Days Of Freezing Research Articles

P-157 Comparative analysis of iDAScore and KIDScore in predicting live birth following single frozen day 5 blastocyst transfer

Abstract Study question How do iDAScore® and KIDScoreTM perform in predicting live birth outcomes after single frozen Day 5 blastocyst transfers? Summary answer In a euploid major cohort, both KIDScoreTM and iDAScore® demonstrated comparable predictive ability for live birth. However, iDAScore® offers the advantage of being fully automated. What is known already In the field of in vitro fertilization (IVF), the escalating use of artificial intelligence (AI) for predicting clinical outcomes is a burgeoning trend. One key contributor to this trend is time-lapse incubation, which captures images of developing images, generating a wealth of data. This data has facilitated the creation of various predictive models for implantation. KIDScoreTM is one such model that uses morphokinetic data to predict the probability of implantation after a Day 5 embryo transfer. Similarly, iDAScore® is an AI-driven automated scoring system specifically designed to automatically identify embryos with the greatest likelihood of implantation. Study design, size, duration This retrospective study included 779 patients (maternal age range: 19-44, mean age: 33.38) who underwent single frozen day 5 blastocyst transfer cycle from January 2021 to January 2023. Of these, 621 patients transferred euploid blastocysts, 48 transferred mosaic blastocysts, and 110 transferred non-PGT-A tested blastocysts. This study comprised of 12.1% (94/779) fair-to-good and fair grade (below AB and BA), 51.5% (401/779) good grade (AB and BA), and 36.4% (284/779) excellent grade (AA) blastocysts. Participants/materials, setting, methods After PIEZO-ICSI, embryos were cultured in EmbryoScopeTM Plus (Vitrolife, Denmark) until they reached the blastocyst stage. Blastocysts graded at least as fair according to Gardner (1999), were either biopsied for PGT-A and vitrified (Cryotec, Japan), or vitrified without biopsy. Clinical pregnancy and the number of gestational sacs were determined using ultrasound and successful pregnancies were tracked until delivery. KIDScoreTM (KIDScoreTM Day 5 v3.2) and iDAScore® (iDAScore® v2.0) were retrospectively generated for each transferred blastocyst. Main results and the role of chance Box plot showed that both KIDScoreTM and iDAScore® have positive correlation with morphology (Excellent vs. Good vs. Fair-to-Good and Fair; KIDScoreTM median: 8.8 vs. 6.8 vs. 5.7; iDAScore® median: 8.57 vs. 7.68 vs. 6.615), ICM (A vs. B; KIDScoreTM median: 7.5 vs. 6.5; iDAScore® median: 8.14 vs. 7.26) and TE (A vs. B vs. C; KIDScoreTM median: 8.7 vs. 6.5 vs. 4.65; iDAScore® median: 8.41 vs. 7.515 vs. 5.2). In this cohort, the overall implantation rate was 67.91% (529/779), and the live birth rate was 59.31% (462/779). Further statistical analysis revealed interesting correlations. A linear regression analysis indicated a statistically significant positive correlation between KIDScoreTM and iDAScore® (F(1,777)=297.6, p < 0.001, adjusted R2=0.276). Wilcoxon rank sum tests showed that KIDScoreTM has significant higher mean scores in blastocysts resulting in live birth compared to those that did not (7.44 vs. 7.22, w = 66577, p < 0.05). iDAScore® also had higher mean scores in blastocysts resulting in live birth but without statistically significant difference (7.56 vs. 7.48, w = 71328, p > 0.05). ROC curve analysis showed that AUC for KIDScoreTM was 54.4 and AUC for iDAScore® was 51.9, indicating no significant difference in their predictive ability for live birth (p = 0.2455). Limitations, reasons for caution The study’s cohort predominantly consisted of blastocysts with good morphology and were euploid, potentially skewing the results. Therefore, the findings may not be representative of all IVF patients and should be interpreted with caution. Additionally, further study should investigate the capability of iDAScore® 2.0 to predict euploidy. Wider implications of the findings While KIDScoreTM had a higher AUC than iDAScore®, there was no statistically significant difference. The major advantage of iDAScore® lies in its automation, eliminating the need for manual embryo evaluation. This can greatly enhance consistency in embryo grading and selection. Trial registration number NOT APPLICABLE

Fresh or frozen day 6 blastocyst transfer: is there still a question?

