Time-lapse imaging of embryo development is increasing in popularity and has been shown to predict an embryo's potential for blastocyst formation (1, 2) and implantation (3). The technology could be used to allow the transfer of fewer embryos through improved and objective selection of viable embryos. Currently, the best embryo selection method is subjective morphological assessment, but this is unreliable since most patients are still transferring 2 or more, resulting in twin pregnancy rates consistently above 30% (4). Greater utilization of eSET requires improved and objective selection methods that can reliably identify viable embryos in a cohort. To evaluate if time-lapse imaging could help identify on day 3 (D3) or day 5 (D5) embryos with higher implantation potential. 77 good prognosis patients (antral follicle count >8 and cycle day 3 FSH < 10 IU/ml) were included in the study, for a total of 135 embryos analyzed. Embryos were imaged from Day 1 to 3 using the Early Embryo Viability Assessment System test (EevaTM; Auxogyn, Menlo Park, CA) at 5 clinical sites. Only patients with 100% or 0% of the transferred embryos implanting after D5 transfer were included. During imaging, automated cell tracking software predicted a "high" or "low" blastocyst development probability for each embryo by the afternoon of D2. The addition of an Eeva high result to the D3 morphology assessment predicted slightly higher implantation rates for good D3 embryos (55%) compared with fair/poor embryos (42%). For morphologically good quality blastocysts, the implantation rate was not impacted by the Eeva prediction (56% without Eeva, 55% with an Eeva high and 55% with an Eeva low score). However, Eeva discriminated well among poor quality blastocysts (Eeva high IR was 47%, Eeva low was 31% and morphology only without Eeva was 27%). Similarly, early and expanding blastocysts had better IR's with an Eeva high score (50% and 43%) compared with those scored low by Eeva (30% and 36%) and those scored by morphology only (33% and 36%).Tabled 1Embryo groupImplantation rate (%) after D5 ETGood D3 morphology + Eeva high55%Good D3 morphology + Eeva low40%Good D5 morphology + Eeva high55%Good D5 morphology + Eeva low55%Fair/Poor D5 morphology + Eeva high47%Fair/Poor D5 morphology + Eeva low27%Early blastocyst + Eeva high50%Early blastocyst + Eeva low30% Open table in a new tab For D3 embryos, the information provided by Eeva was a good indicator of which embryos would make blastocysts and which would implant after transfer. If blastocyst morphology was poor, or the blastocysts were not fully expanded, Eeva provided a valuable additional parameter indicating which embryos would implant. Eeva, a computer automated predictor of blastocyst formation, may be effective in selecting embryos for transfer and allowing for fewer embryos to be transferred.

Since many transferred embryos with "good morphology" fail to implant, technologies are needed to distinguish embryos with highest developmental competence. We developed and validated an integrated time-lapse and automated image analysis system which measures specific cell division timings and predicts blastocyst development by day 2. The objective of this study was to determine the degree of improvement to viable embryo selection when using our prediction model with traditional morphology. Multi-site, prospective, cohort study. The study included 43 patients (≤42 years old) undergoing in vitro fertilization at 3 clinics. Day 3 morphology and time-lapse image data captured by an integrated imaging system, Eeva (Early Embryo Viability Assessment), were prospectively collected. Eeva generated a blastocyst probability score (Low, High) based on specific cell division timings. Eeva blastocyst probability was determined to be High when all cell division timings were within the defined ranges. Five experienced embryologists made a "blastocyst" or "arrest" prediction for each embryo using Day 3 Morphology only, and then using Morphology and Eeva scores. The prediction results were compared to true blastocyst outcomes. A total of 343 embryos were evaluated. By combining Morphology+Eeva, the average blastocyst prediction accuracy significantly improved. The degree of improvement was augmented for embryos with "good morphology" on day 3.Tabled 1Blastocyst Prediction on Day 3 (% Accuracy, Mean±SD)Embryo GroupsMorphology onlyMorphology+EevaP valueTotal Embryos (n=343)62±680±2<0.0056 to 10-cell, ≤10% Frag, Perfect Symmetry (n=135)37±466±4<0.005 Open table in a new tab Adding Eeva to traditional morphology dramatically improved day 3 blastocyst prediction, particularly among "good morphology embryos". Predictions are non-invasive and available by day 2 using automated analysis. Eeva is a uniquely effective and efficient tool for viable embryo selection that may ultimately improve implantation rates.

