and in striatal glutamatergic function. We found that ßarr1 KO mice were both slower in acquiring cocaine self-administration and in extinguishing this behavior. They also showed deficits in learning tasks supported by a natural food reward, suggesting a general alteration in reward processing. We then examined glutamatergic synaptic strength in WT and KO medium spiny neurons (MSNs) of the Nucleus Accumbens (NAc) shell in naïve animals, and from those that underwent cocaine self-administration. An increase in the AMPA/NMDA (A/N) ratio and a relative lack of GluN2B-enriched NMDARs was found in naïve KO vs WT MSNs. Applying Lim Domain Kinase (LIMK1), the kinase that phosphorylates and inactivates cofilin, to these cells, showed that both ßarr1 and LIMK regulate the A/N ratio and GluN2B-NMDARs. Cocaine self-administration increased the A/N ratio and GluN2B-NMDARs in WT MSNs and, although the A/N ratio also increased in KO MSNs, this was accompanied by fewer GluN2B-NMDARs and an appearance of calcium-permeable AMPARs. Finally, to examine the consequences of reduced basal GluN2B-NMDARs in reward-processing seen in KO mice, we chronically infused ifenprodil, a GluN2B antagonist, into the NAc shell of WT mice. This intervention substantially reduced food-motivated behavior. Together these findings identify a previously unknown role of ßarr1 in regulating specific reward-motivated behaviors and glutamatergic function.

Delayed blastulation, multinucleation, and expansion grade are independently associated with live-birth rates in frozen blastocyst transfer cycles.

OBJECTIVE: To identify blastocyst features independently predictive of successful pregnancy and live births with vitrified-warmed blastocysts. DESIGN: Retrospective study. SETTING: Academic hospital. PATIENT(S): Women undergoing a cycle with transfer of blastocysts vitrified using the Rapid-i closed carrier (n = 358). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Clinical pregnancy and live-birth rates analyzed using logistic regression analysis. RESULT(S): A total of 669 vitrified-warmed blastocysts were assessed. The survival rate was 95%. A mean of 1.7 ± 0.5 embryos were transferred. The clinical pregnancy, live-birth, and implantation rates were 55%, 46%, and 43%, respectively. The odds of clinical pregnancy (odds ratio [OR] 3.08; 95% confidence interval [CI], 1.88-5.12) and live birth (OR 2.93; 95% CI, 1.79-4.85) were three times higher with day-5 blastocysts versus slower-growing day-6 vitrified blastocysts, irrespective of patient age at cryopreservation. Blastocysts from multinucleated embryos were half as likely to result in a live birth (OR 0.46; 95% CI, 0.22-0.91). A four -fold increase in live birth was observed if an expanded blastocyst was available for transfer. The inner cell mass-trophectoderm score correlated to positive outcomes in the univariate analysis. The implantation rate was statistically significantly higher for day-5 versus day-6 vitrified blastocysts (50% vs. 29%, respectively). CONCLUSION(S): The blastocyst expansion grade after warming was predictive of successful outcomes independent of the inner cell mass or trophectoderm score. Delayed blastulation and multinucleation were independently associated with lower live-birth rates in frozen cycles. Implantation potential of the frozen blastocysts available should be included in the decision-making process regarding embryo number for transfer.

Ovarian Tissue Cryopreservation for Benign Gynecologic Disease: A Case of Ovarian Torsion and Review of the Literature.

Ovarian tissue cryopreservation (OTC) is an emerging method for fertility preservation. Although OTC has been previously proposed for benign indications, to our knowledge this is the first report highlighting the use of OTC for the indication of ovarian torsion. A 36-year-old nulligravid woman with a history of recurrent ovarian torsion presented with an acute episode of ovarian torsion confirmed by ultrasound. She requested a laparoscopic oophorectomy because her previous oophoropexy had failed, and in light of this was counseled to undergo concurrent OTC. On laparoscopy, 10 strips of ovarian cortex were obtained. A portion of this tissue was sent for pathological analysis, which revealed a primordial follicle density of 167 follicles/mm(3), a primary follicle density of 38 follicles/mm(3), and minimal ischemic damage. Although the clinical application of OTC continues to evolve and requires further investigation, the possibility of expanding the indications for benign gynecologic conditions is promising.

Does the addition of time-lapse morphokinetics in the selection of embryos for transfer improve pregnancy rates? A randomized controlled trial.