BackgroundThe Live Birth Rate (LBR) after day 5 (D5) blastocyst transfer is significantly higher than that with D6 embryos in both fresh and frozen-vitrified embryo transfer cycles, according to the most recently published meta-analyses. Therefore, for women obtaining only D6 blastocysts, the chances of pregnancy may be lower but nonetheless sufficient to warrant transferring such embryos. The best strategy for transfer (i.e., in fresh versus frozen cycles) remains unclear and there is a paucity of data on this subject.MethodsA total of 896 couples with D6 single blastocyst transfers were retrospectively analyzed: patients receiving a fresh D6 embryo transfer (Fresh D6 transfer group, n = 109) versus those receiving a frozen-thawed D6 embryo transfer (Frozen D6 transfer group, n = 787). A subgroup comprising a freeze-all cycle without any previous fresh or frozen D5 embryo transfers (Elective frozen D6, n = 77) was considered and also compared with the Fresh D6 transfer group. We compared LBR between these two groups. Correlation between D6 blastocyst morphology according to Gardner’s classification and live birth occurrence was also evaluated. Statistical analysis was carried out using univariate and multivariate logistic regression models.ResultsThe LBR was significantly lower after a fresh D6 blastocyst transfer compared to the LBR with a frozen-thawed D6 blastocyst transfer [5.5% (6/109) vs. 12.5% (98/787), p = 0.034]. Comparison between LBR after Elective frozen D6 group to the Fresh D6 blastocyst transfers confirmed the superiority of frozen D6 blastocyst transfers. Statistical analysis of the blastocyst morphology parameters showed that both trophectoderm (TE) and inner cell mass (ICM) grades were significantly associated with the LBR after D6 embryo transfer (p < 0.001, p = 0.037). Multiple logistic regression revealed that frozen D6 thawed transfer was independently associated with a higher LBR compared with fresh D6 transfer (OR = 2.54; 95% CI: [1.05–6.17]; p = 0.038). Our results also show that transferring a good or top-quality D6 blastocyst increased the chances of a live birth by more than threefold.ConclusionsOur results indicate that transferring D6 blastocysts in frozen cycles improves the LBR, making it the best embryo transfer strategy for these slow-growing embryos.Clinical trial numberNot applicable.

P-511 Prolonging the time of vaginal progesterone supplementation does not improve pregnancy outcomes after day 6 blastocyst transfer

