Deep technology for the optimization of cryostorage.

Cryopreservation, for many reasons, has assumed a central role in IVF treatment cycles, which has resulted in rapidly expanding cryopreserved oocyte and embryo inventory of IVF clinics. We aspire to consider how and with what resources and tools "deep" technology can offer solutions to these cryobiology programs. "Deep tech" has been applied as a global term to encompass the most advanced application of big data analysis for the most informed construction of algorithms and most sophisticated instrument design, utilizing, when appropriate and possible, models of automation and robotics to realize all opportunities for highest efficacy, efficiency, and consistency in a process.

Two microcephaly-associated novel missense mutations in CASK specifically disrupt the CASK-neurexin interaction.

Deletion and truncation mutations in the X-linked gene CASK are associated with severe intellectual disability (ID), microcephaly and pontine and cerebellar hypoplasia in girls (MICPCH). The molecular origin of CASK-linked MICPCH is presumed to be due to disruption of the CASK-Tbr-1 interaction. This hypothesis, however, has not been directly tested. Missense variants in CASK are typically asymptomatic in girls. We report three severely affected girls with heterozygous CASK missense mutations (M519T (2), G659D (1)) who exhibit ID, microcephaly, and hindbrain hypoplasia. The mutation M519T results in the replacement of an evolutionarily invariant methionine located in the PDZ signaling domain known to be critical for the CASK-neurexin interaction. CASKM519T is incapable of binding to neurexin, suggesting a critically important role for the CASK-neurexin interaction. The mutation G659D is in the SH3 (Src homology 3) domain of CASK, replacing a semi-conserved glycine with aspartate. We demonstrate that the CASKG659D mutation affects the CASK protein in two independent ways: (1) it increases the protein's propensity to aggregate; and (2) it disrupts the interface between CASK's PDZ (PSD95, Dlg, ZO-1) and SH3 domains, inhibiting the CASK-neurexin interaction despite residing outside of the domain deemed critical for neurexin interaction. Since heterozygosity of other aggregation-inducing mutations (e.g., CASKW919R) does not produce MICPCH, we suggest that the G659D mutation produces microcephaly by disrupting the CASK-neurexin interaction. Our results suggest that disruption of the CASK-neurexin interaction, not the CASK-Tbr-1 interaction, produces microcephaly and cerebellar hypoplasia. These findings underscore the importance of functional validation for variant classification.

Accuracy of preimplantation genetic screening (PGS) is compromised by degree of mosaicism of human embryos.

BACKGROUND: To preclude transfer of aneuploid embryos, current preimplantation genetic screening (PGS) usually involves one trophectoderm biopsy at blastocyst stage, assumed to represent embryo ploidy. Whether one such biopsy can correctly assess embryo ploidy has recently, however, been questioned. METHODS: This descriptive study investigated accuracy of PGS in two ways. Part I: Two infertile couples donated 11 embryos, previously diagnosed as aneuploid and, therefore, destined to be discarded. They were dissected into 37 anonymized specimens, and sent to another national laboratory for repeat analyses to assess (i) inter-laboratory congruity and (ii) intra-embryo congruity of multiple embryo biopsies in a single laboratory. Part II: Reports on human IVF cycle outcomes after transfer of allegedly aneuploid embryos into 8 infertile patients. RESULTS: Only 2/11 (18.2 %) embryos were identically assessed at two PGS laboratories; 4/11 (36.4 %), on repeat analysis were chromosomally normal, 2 mosaic normal/abnormal, and 5/11 (45.5 %) completely differed in reported aneuploidies. In intra-embryo analyses, 5/10 (50 %) differed between biopsy sites. Eight transfers of previously reported aneuploid embryos resulted in 5 chromosomally normal pregnancies, 4 delivered and 1 ongoing. Three patients did not conceive, though 1 among them experienced a chemical pregnancy. CONCLUSIONS: Though populations of both study parts are too small to draw statistically adequately powered conclusions on specific degrees of inaccuracy of PGS, here presented results do raise concerns especially about false-positive diagnoses. While inter-laboratory variations may at least partially be explained by different diagnostic platforms utilized, they cannot explain observed intra-embryo variations, suggesting more frequent trophectoderm mosiaicsm than previously reported. Together with recentl published mouse studies of lineages-specific degrees of survival of aneuploid cells in early stage embryos, these results call into question the biological basis of PGS, based on the assumption that a single trophectoderm biopsy can reliably determine embryo ploidy.