BACKGROUND: Cleavage stage embryos that are mosaic euploid-aneuploid are presumably more likely to be euploid by blastocyst stage than those with no euploid cells. While cleavage stage Preimplantation Genetic Screening (PGS) enables Day 5 transfer, this may result in unnecessary exclusion of mosaic euploid-aneuploid embryos that have an aneuploid result on single blastomere biopsy but which develop into fully euploid blastocysts. Meiotic aneuploidy is more likely to affect all of the cells in an embryo than mitotic aneuploidy. For patients without any euploid blastomere results in a given cycle, transferring embryos with possible mitotic aneuploidy may be an alternative to no transfer. OBJECTIVE(S): To determine whether transfer of an embryo with paternal aneuploidy on a chromosome in which full aneuploidy would be non-viable could result in a healthy fetus. MATERIALS AND METHOD(S): In vitro fertilization cycle from a 46-year-old male patient and egg donor. Single blastomere biopsies from eleven cleavage stage embryos were shipped to a microarray lab for 24-chromosome PGS. Genetic data from the biological parents was used to determine parental source of aneuploidy and distinguish potential mitotic from meiotic trisomy. For each aneuploid blastomere, we computed the probability that it was derived from a mosaic euploid-aneuploid embryo, based on the type of aneuploidy observed. RESULT(S): One 46,XY embryo was transferred, along with a 47,XY,+3 embryo with an extra paternal copy of Chromosome 3, suggesting that the embryo had a significant probability for still containing euploid cells. Trisomy 3 has not been reported in livebirths or beyond the first trimester and is thus “non-viable”. Two healthy boys were born in July 2010. Cheek swab samples were obtained and DNA fingerprinting was performed with livebirth samples matching blastomere samples for both babies. CONCLUSION(S): An embryo transferred following aneuploid PGS results revealing paternal aneuploidy on a “non-viable” chromosome of likely mitotic origin became a chromosomally normal, healthy male infant, presumably because only the euploid cells in the embryo developed. This outcome supports transfer of embryos with mitotic errors on chromosomes in which aneuploidy would be non-viable, especially when no euploid embryos of good morphology are available. SUPPORT: None.

To evaluate the clinical utility and outcome of a novel protocol in which the single dose GnRH antagonist is administered in the office by the clinical team during controlled ovarian hyperstimulation (COH) and IVF-ET. Case control study. During calendar year 2004 seventy-two patients undergoing COH and IVF received the administration of 3 mg of Cetrorelix in the office immediately after the morning ultrasound evaluation for follicular growth. A lead follicle of 14 mm was used as the sole criteria for administering the antagonist. Estradiol levels were concomitantly measured and patients were instructed to add 75 units of HMG on the day the antagonist was given. Oocyte retrieval was performed 35 hours after hCG, followed by ultrasound guided embryo transfer on day 3 or day 5. Our control group consisted of 119 patients during the same time period who self administered either a GnRH agonist or daily dose 0.25 mg GnRH antagonist in the evening. Ultrasounds, retrievals and transfers were performed by a single investigator to ensure uniformtiy of findings and procedures. Statistical analysis was performed by t-test and Fisher's exact test where applicable. Our study group consisted of 72 patients, of which 70 underwent embryo transfer. The mean age of our study group was 34.9 +5.0 years, with a B.M.I. of 24.0 ± 5.5 kg/m2 and a basal FSH/E2 6.7 ± 2.2 mIU/ml/ 42.4 +15.2 pg/ml. There were no significant differences between the baseline study characteristics in our study group (N= 70) as compared to our control group (N=119). The mean FSH start dose was 224.7 ± 87.2 units and the mean HMG start dose was 105.2 ± 115.1 units. On average, our study group required 8.8 ± 1.5 days of stimulation and 2683.3 ± 1048.5 units of total gonadotropins, which resulted in a mean peak estradiol level of 1931.8 ± 899.8 pg/ml. The mean total number of oocytes were 12.2 ± 5.3 and among patients that underwent ICSI a mean of 9.3 ± 4.3 MII oocytes were noted. The mean total number of embryos from standard insemination and ICSI were 7.7 ± 4.4 and 8.2 ± 3.9 respectively (P > 0.05). The clinical and ongoing pregnancy rates per transfer were as follows; ≤ 34 years of age (N=31) 64.5% (20/31) and 54.8% (17/31), 35-37 (N=17) 58.8% (10/17) and 47.1% (8/17), 38-39 (N=9) 44.4% (4/9) and 33.3% (3/9), 40-43 (N=13) 38.5% (5/13) and 23.1% (3/13). The total ongoing pregnancy rate per transfer in this study group was 44.3% (31/70), compared to 39.5% (47/119) in our control group (P > 0.05). Single dose GnRH antagonist administration in the office provides a convenient and simple approach to minimize compliance issues for patients undergoing COH and IVF. Our data suggests that aside from the advantages of a more injection friendly stimulation, the use of a single dose GnRH antagonist on the morning of the lead follicle reaching 14 mm results in comparable ongoing pregnancy rates in this case control study. Overall, clinical parameters of ovarian stimulation and outcome were consistent with an efficient and effective protocol for patients undergoing COH and IVF.

