Research question: Will ultra-fast vitrification (UFV) and rapid elution of mature human oocytes retain the reliable, high survival rates and meiotic spindle normality seen in the germinal vesicle model, and will these oocytes maintain their developmental competence to form blastocyst-stage embryos following artificial oocyte activation (AOA)? Design: Conventional vitrification treatment was compared with UFV treatment in mature, germinal-vesicle-derived oocytes (Phase 2, Expt. 2, n = 50) and substandard donor oocytes, metaphase I- metaphase II (MII) oocytes and poor-quality MII oocytes (n = 222). Post-warming survival, the integrity of the meiotic spindle and AOA-related development were assessed. Results: Overall survival rates were higher (P = 0.003) for UFV/rapid elution treatment (94-100%, mean = 98%) compared with conventional vitrification/control dilution treatment (80-90%, mean = 83.3%). MII oocytes derived from immature germinal vesicles following conventional vitrification/control dilution or UFV/rapid elution treatments proved to be capable of activated development (54-71% cleavage rate), with four blastocysts produced. AOA treatment with DMAP exposure yielded optimal activated development. When vitrifying mature oocytes, both UFV and conventional vitrification treatments exhibited normal activated development and blastocyst production (34.9% and 31.7%, respectively). Conclusions: Considering that oocyte freezing was deemed non-experimental based primarily on healthy live births from frozen-oocyte-derived embryo transfer the validation of normal blastocyst formation using the novel UFV approach is a critical accomplishment. The UFV method for oocyte cryopreservation represents a strategic deviation from traditional semi- equilibration vitrification protocols. UFV is a more time-efficient approach that consistently yields a higher survival rate, and thus has the potential to create more embryos. These fi ndings justify proceeding with strategic clinical trial applications.

The ZyMōt sperm separation device has proven favorable for use in elevated DNA fragmentation index (DFI) male factor patients, as an alternative to density gradient (DG) washing or surgically attained testicular sperm. In 2020, without fully understanding the infectivity and transmission potential of SARS-CoV2 in semen, a more liberal application of a timed ZyMōt microfluidic swim-up was applied to our IVF patients to dilute out and minimize potential pathogens. This study aimed to evaluate whether the use of ZyMōt sperm improved normal embryo development. Retrospective analysis of PGT-A/ICSI cycles (N= 3219) between 2016-2020 was conducted to assessed fertilization rates (FR), blastocyst development/utilization rates (BUR) and genetic outcomes. Sperm preparations were performed per standard operating or manufacturer advised (i.e., ZyMōt) procedures. Cumulus oocyte complexes were harvested 36h post-hCG, stripped and ICSI performed 3-5hr later. Zygotes were assessed at 16-18hr post-ICSI, and embryos cultured under humidified tri-gas incubation for up to 7 days. Blastocyst (BL) development as evaluated, and expanded BL or greater were biopsied on Days 5, 6 or 7. All BL were vitrified and genetics determinations for euploidy, aneuploidy and mosaicism were contrast. Applying Chi-squared analysis, we compared potential differences (p<0.05) between oocytes inseminated by DG wash (n=23,549), ZyMōt wash (n=7,331) or testicular sperm (n=815). No difference in FR (76%), D5 BL formation (52-56%) or BUR (52-53%) was detected between DG and ZyMōt washed sperm, respectively. Meanwhile, testicular sperm had a lower FR (70%; p<0.05), fewer BL forming on D5 (48%; p<0.05) and a lower overall BUR (41%; p<0.05). In addition, fewer testicular-derived BL were euploid (39%; p<0.05) with more aneuploidy (54%; p<0.05) than DG wash (50%, 39%; respectively) or ZyMōt swim-up (45%, 37%; respectively) derived-embryos. No difference in potentially viable BL (Euploidy+Mosiac outcome) was observed between DG or ZyMōt wash groups (63-64%). Application of the ZyMōt device in the general IVF population offered no benefit to embryo development outcomes compared to standard sperm wash procedures. Our data does support that microfluidic separation of sperm using ZyMōt for male factor patients with elevated DFI is a more favorable and cost-effective approach to surgically attaining testicular sperm when ejaculated sperm is possible. However, when insufficient motile and or morphologically normal sperm are available in an ejaculate further analysis is needed to elucidate the benefit of testicular biopsy treatment, as our assessments in this study may be biased by including men with non-obstructive azoospermia.