The risk of embryo-endometrium asynchrony increases with maternal age after ovarian stimulation and IVF.

This retrospective cohort analysis examined the effects of maternal age on the incidence of factors associated with embryo-endometrium asynchrony in fresh autologous blastocyst transfer. The study included 1169 routine fresh autologous blastocyst transfers. The main outcome measure was asynchronous transfer defined by delayed (day 6) blastocyst transfer or elevated pre-ovulatory serum progesterone level. Compared with patients younger than 35 years, patients 35 years or older had increased risk of having at least one risk factor for asynchronous transfer, including premature progesterone elevation or delayed blastocyst transfer (RR 1.36; 95% CI 1.24 to 1.50). The older group had increased risk of simultaneously having both risk factors (RR 1.61, 95% CI 1.17 to 2.21) compared with the younger group. In patients younger than 35 years, live birth rate per transfer was 62.9% with day 5 transfer and low progesterone, declining to 27.9% for day 6 transfer combined with elevated progesterone. In patients 35 years or older, live birth rate per transfer was 38.0% with day 5 transfer and low progesterone, declining to 18.1% for day 6 transfer combined with elevated progesterone. Indicators of embryo-endometrium asynchrony increase in prevalence as women age and asynchrony disproportionately decreases birth rates in older patients.

Freeze-all at the blastocyst or bipronuclear stage: a randomized clinical trial.

OBJECTIVE: To compare outcomes for patients randomized to have all embryos cryopreserved at the blastocyst stage or at the bipronuclear stage with subsequent post-thaw culture to the blastocyst stage. DESIGN: Randomized controlled trial. SETTING: Private fertility center. PATIENT(S): This study included 140 women, age 18-40 years, with at least eight antral follicles, and day 3 FSH <10 IU/L undergoing IVF. INTERVENTION(S): After oocyte retrieval, subjects were randomized to have entire embryo cohorts cryopreserved at either the bipronuclear stage (2PN Cryo group) or at the blastocyst stage (Blast Cryo group). MAIN OUTCOME MEASURE(S): Ongoing pregnancy (viable fetal heart motion at 10 weeks' gestation) per oocyte retrieval through the first transfer attempt. RESULT(S): No significant differences were observed between the two study groups in age at retrieval, body mass index, antral follicle count, day 3 FSH level, or IVF cycle parameters. No significant differences were observed in ongoing pregnancy rate per retrieval (62.0%; 95% confidence interval [CI], 50.3%-72.4%) in the 2PN Cryo group; and 55.1%; 95% CI, 42.6%-67.1% in the Blast Cryo group), implantation rate (60.0% vs. 62.7%), ongoing pregnancy rate per thaw (62.0% vs. 59.4%), ongoing pregnancy rate per transfer (67.7% vs. 69.1%), and the cumulative ongoing pregnancy rate per retrieval from all thaws to date of embryos derived from the study retrieval cycle (64.8% vs. 60.9%). CONCLUSION(S): Freeze-all at the blastocyst stage or at the bipronuclear stage has similar efficacy and IVF outcomes. The choice between them may depend primarily on logistical factors. CLINICAL TRIAL REGISTRATION NUMBER: NCT01247987.

Freeze-all can be a superior therapy to another fresh cycle in patients with prior fresh blastocyst implantation failure.