Abstract Study question Does the administration of vaginal progesterone for 6 days improve pregnancy outcomes when transferring day 6 blastocysts, as opposed to 5 days of progesterone treatment? Summary answer We observed similar pregnancy outcomes after 5 and 6 days of vaginal progesterone supplementation following day 6 blastocyst transfer in oocyte recipient cycles. What is known already Progesterone administration promotes the final phase of endometrial preparation for embryo transfer. However, the optimal duration of progesterone transformation still remains unknown. The implantation potential of blastocysts formed at different timepoints is also contentious, as blastocysts on day 6 have been linked to poorer clinical outcomes compared to their day 5 counterparts. It is unclear whether this effect stems from an intrinsic impaired implantation capacity of slow-developing blastocysts, or a displaced window of implantation (WOI). It has been hypothesized that 6 days of progesterone administration may improve clinical outcomes after frozen day 6 blastocyst transfer. Study design, size, duration Retrospective cohort study of oocyte recipients undergoing single day 6 blastocyst transfer, between January 2020 and December 2022. This study compared two groups: day 6 blastocysts transferred after 5 days of progesterone administration (n = 284) and day 6 embryo transfers performed after 6 days of progesterone administration (n = 69). Participants/materials, setting, methods Our study included oocyte recipients who underwent a single embryo transfer of a day 6 blastocyst and hormonal replacement treatment for endometrial preparation with vaginal external progesterone administration (800mg/24h) for either 5 or 6 days prior to embryo transfer. Outcomes were analyzed using unpaired t-test or Fisher’s test and logistic regression. Multivariate models were adjusted for oral or transdermal estrogen administration and endometrial thickness on the day of embryo transfer. P-values &amp;lt;0.05 were considered significant. Main results and the role of chance Mean maternal age was 43.5 years, while patients had a mean BMI of 25.1. Mean days of estrogen therapy prior to embryo transfer were 22.4, with 75.3% of patients using transdermal estrogen administration, and achieving a mean endometrial thickness of 9.6 mm on the day of transfer. Mean progesterone levels on the day of transfer were 11.9 ng/ml. Thirty-seven percent of the day 6 blastocysts were of high quality. None of the demographic or cycle characteristics were significantly different between the study groups. When considering outcomes of day 6 blastocyst transfers, we found no significant differences in biochemical (26.33% vs 20.13%, p = 0.343), clinical (22.06% vs 17.46%, p = 0.49) and ongoing (14.64% vs 9.52%, p = 0.41) pregnancy rates between 5 and 6 days of progesterone administration. Our adjusted analysis further corroborated these results. Limitations, reasons for caution The retrospective nature of the study and low number of transfers performed after 6 days of progesterone treatment warrant careful interpretation. This study only evaluated endometrial preparation by vaginal progesterone administration in oocyte recipients and cannot be extrapolated to other routes of progesterone administration or other patient populations. Wider implications of the findings Poorer reproductive outcomes associated with the transfer of day 6 blastocysts appear to be related to a reduced implantation capacity of slow-developing embryos rather than a displaced WOI due to rising progesterone. WOI appears wider than assumed, as prolonged progesterone transformation times had no effect on pregnancy outcomes. Trial registration number Not applicable

A Study to Evaluate the Implantation and Clinical Pregnancy Rates in Patients Undergoing Sequential Frozen Day 3 Embryo and Day 5 Blastocyst Transfer

A Study to Evaluate the Implantation and Clinical Pregnancy Rates in Patients Undergoing Sequential Frozen Day 3 Embryo and Day 5 Blastocyst Transfer

Characteristic Analysis of the Spatio-Temporal Distribution of Key Variables of the Soil Freeze–Thaw Processes over Heilongjiang Province, China

The soil freeze–thaw phenomenon is one of the most outstanding characteristics of the soil in Heilongjiang Province. Quantitative analysis of the characteristics of changes in key variables of the soil freeze–thaw processes is of great scientific importance for understanding climate change, as well as ecological and hydrological processes. Based on the daily surface temperature and air temperature data in Heilongjiang Province for the past 50 years, the spatial–temporal distribution characteristics of key variables and their correlations with air temperature and latitude in the freeze–thaw process of soil were analyzed using linear regression, the Mann–Kendall test, the local thin disk smooth spline function interpolation method, and correlation analysis; additionally, the spatial–temporal distribution of key variables and the changes in the surface temperature during the freeze–thaw process are discussed under different vegetation types. The results show that there is a trend of delayed freezing and early melting of key variables of the soil freeze–thaw process from north to south. From 1971 to 2019 a, the freezing start date (FSD) was delayed at a rate of 1.66 d/10 a, the freezing end date (FED) advanced at a rate of 3.17 d/10 a, and the freezing days (FD) were shortened at a rate of 4.79 d/10 a; with each 1 °C increase in temperature, the FSD was delayed by about 1.6 d, the FED was advanced by about 3 d, and the FD was shortened by about 4.6 d; with each 1° increase in latitude, the FSD was delayed by about 2.6 d, the FED was advanced by about 2.8 d, and the FD was shortened by about 5.6 d. The spatial variation in key variables of the soil freeze–thaw process under the same vegetation cover was closely related to latitude and altitude, where the lower the latitude and altitude, the more obvious the variation trend; among them, the interannual variation trend of key variables of soil freeze–thaw under meadow cover was the most obvious, which varied by 9.65, 16.86, and 26.51 d, respectively. In addition, the trends of ground temperature under different vegetation types were generally consistent, with the longest period of unstable freeze–thaw and the shortest period of stable freeze in coniferous forests, compared to the shortest period of unstable freeze–thaw and the longest period of stable freeze in meadows. The results of the study are important for our understanding of soil freeze–thaw processes and changes in Heilongjiang Province, as well as the evolution of high-latitude permafrost; they also promote further exploration of the impact of soil freeze–thaw on agricultural production and climate change.