OBJECTIVE: To determine if the addition of continuous morphokinetic data improves reproductive outcomes when all embryos are cultured in a closed system. DESIGN: Prospective, randomized, controlled study. SETTING: Single academic center. PATIENT(S): A total of 235 patients undergoing fresh autologous IVF cycles with at least four embryos, cultured in the Embryoscope: 116 patients randomized to conventional once-daily morphologic embryo screening (CS) and 119 to additional time-lapse kinetic monitoring (TLM) for selection. INTERVENTION(S): TLM versus CS. MAIN OUTCOME MEASURE(S): Intrauterine clinical pregnancy (CPR) and implantation (IR) rates. RESULT(S): CPR and IR were similar overall (TLM vs. CS, respectively: CPR 68% vs. 63%; IR 51% vs. 45%) and with blastocyst transfers (CPR 74% vs. 67%; IR 56% vs. 51%). CPR with day 5 transfer was threefold higher than day 3 transfer, but group (TLM vs. CS) was not a significant predictor of clinical pregnancy or implantation. Significantly more multinucleation was detected when CS embryos were retrospectively reviewed with the use of TLM (7.0% vs. 35.3%), and multinucleation was independently associated with decreased rates of implantation. Time to the start of blastulation of <100 hours after insemination and the morphokinetic scoring system used in the TLM group were independently associated with implantation. CONCLUSION(S): The addition of time-lapse morphokinetic data did not significantly improve clinical reproductive outcomes in all patients and in those with blastocyst transfers. Absence of multinucleation, timing of blastulation, and morphokinetic score were found to be associated with blastocyst implantation rates. CLINICAL TRIAL REGISTRATION NUMBER: NCT02081859.

Human embryonic stem cell cultivation: historical perspective and evolution of xeno-free culture systems.

Human embryonic stem cells (hESC) have emerged as attractive candidates for cell-based therapies that are capable of restoring lost cell and tissue function. These unique cells are able to self-renew indefinitely and have the capacity to differentiate in to all three germ layers (ectoderm, endoderm and mesoderm). Harnessing the power of these pluripotent stem cells could potentially offer new therapeutic treatment options for a variety of medical conditions. Since the initial derivation of hESC lines in 1998, tremendous headway has been made in better understanding stem cell biology and culture requirements for maintenance of pluripotency. The approval of the first clinical trials of hESC cells for treatment of spinal cord injury and macular degeneration in 2010 marked the beginning of a new era in regenerative medicine. Yet it was clearly recognized that the clinical utility of hESC transplantation was still limited by several challenges. One of the most immediate issues has been the exposure of stem cells to animal pathogens, during hESC derivation and during in vitro propagation. Initial culture protocols used co-culture with inactivated mouse fibroblast feeder (MEF) or human feeder layers with fetal bovine serum or alternatively serum replacement proteins to support stem cell proliferation. Most hESC lines currently in use have been exposed to animal products, thus carrying the risk of xeno-transmitted infections and immune reaction. This mini review provides a historic perspective on human embryonic stem cell culture and the evolution of new culture models. We highlight the challenges and advances being made towards the development of xeno-free culture systems suitable for therapeutic applications.

Analysis of embryo morphokinetics, multinucleation and cleavage anomalies using continuous time-lapse monitoring in blastocyst transfer cycles.

BACKGROUND: Time-lapse imaging combined with embryo morphokinetics may offer a non-invasive means for improving embryo selection. Data from clinics worldwide are necessary to compare and ultimately develop embryo classifications models using kinetic data. The primary objective of this study was to determine if there were kinetic differences between embryos with limited potential and those more often associated with in vitro blastocyst formation and/or implantation. We also wanted to compare putative kinetic markers for embryo selection as proposed by other laboratories to what we were observing in our own laboratory setting. METHODS: Kinetic data and cycle outcomes were retrospectively analyzed in patients age 39 and younger with 7 or more zygotes cultured in the Embryoscope. Timing of specific events from the point of insemination were determined using time-lapse (TL) imaging. The following kinetic markers were assessed: time to syngamy (tPNf), t2, time to two cells (c), 3c (t3), 4c ( t4), 5c (t5), 8c (t8), morula (tMor), start of blastulation (tSB); tBL, blastocyst (tBL); expanded blastocyst (tEBL). Durations of the second (cc2) and third (cc3) cell cycles, the t5-t2 interval as well as time to complete synchronous divisions s1, s2 and s3 were calculated. Incidence and impact on development of nuclear and cleavage anomalies were also assessed. RESULTS: A total of 648 embryos transferred on day 5 were analyzed. The clinical pregnancy and implantation rate were 72% and 50%, respectively. Morphokinetic data showed that tPNf, t2,t4, t8, s1, s2,s3 and cc2 were significantly different in embryos forming blastocysts (ET or frozen) versus those with limited potential either failing to blastulate or else forming poor quality blastocysts ,ultimately discarded. Comparison of embryo kinetics in cycles with all embryos implanting (KID+) versus no implantation (KID-) suggested that markers of embryo competence to implant may be different from ability to form a blastocyst. The incidence of multinucleation and reverse cleavage amongst the embryos observed was 25% and 7%, respectively. Over 40% of embryos exhibiting these characteristics did however form blastocysts meeting our criteria for freezing. CONCLUSIONS: These data provide us with a platform with which to potentially enhance embryo selection for transfer.