Implantation failure has various causes, including impaired uterine receptivity following ovarian stimulation. This retrospective cohort study compared outcomes in patients with prior implantation failure who elected to undergo another fresh cycle versus those who opted for embryo cohort cryopreservation (freeze-all) and subsequent thaw. There were 269 patients with implantation failure following fresh autologous blastocyst transfer opting to undergo a subsequent cycle, with 163 choosing another fresh cycle and 106 electing freeze-all and subsequent thaw. Multiple logistic regression analysis indicated that cohort cryopreservation was associated with greater chance of live birth when compared with another fresh cycle (P < 0.0001). The odds ratio for live birth with freeze-all relative to a fresh cycle was 3.8 (95% CI 2.1-7.2). A second analysis was then performed using cumulative live birth rate as the outcome measure. Multiple logistic regression indicated freeze-all was associated with greater cumulative live birth rate than was a fresh cycle (OR 1.9, 95% CI 1.1-3.3, P = 0.0287). These findings suggest that, following implantation failure with fresh blastocysts, patients have a significantly greater chance of live birth with freeze-all and subsequent thaw than with another fresh cycle.

Clinical rationale for cryopreservation of entire embryo cohorts in lieu of fresh transfer.

Recent dramatic increases in success rates with frozen-thawed embryo transfer (FET) are encouraging, as are numerous findings of several reduced risks with FET when compared with fresh transfer. These reduced risks include low birth weight and prematurity, among others. However, FET is also associated with increased risks of macrosomia and large for gestational age. There have been reports of greater implantation and pregnancy rates with FET than with fresh autologous embryo transfer, suggesting superior endometrial receptivity in the absence of ovarian stimulation. As cryo-technology evolves, there is potential for further increase in FET success rates, but for now it may be best to follow an individualized approach, balancing fresh transfer and embryo cohort cryopreservation options while considering patient characteristics, cycle parameters, and clinic success rates.

Matched-cohort comparison of single-embryo transfers in fresh and frozen-thawed embryo transfer cycles.

OBJECTIVE: To discern the potential effect of ovarian stimulation on implantation potential by comparing ongoing pregnancy rates from matched blastocysts in fresh and frozen-thawed single-embryo-transfer cycles. DESIGN: Matched cohort study. SETTING: Private fertility center. PATIENT(S): Ninety-three matched pairs of single-blastocyst transfer. INTERVENTION(S): Fresh and frozen-thawed embryo transfers were matched on embryo parameters and patient age. MAIN OUTCOME MEASURE(S): Ongoing pregnancy at 10 weeks' gestation. RESULT(S): The fresh and frozen-thawed groups did not differ significantly in blastocyst diameter, inner cell mass size, trophectoderm cell count, patient age, use of genetic screening, or presence of supernumerary embryos. The ongoing pregnancy rate was significantly greater in the frozen-thawed group than in the fresh group for transfers of day 6 blastocysts (54.3% vs. 17.1%, respectively), but not for day 5 blastocysts (60.9% vs. 56.5%, respectively). This resulted in the overall ongoing pregnancy rate to be significantly greater in the frozen-thawed group than in the fresh group (55.9% vs. 26.9%, respectively). CONCLUSION(S): Autologous day 6 blastocysts transferred in frozen-thawed cycles have significantly greater chance of viable implantation than morphologically equivalent embryos transferred in fresh cycles. This advantage appears to result from impaired implantation of day 6 blastocysts in fresh transfers after ovarian stimulation, suggesting that embryo-endometrium asynchrony is a major cause of impaired endometrial receptivity after ovarian stimulation.

Frozen-thawed embryo transfer is associated with a significantly reduced incidence of ectopic pregnancy.