MATERNAL PHYSIOLOGY AND BLASTOCYST MORPHOLOGICAL GRADE CORRELATE WITH AN INHERENT DIFFERENCE IN HUMAN PERI-IMPLANTATION DEVELOPMENTAL POTENTIAL

Our group has been using an extended embryo culture (EEC) system to culture human embryos for up to an additional 7 d to study peri-implantation over the past three years. Since similar EEC parameters were measured for these projects, we performed a retrospective analysis of 653 human embryos to determine what maternal factors correlate with peri-implantation developmental outcomes. Frozen day five or day six human blastocysts donated for research (WIRB study no. 1179872) were warmed, dezonated, and cultured in vitro up to EEC day seven following an established protocol (Deglincerti et al., Nature 2016). The trophectoderm outgrowth area, total cell number, epiblast cell number, and human chorionic gonadotropin (hCG) production in spent media were measured during EEC. Patient information including maternal BMI, age, infertility diagnosis, FSH, LH, FSH: LH ratio, estradiol, and anti-müllerian hormone (AMH) levels on menstrual cycle day 3, and an antral follicle count (AFC) were used in our analysis. Additionally, blastocysts with the highest morphological grade that typically yield live birth rates above 65% (Good Quality) were compared with those of lower morphological grade that typically yield less than 55% live birth rate (Fair Quality), based on our historical data. We then used a spearman correlation coefficient to assess the strength of the association between the aforementioned maternal factors and extended culture parameters. A chi-squared analysis was used to compare binomial outcomes with categorical predictors. T tests or ANOVA’s were used for comparing two or multiple continuous variables from categorical predictors respectively. Several maternal physiological influences during oocyte development (age, BMI, and FSH: LH, AMH, AFC) were significantly correlated with extended culture outcomes (p<0.05). Embryos with high morphological grade had significantly increased outgrowth areas compared to those with low morphological grade (Good Quality: 0.17 ± 0.02; Fair Quality: 0.12 ± 0.02, p<0.05). Maternal age was correlated with hindered EEC measurements in both the non-donor fertility patients as well as in healthy donors. When comparing embryos with the highest morphological grade only, advanced maternal age (35+ years old) still contributed to decreased hCG production (p<0.05) and hindered epiblast formation compared to those from younger patients (<35 years old, p<0.01). In conclusion, we show that human EEC may be valuable in predicting post-transfer developmental potential. Additionally, maternal physiology during oocyte development including age, BMI, and FSH: LH, AFC, and AMH significantly correlates with the peri-implantation developmental potential of human embryos.

The Effect of Additional Embryo Transfer on the Pregnancy Rate in Young Women Receiving in vitro Fertilization: A Natural Experiment Study.

PurposeTo assess the impact of additional embryo transfer (ET) on pregnancy in young women, we used a natural-experiment approach.Patients and MethodsThe design was based on the national policy of South Korea limiting the number of embryos transferred in vitro fertilization (IVF):≦2 embryos on day 2–4 or one on day 5–6 for patients aged <35, with one extra embryo allowed for patients aged ≥35. Using the data from 1909 ET cycles of 1287 women aged ≥34 and ≤35, we calculated adjusted risk ratios (RRs) for pregnancy.ResultsHalf of cycles were undertaken by women aged 35, and additional ET was performed in 68.7% of them. Intrauterine pregnancy (45.2% vs 51.3%) and multiple gestation (30.5% vs 6.9%) were more common in women aged 35 than in those aged 34. The RR for intrauterine pregnancy was 1.34 (95% confidence interval: 1.12–1.59) when comparing double ET to single ET in frozen day 5–6 cycles.ConclusionWe observed no evidence of a higher probability of pregnancy with additional ET in fresh or frozen day 3–4 ET, or in fresh day 5–6 ET of women aged 35. Additional ET may not increase the successful pregnancy rate in the 35-year-old group, unless it is a frozen day 5–6 ET cycle.