To determine whether the degree of blastocyst re-expansion post warming affects the Clinical Pregnancy Rate (CPR) in PGT-A tested Embryo Transfer cycles. Retrospective analysis of data from a single private fertility clinic CPR of 519 frozen PGT-A tested euploid blastocyst was evaluated for sFET cases during 2018-2019. CPR was determined by the presence of a positive fetal heartbeat (FHB) at 7 weeks. As a standard lab protocol, all fertilized oocytes underwent uninterrupted extended culture in FujiFilm Irvine Scientific Media CSCM-NXC and CSCM-NX until biopsy on day 5, 6 or 7. Transfers were performed using vitrified/warmed blastocysts. Blastocyst warming was performed on the day of the sFET and re-expansion was checked at the time of transfer. A standardized expansion guide was created, and embryologists were trained to use it to examine and document the degree of re-expansion post warming. Re-expansion evaluations were subdivided into five intervals; 0%, 25%, 50%, 75% and 100%. Data was analyzed by mean time difference for blastocyst re-expansion (time difference between the first and the second check), the patients’ age, the egg source (donor vs patient) and the embryo recipient (gestational carrier vs patient). Statistical analysis was performed using Chi-square (P <0.05). A multivariable linear regression analysis was also performed to adjust for confounders such as age. Our data show that embryos with > 50% re-expansion post warming have a significantly higher CPR when compared to embryos with < 50% re-expansion (63% vs 21% respectively, P= 0.002). Furthermore, the difference was more substantial with embryos with < 25% re-expansion compared to embryos that achieved >= 50% re-expansion (25% vs 71%, p=0.001) Table 1. There was no correlation between patients’ age and the degree of re-expansion (P=0.995). In addition, there was not an association between the degree of expansion and the egg source (P= 0.913) or embryo recipient (P= 0.879) categories. Finally, the mean time difference from thaw did not influence the CPR when correlated with the degree of expansion (P=0.824). Our data suggest that the degree of embryo re-expansion post warming could serve as a strong predictor of clinical pregnancy in frozen embryo transfers of euploid blastocysts. Furthermore, our data suggests that a minimum of 25% of re-expansion may be a good indicator to predict the embryo’s implantation potential.Table 1The clinical pregnancy rate in each expansion interval. Each subscript letter denotes a subset of Expansion (%) categories that do not differ significantly from each other at the 0.05 level.Expansion (%)Transfer Count(n)FHB(n)CPR014214%a2520525%a,b50282071%c75493061%c10040825663%cTotal519 Open table in a new tab

To determine and compare the clinical outcomes of transferring a trophectoderm biopsied fully hatched blastocyst to other blastocyst stages in Single Embryo Transfer (SET) cycles. Retrospective analysis of private clinic data outcomes. Pregnancy Rate (PR), Implantation Rate (IR) and Clinical Pregnancy Rate (CPR) of PGT-tested blastocyst SETs during 2017-2018 were analyzed. All fertilized oocytes underwent uninterrupted extended culture until the day of biopsy. Trophectoderm biopsy was performed on culture day 5, 6, or 7 using a single pulse laser breach of the zona pellucida, followed by the insertion of a beveled needle with excision of 3-5 cells. PGT testing was performed utilizing NexGen sequencing. All transfers were performed with vitrified/warmed blastocysts. PR was determined by bHCG level of > 5mIU/ml. IR was determined by the number of sacs present at 3 weeks after positive pregnancy, and CPR by the presence of a positive fetal heart beat (FHB) at 7 weeks gestation. Statistical analysis was performed using Chi-square (P <0.05). Outcomes from 651 transfers utilizing euploid SET were analyzed. Fully hatched blastocysts (n=73) showed a significantly lower PR (42%) when compared to blastocysts with a blastocoel of more than or equal to half the volume of the embryo (n=168) (58%) (p=0.02), expanded blastocysts (n=260) with a full blastocoel (60%) (p=0.009) and hatching blastocysts (n=150) (65%) (p=0.001). SETs with fully hatched blastocysts showed the lowest IR% (29%) when compared to full blastocysts (51%) (p=0.002), expanded blastocysts (53%) (p=0.0002) and hatching blastocysts (56%) (p=0.0004). Moreover, CPR% was significantly impacted after the transfer of fully hatched blastocysts (27%) when compared to full blastocysts (47%) (p=0.004), expanded blastocysts (53%)(p=0.002), and hatching blastocysts (56%) (p=0.0006). Day of development did not influence the clinical outcomes between the different stages of blastocysts (p= 0.18). Also the analysis showed no significant difference among the stages of development within the same category for neither the performing physician nor the transferring embryologist (p=0.94, p=0.65 respectively). It has been recently reported that the transfer of fully hatched blastocysts results in significantly lower success rates when compared to other stages of blastocyst development (James, R. M et al, 2018). It has also been suggested that the complete removal of the zona pellucida increases the implantation potential. However, these studies lack evidence to support the hypothesis (Alteri, A et al, 2018). Despite the striking differences in the outcomes, other factors such as age and day of development did not influence the final result. These findings suggest that the zona pellucida confers some level of protection during transfer, and its absence may contribute to lower clinical outcomes.