OBJECTIVE: To compare the incidence of ectopic pregnancy (EP) after fresh ET and thawed ET. DESIGN: Retrospective cohort study. SETTING: Private fertility center. PATIENT(S): This retrospective study included 2,150 blastocyst transfers, including all 1,460 fresh autologous blastocyst transfers and all 690 transfers of autologous blastocysts derived from post-thaw extended culture of thawed bipronuclear oocytes in the 8-year study period 2004-2011. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Visualized EP and treated persistent pregnancy of unknown location. RESULT(S): The rate of visualized EP was 1.5% in pregnancies in fresh autologous cycles, which was significantly more than the rate of 0 with autologous post-thaw extended culture. The rates of treated persistent pregnancy of unknown location were 2.5% and 0.3% in these two groups, respectively, a difference that was also statistically significant (relative risk 7.3, 95% confidence interval 1.7-31.0). CONCLUSION(S): Relative to fresh transfer, thawed ET was associated with significantly reduced incidence of EP. These findings are consistent with ovarian stimulation increasing the risk of EP.

Evidence of impaired endometrial receptivity after ovarian stimulation for in vitro fertilization: a prospective randomized trial comparing fresh and frozen-thawed embryo transfers in high responders.

Clinical pregnancy rates of 80% and 65% were observed in cycles using thawed and fresh embryos, respectively, although embryo quality indicators revealed morphologically and numerically inferior embryo cohorts after cryopreservation. Subsequent logistic regression analysis controlled for differences in embryo quality and revealed significantly greater probability of clinical pregnancy with thawed embryos when compared with fresh, suggesting a negative effect of ovarian stimulation on endometrial receptivity.

Evidence of impaired endometrial receptivity after ovarian stimulation for in vitro fertilization: a prospective randomized trial comparing fresh and frozen-thawed embryo transfer in normal responders.

OBJECTIVE: To compare success rates between fresh ETs after ovarian stimulation and frozen-thawed ETs (FET) after artificial endometrial preparation, to compare endometrial receptivity. DESIGN: Randomized, controlled trial. SETTING: Private fertility center. PATIENT(S): There were 53 patients completing fresh blastocyst transfer (fresh group) and 50 patients completing FET (cryopreservation group). All were first-time IVF patients aged <41 years, with cycle day 3 FSH <10 mIU/mL and 8-15 antral follicles. INTERVENTION(S): Randomized to fresh or thawed ET. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate per transfer. RESULT(S): The clinical pregnancy rate per transfer was 84.0% in the cryopreservation group and 54.7% in the fresh group. The implantation rates were 70.8% and 38.9%, respectively. The ongoing pregnancy rates per transfer (at 10 weeks' gestation) were 78.0% and 50.9%, respectively. The attributable risk percentage of implantation failure due to reduced endometrial receptivity in the fresh group was 64.7%. CONCLUSION(S): The clinical pregnancy rate per transfer was significantly greater in the cryopreservation group than in the fresh group. These results strongly suggest impaired endometrial receptivity in fresh ET cycles after ovarian stimulation, when compared with FET cycles with artificial endometrial preparation. Impaired endometrial receptivity apparently accounted for most implantation failures in the fresh group. ClinicalTrials.gov Identifier: NCT00963625.

Comparison of "triggers" using leuprolide acetate alone or in combination with low-dose human chorionic gonadotropin.

This retrospective study of fresh autologous blastocyst transfers in high responders compared ongoing pregnancy rates in cycles that followed trigger with GnRH agonist (GnRHa) alone with standard luteal support, GnRHa alone with enhanced luteal support, or GnRHa with concomitant low-dose hCG (dual trigger). Ongoing pregnancy rates were significantly increased with the dual trigger or with enhanced luteal support, whereas the incidence of clinically significant ovarian hyperstimulation syndrome was 0.0% in the groups receiving only GnRHa and 0.5% (1 of 182) in patients receiving GnRHa with concomitant low-dose hCG.

Efficacy of induced luteinizing hormone surge after "trigger" with gonadotropin-releasing hormone agonist.

The magnitude of the LH surge after GnRH agonist "trigger" was correlated with oocyte yield and maturity and was suboptimal in approximately half of the cycles. A modest reduction in oocyte yield and maturity was observed when the serum level of LH 12 hours after GnRH agonist trigger was less than the median value (52 IU/L), and a dramatic reduction in yield and maturity was observed when that level was less than 12 IU/L.