Characteristics of the active-layer under the China-Russia Crude Oil pipeline

Active layer is a key component for permafrost environment studies as many subsurface biological, biogeochemical, ecological, and pedogenic activities prevail in this layer. This study focuses on active layer temperature monitoring in an area with sporadic permafrost at two adjacent sites along China-Russia Crude Oil Pipeline (CRCOP), North-East China. Site T1 is located in disturbed ground on the right-of-way (on-ROW) 2 m away from the center of the oil pipeline. T2 is located in a natural and undisturbed site, around 16.6 m off-ROW. Our objective was to study seasonal variability of the active layer depth and thermal regime from October 2017 to September 2018. The monitoring sites consist of soil temperature probes arranged in a vertical array at different depths at both sites. The following parameters were computed: number of isothermal days (ID), freezing days (FD), thawing days (TD), freezing degree days (FDD), thawing degree days (TDD), number of freeze-thaw days (FTD). The mean air temperature in the monitoring period reached - 3.2°C. The temperature profile indicates that the maximum active layer thickness observed during the study period was 10 m at T1 and 2 m at T2. The majority of the soil temperatures were above or close to 0°C, resulting in great values of TDD, especially in the first 4 m depth. TDD for T1 were predominant and ranged between 600–1160°C·days (0–4 m depth) reflecting the influence of oil temperature from the pipeline. In T2 borehole FDD were predominant for all the soil layer depths resulting in less permafrost degradation. This comparison emphasizes the significant influence of vegetation removal and the dispersed heat from the pipeline on the active layer thickness.

Clinical Outcomes of Transfer Vitrified-Thawed Day 4, 8-Cell Embryos into Endometrium Prepared for Day 3 Embryos

Objective: To determine the clinical outcomes of transfer of day 3, 8-cell embryos into endometrium prepared for day 3 embryos. Methods: This study performed a retrospective analysis of 1,190 retarded embryos. These embryos underwent extended culture in vitro to select and vitrify day 4, 8-cell embryos, followed by 176 frozen embryo transfer cycles (study group), matching 660 transfer cycles with single frozen day 3, 8-cell embryos as the control (control group). Results: The study group achieved successful implantation rates, clinical pregnancy rate, and live-birth rates (20.45%, 15.91%, 14.20%, respectively), although these were lower than that in the control group (30.45%, 27.42%, and 20.91%; P = 0.009, 0.002, and 0.046, respectively). The miscarriage rate was similar to that in the control group (4.55% vs. 3.33%, P > 0.05), and the mean birth weight of the study group was higher than that in the control group (3,556 ± 381 g vs. 3,311 ± 570 g, P = 0.012). Conclusions: Transfer of frozen day 4, 8-cell embryos into endometrial prepared for day 3 embryos can be a new and safe alternative for patients with delayed embryos.

Euploid single frozen day 5 versus day 6: can we expect equal outcome?

Euploid single frozen day 5 versus day 6: can we expect equal outcome?

Long term active layer monitoring at a warm-based glacier front from maritime Antarctica

Knowledge on active-layer dynamics and permafrost distribution is of especial importance in Maritime Antarctica, where dramatic climate warming occurred in the last decades. Few long-term studies of active-layer temperatures in this region, and no one focus on recently deglaciated areas under paraglacial conditions. This paper analyses the long-term soil thermal regime of a warm-based glacial front site located at Low Head, King George Island. The monitoring system consists of soil temperature probes connected to a datalogger that recorded data at hourly intervals. We calculated the thawing days (TD), freezing days (FD), number of isothermal days (ID), number of freeze-thaw days (FTD), thawing degree days (TDD), freezing degree days (FDD), and the apparent thermal diffusivity (ATD). The results indicate that active layer thermal regime at Low Head is similar to other periglacial environments from Maritime Antarctica, with differences associated with the influence from the nearby warm-based glacier. Surface temperatures show greater variations during the summer resulting in frequent freeze and thaw cycles, mainly (1cm and 10cm). The temperature profile during the studied period indicates that the active layer thickness reached a maximum of 106cm on February 7th 2015. Soil temperature buffering was limited by the low snow cover, low soil moisture, and absence of vegetation. Based on the high interannual variability detected during the five years monitoring run, we stress that longer monitoring periods are necessary for a more detailed knowledge on how permafrost respond to climate changes in this rapidly warming zone.