Objective: To identify determinants of blastocyst yield, implantation rate, and pregnancy outcome.Design: Retrospective analysis of outcomes of 1,653 cycles of IVF.Setting: Private infertility clinic.Patient(s): Couples presenting to an infertility clinic for IVF.Intervention(s): None.Main Outcome Measure(s): Blastocyst yield, implantation rate, and pregnancy.Result(s): Of a broad array of potential determinants, only the total numbers of oocytes retrieved and properties of day 3 embryos were consistently predictive of blastocyst formation. Relative to numbers of oocytes fertilized by intracytoplasmic sperm injection (ICSI), yields of quality blastocysts were highest in cycles in which <10 oocytes were retrieved. Blastocyst yield was closely linearly correlated with average numbers of blastomeres in embryos on day 3. As oocyte yields rose, average grades and the implantation potential of the blastocysts selected for transfer increased by approximately 0.015 and 0.15%, respectively, for each additional oocyte. Independently, the implantation potential of blastocysts decreased 1.1% for each advancing year in age of the oocyte provider, and, for autologous transfers, uterine receptivity declined an additional 0.6% per year. Higher yields of blastocysts from cycles with high oocyte numbers afforded better selection of blastocysts for transfer, supporting higher overall implantation and pregnancy rates.Conclusion(s): While the proportion of fertilized oocytes that progressed to quality blastocysts diminished as numbers of recovered oocytes rose, rates of implantation and pregnancy after transfer of the selected best blastocysts increased. The age of the oocyte provider and oocyte yields independently impacted blastocyst implantation potential and uterine receptivity after controlled ovarian hyperstimulation, ICSI, and blastocyst transfer. (C) 2014 by American Society for Reproductive Medicine.

OBJECTIVE:To determine whether elements of treatment associated with faster doubling times of total beta-hCG in serum (beta-t2) in pregnant patients are also associated with a higher likelihood of pregnancy in all patients.DESIGN:Retrospective analysis of beta-t2 values, elements of ovarian stimulation (COH), and outcomes.SETTING:Private assisted reproductive technology (ART) center.PATIENT(S):Initial analysis of data from 432 cycles in which conception occurred after COH and embryo transfer, followed by analysis of pregnancy outcomes after 1,287 cycles of COH/ embryo transfer.INTERVENTION(S):No interventions.MAIN OUTCOME MEASURES:The beta-t2 values initially computed from consecutive serum beta-hCG levels in ongoing pregnancies were correlated with multiple properties of the patients and their treatment cycles.RESULT(S):The beta-t2 values during early pregnancy increased exponentially from about 1.6 days at 12 days to about 3.0 days at 24 days after embryo transfer. In those pregnancies which spontaneously aborted, early average beta-t2 values were higher than those for ongoing pregnancies; absolute beta-hCG levels did not differ. Positive correlations were established between beta-t2 values, the number of days of stimulation, and the number of ampules of drug administered per oocyte retrieved. The beta-t2 values were inversely related to average numbers of blastomeres in transferred embryos. Ongoing pregnancy rates (PR) were higher for cycles with lower gonadotropin dosages per oocyte retrieved, and when the average number of blastomeres in transferred embryos was higher.CONCLUSION(S):Steeper beta-hCG doubling times in early pregnancy were associated with lower gonadotropin dosages during ovarian stimulation and with higher numbers of blastomeres in transferred embryos. The latter variables were, in turn, associated with a higher likelihood of pregnancy after embryo transfer.

There is little information on the molecular events underlying the effects of cAMP on human chorionic gonadotropin (hCG) and particularly steroidal hormone production in normal trophoblasts. We examined the effects of 8-bromo-cAMP on mRNAs encoding two components of the cholesterol side-chain cleavage system, cytochrome P-450scc and adrenodoxin, and the α and β subunits of hCG in cultured cytotrophoblasts. cAMP caused an increase in all of these mRNAs within 24 h, whereas actin mRNA declined. α-hCG mRNA increased first, followed by adrenodoxin, β hCG and cytochrome P-450scc mRNAs.The effects of 8-bromo-cAMP on α- and β-hCG, adrenodoxin, and cytochrome P-450scc mRNAs, in cytotrophoblasts and JEG-3 choriocarcinoma cells, required the catalytic unit of protein kinases since H-7, a kinase inhibitor, blocked the increase in the mRNAs and prevented the stimulation of hCG and progesterone secretion. 8-Bromo-cAMP promoted a rapid increase in α-hCG mRNA in cytotrophoblasts in the presence of cycloheximide, an inhibitor of protein synthesis. In cytotrophoblasts, cycloheximide reduced basal and 8-bromo-cAMP-stimulated adrenodoxin mRNA abundance. In contrast, basal and cAMP-stimulated adrenodoxin mRNA was augmented by cycloheximide in JEG-3 cells. These data indicate that: (1) cAMP regulates expression of endocrine functions of normal human trophoblast cells at least in part by controlling the abundance of mRNAs encoding steroidogenic enzymes and hCG subunits; (2) the actions of cAMP require the intermediacy of protein kinases; (3) there are differences in the sensitivity of the cAMP regulation of various mRNAs to inhibitors of protein synthesis; and (4) there are differences between normal and transformed trophoblasts with respect to the control of certain transcripts.