Similar ongoing pregnancy rates after blastocyst transfer in fresh donor cycles and autologous cycles using cryopreserved bipronuclear oocytes suggest similar viability of transferred blastocysts.

This retrospective cohort study compared blastocyst transfers in 136 fresh oocyte donor cycles and 69 autologous cycles using blastocysts derived from culture of thawed bipronuclear oocytes, all with oocytes derived from patients or donors less than 35 years old. The autologous cycles and oocyte donor cycles had similar rates of implantation (65.9% vs. 62.1%, respectively) and ongoing pregnancy (79.7% vs. 75.0%, respectively), suggesting that autologous blastocysts transferred after post-thaw extended culture have viability and implantation potential that are comparable with those of blastocysts transferred in fresh oocyte donor cycles.

Embryo cryopreservation rescues cycles with premature luteinization.

OBJECTIVE: To determine whether embryo cryopreservation in cycles with elevated preovulatory P followed by thaw, extended culture, and transfer results in greater ongoing pregnancy rates than fresh blastocyst transfer. DESIGN: Retrospective matched cohort study. SETTING: Private fertility center. PATIENT(S): The study group consisted of 118 consecutive thaws of bipronucleate (2PN) oocytes derived from autologous cycles with elevated preovulatory P, resulting in 95 blastocyst transfers. The control group was selected by matching on the number of 2PN oocytes and patient age and consisted of 118 fresh cycles with elevated preovulatory P, including 108 fresh autologous blastocyst transfers. All patients were <41 years old at the time of stimulation. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Implantation and ongoing pregnancy rates. RESULT(S): The study group had significantly fewer blastocysts per 2PN oocyte than the control group (19.7% vs. 36.8%, respectively) and a significantly greater cancellation rate (19.5% vs. 8.5%, respectively). However, the ongoing pregnancy rate per cycle was significantly higher in the study group than in the control group (56.8% vs. 32.2%, respectively), resulting from greater rates of implantation (56.8% vs. 26.9%, respectively) and of ongoing pregnancy per transfer (70.5% vs. 35.2%, respectively). CONCLUSION(S): In cycles with elevated preovulatory P, the probabilities of implantation and ongoing pregnancy are increased if all 2PN oocytes are cryopreserved and subsequently thawed and cultured to the blastocyst stage before transfer.

High ongoing pregnancy rates after deferred transfer through bipronuclear oocyte cryopreservation and post-thaw extended culture.

OBJECTIVE: To describe success rates with bipronuclear (2PN) oocyte cryopreservation, followed by thaw, extended culture, and blastocyst transfer. DESIGN: Retrospective study. SETTING: Private fertility center. PATIENT(S): There were 48 blastocyst transfers after post-thaw extended culture in patients less than 35 years old and 43 transfers in patients 35-40 years old. INTERVENTION(S): Patients opted for cryopreservation of their entire cohorts at the 2PN stage. Thawed 2PN oocytes were cultured to the blastocyst stage before transfer. MAIN OUTCOME MEASURE(S): Implantation, ongoing pregnancy. RESULT(S): Among patients less than 35 years old at oocyte retrieval, there were 59 thaws of 2PN oocytes, 48 blastocyst transfers, and 40 ongoing pregnancies (79.2% per transfer), the implantation rate was 64.2%, and the ongoing pregnancy rate (PR) per thaw was 64.4%. Among patients 35-40 years of age at retrieval, there were 58 thaws, 43 blastocyst transfers, and 22 ongoing pregnancies (51.2% per transfer), the implantation rate was 44.0%, and the ongoing PR per thaw was 39.3%. Patients less than 35 years old had significantly greater rates of implantation and ongoing pregnancy than did patients 35-40 years of age. Patients with 12 or more 2PN oocytes had significantly greater rate of ongoing pregnancy than those with fewer than 12 2PN oocytes. CONCLUSION(S): Post-thaw extended culture and blastocyst transfer yield high rates of implantation and ongoing pregnancy. Ongoing PRs decline with increasing age.