Building a floating palace [superyachts

It's A windswept frozen day in the small town of Oss where, in a colossal enclosed boatbuilder's yard, the sights and sounds of good old-fashioned metal bashing are in the air. A 50-metre-long skeletal aluminium framework forms the bones of what will become one of the most luxurious yachts the world has ever seen. For those inspired by the fabrication of big shiny things, these construction areas (the size of aircraft hangars) at Netherlands-based boatbuilder Heesen are a joy to behold. However, for those who like their superyachts sleek and finished, elegantly cruising the Caribbean main, it appears that - on the surface at least - this primer-pink carcass of a ship has a long journey ahead of it. It will be months before the client will even set foot on what is ultimately a floating private palace that displays the wealth of Croesus. Founded in 1978, Heesen is one of the world's leading brands in superyacht design and manufacture. Earlier this year, the boatbuilder scooped no fewer than three gongs at the 10th annual World Superyacht Awards. MySky (51 metres), Elena (47m) and Alumercia (37.9m) all made their ostentatious mark on the event.

Comparison of clinical pregnancy rates with single blastocyst transfer when using frozen day 5 or day 6 embryos

Comparison of clinical pregnancy rates with single blastocyst transfer when using frozen day 5 or day 6 embryos

Differences in embryo weight and chemical composition, and their implications for cryopreservation survival

OBJECTIVE: Cryopreservation is considered a standard of care in ART; however, great variability still exists in embryo survival rates. The process assumes that water is highly conserved across embryos of the same species. Therefore, any dramatic shift in the embryos' chemical nature might lead to failure of the cryopreservation process. The objective of the present study was to determine if embryos from the same species have significantly different chemical composition. Embryo weight and lipid content were compared between Jersey cattle, a breed with significant body fat content and considered difficult to cryopreserve, and beef cattle breeds with known high success rates for cryopreservation. DESIGN: A laboratory based study of cryopreservation using cattle embryos of various breeds. MATERIALS AND METHODS: Frozen day 6 cattle blastocysts of Jersey and beef breeds were thawed using standard procedures, and equilibrated into P-1 medium. Specific gravities were then determined by “dropping” the embryos through two solutions (50 embryos /solution/breed) of known osmolarity and timing their decent over a standard distance. The system had been previously standardized using glass spheres of known size and weight. Additionally, ten embryos of both Jersey and beef cattle (20 embryos total) were thawed, equilibrated into P-1 and then fixed for electron microscopy (EM) using standard techniques. Random sections were then examined using a Hitachi H-7650 TEM (1500X). Resulting micrographs were then examined to quantify the lipid content of the embryo. Data between groups were analyzed by student's T test using SPSS. RESULTS: Specific gravity data suggest the beef cattle embryos to be 20-27% heavier than Jersey embryos (P < .001). EM data suggest these differences are due to differing lipid content; with Jersey embryos having approximately 36% lipid/unit area as compared to only 8% in beef cattle embryos (P < .01); thus, explaining the Jersey embryos lighter weight. CONCLUSIONS: The data suggest that subtle differences in embryo weight can be quantified using a simple specific gravity technique. In the present study, animals with a known higher body fat content were shown to have embryos of lighter weight. Electronic microscopy appeared to confirm these embryos had higher lipid content. These data would appear to suggest that “standard” cryopreservation protocols might not be effective with all embryos and selective cohorts might require modification of the cryoprotectants.

Compaction and blastulation after thawing and overnight culture of frozen day 3 embryos are reliable predictors of clinical pregnancy

OBJECTIVE: Single embryo transfer (SET) has become daily practice in Belgium in view of a government funded program to reduce multiple pregnancies. This attitude tends to extend now to transfer of frozen embryos. Characterization of the single best frozen-thawed embryo for transfer has become a necessity. We analysed impact of embryo stage before freezing, cell loss and embryo development between thawing and transfer on clinical pregnancy rates in single frozen embryo transfers. DESIGN: Retrospective analysis of 526 single frozen day 3 embryo transfers carried out between January 2004 and October 2007. MATERIALS AND METHODS: Day 3 embryos with at least 6 blastomeres and ≤20% fragmentation were frozen using a slow-controlled DMSO protocol. Embryos surviving thawing were cultured overnight in Medicult BlastAssist medium. Embryo stage before freezing, immediately after thawing and at the moment of transfer was recorded. Clinical pregnancy was defined as a pregnancy with at least one gestational sac at seven weeks of gestation. Pregnancy rates were compared using Chi-squared test (p< 0.05). RESULTS: The overall clinical pregnancy rate was 20.0% (105/526). Embryo stage before freezing did not influence clinical pregnancy rates: 18.9% (14/74) for 6-cell embryos, 23.7% (27/114) for 7-cell embryos, 17.6% (43/245) for 8-cell embryos and 22.6% (21/93) for >8 cell embryos. The clinical pregnancy rate after transfer of frozen embryos with 100% intact cells was 19.9% (76/382). This was not significantly different compared to embryos with 1 blastomere (23.3%, 20/86) or 2 blastomeres (17.6%, 6/34) damaged. Three clinical pregnancies were obtained in the group of embryos with > 2 cells damaged (3/24, 12.5%) originating from transfer of embryos with 50% blastomere damage. Significantly higher clinical pregnancy rates were obtained after transfer of embryos with signs of compaction (71/317, 22.4%, p<0.001) or blastulation (21/62, 33.9%, p<0.0001) than embryos with >8 cells but without compaction (8/109, 7.3%). Embryos with ≤8 cells at transfer had a clinical pregnancy rate of 13.2% (5/38). CONCLUSIONS: Compaction and blastulation after thawing and overnight culture of frozen day 3 embryos are reliable predictors of clinical pregnancy. Loss of 1 or 2 blastomeres did not have a negative impact on clinical pregnancy rates. It is possible to obtain a clinical pregnancy after transfer of embryos with up to 50% of cell loss.

Quantitative RT-PCR reveals tuberous sclerosis gene, TSC2, mRNA degradation following cryopreservation in the human preimplantation embryo.

The use of cryopreserved human embryos in gene expression studies provides an additional source to the scarce embryos available for research. To validate their use we have implemented a quantitative RT-PCR to characterize the levels of the tuberous sclerosis, TSC2 gene in fresh and frozen-thawed human embryos. Frozen embryos were thawed using two different clinical protocols. In fresh embryos 9.95 fg of TSC2 cDNA was present in the unfertilized oocyte, which was comparable to the level on day 2 of preimplantation development. On day 3 there was a significant drop (P<0.001) to 6.8 fg, followed by an increase in cDNA levels to 10.8 fg (P<0.01) on day 6 at the expanded blastocyst stage. Day 2 frozen embryos possessed 50% less (P<0.001) TSC2 mRNA in comparison to the fresh embryos using thawing protocol one (from frozen to 37 degrees C) and 25% less TSC2 mRNA (P<0.01) with thawing protocol 2 (from frozen to room temperature). After culturing day 2 frozen embryos for an additional day they showed mRNA levels comparable with fresh day 3 embryos. There was no significant difference in the levels of TSC2 mRNA between fresh and frozen day 3 human embryos with either thawing protocol. This study demonstrates that cryopreservation does affect the normal pattern of gene expression during human preimplantation development, and that intact frozen-thawed embryos are not equivalent to their non-frozen counterparts. Furthermore human embryos frozen on day 2 appear to be more susceptible to temperature change than embryos frozen on day 3.