Background: Our previous study showed that in vivo GM-CSF treatment promotes preantral follicle development, as well as modulating the ovarian expression of CYP17, a marker specific for theca-interstitial (T-I) cells. Theca-interstitial cells control follicle growth and atresia, regulates ovarian steroidogenesis, and may provide mechanical support for ovarian follicles. It has not been well established whether CYP17 expression in T-I cells is regulated by GM-CSF directly or indirectly. Objective: To investigate the possible mechanism of GM-CSF action on follicular development using separated then cultured theca-interstitial cells (T-I). Design: Prospective study Material and methods: Theca-interstitial cells were isolated from 25-day-old rats by a modified method of Foghi et al (Foghi et al., 1998). After 4 days culture, T-I cells were treated with different concentrations of GM-CSF (0, 1, 3, 10, 30ng/ml) for 8hrs. Total RNA was isolated from the (T-I) cell cultures using TRIzol reagent (Life Technologies, Inc.) 8h after GM-CSF treatment. For protein extraction, Cells were harvested in ice-cold lysis buffer. We performed an analysis of CYP mRNA by semiquantitative reverse transcriptase–PCR as it has been reported in previous studies. CYP17 protein expression was evaluated by Western blotting. The relative expression of CYP17 mRNA and protein between treatment groups were evaluated by ANOVA and followed by Tukey HSD test. Significant differences were assigned at p<0.05. Results: Total RNA was extracted from T-I cells for semi-quantitative RT-PCR analysis of CYP17. The results showed that CYP17 mRNA was increased by 4.8-fold in the presence of 1ng/ml GM-CSF in the culture medium compared to the group without GM-CSF (P<0.05). Similarly, increased CYP17 protein was found in the presence of 1ng/ml GM-CSF (P<0.05) compared to the group without GM-CSF treatment. Conclusion: The present studies show that GM-CSF regulated CYP17 expression in T-I cells directly in vitro. It may suggest that GM-CSF may promote follicle development through the increased steroidogenic activity of T-I cells by up-regulating CYP17 expression.

Although cryopreservation of supernumerary embryos has increased the overall success rate in human IVF-ET, the survival and implantation rate of frozen-thawed embryos is directly related to the quality of embryos at the time of freezing. Overnight incubation improves selection of good quality embryos for transfer. Studies showed that there was a ∼45% implantation rate for day 4 embryos after fresh morula ET. This study is designed to investigate the predictors of postthaw embryo compaction and the relationship to the outcome of frozen-thawed embryo transfer (FET) cycles. Retrospective analysis of frozen embryo transfer (FET) cycles conducted during 2001-2003 at a clinical IVF center. A total of 186 human pronuclear embryos from 35 patients and 754 day 3 embryos from 137 patients were cryopreserved using a Freeze Control (CL-863) freezing unit (Biogenics, Napa Ca) and the standard Testart/Lasalle 1.5M PrOH/0.1M Suc freeze/thaw procedure, with the modification of starting the freeze at-6.5°C. On thawing, embryos were cultured in vitro in Cleavage (for pronulcear embryos) or Blastocyst medium (for cleavage embryos) (Sage) with 10% Serum Substitute Supplement (Irvine Scientific). Embryo transfer was performed at 48∼54 hours and 24∼30 hours, respectively, for the pronuclear and cleavage stage from time of thaw into a recipient uterus after standard estradiol/progesterone preparation. Significant differences were assigned at p<0.05. The survival rates of the pronuclear and cleavage embryo were 91% and 86.5%, respectively in all age groups. Embryo transfer was performed in 160 patients and produced a clinic pregnancy rate of 53.1% in various age women. The clinic pregnancy rate of patients who had compacted embryos at the time of transfer was 77.4% (24/31), while the clinical pregnancy rate was only 47.3% (61/129) for patients who had no compacted embryos for transfer (P<0.01). For the embryo survival rate, and the number of embryo transfer, there were no significant differences between these two groups (P>.05). Extending the culture of thawed pronuclear and cleavage stage embryos allows for better embryo selection, resulting in higher pregnancy and implantation rates per FET. Postthaw embryo compaction is strong positive predictor for the success of frozen-thawed transfer.

Background: In the last few years a significant resurgence of interest in the potential benefits of human oocyte cryopreservation has immerged. There is, however, little/no information regarding the fertilization and development potential of cryopreserved oocytes compared to fresh oocytes from the same women in the same cycle or comparison of freezing protocols. Objective: To compare oocyte survival, fertilization and cleavage rate between 2 different oocyte cryopreservation protocols in consenting patients undergoing IVF and ICSI. A comparison of the sibling fresh oocyte embryo development with the embryos derived from cryo will be compared at the completion of the study. Materials and Methods: Following standard controlled ovarian hyperstimualtion (Follistim AQ and Ganirelix Acetate, Organon Pharmacueticals USA, Inc.) for IVF, patients who had >10 MII oocytes, had 30% of their oocytes selected for immediate freezing and thawing using either the Boldt method (Sage Invitro Fertilization) or the Fabri method as published in the literature(HR'01). Oocytes were thawed after at least 15min in LN2 and surviving oocytes were injected by a standard ICSI technique 2-4hrs later, along with the remaining 70% of the patient's fresh, mature oocytes. Cryopreserved-thawed oocytes were cultured separately from untreated oocytes under oil in 5%CO2 in Sage cleavage medium. The best embryos, regardless of the treatment were considered for D3 transfer. Supernumerary embryos were cryopreserved on the day of ET or at Blastocyst stage. Results: The data represent the initial 19 patients, 10 in Fabri and 9 in Boldt. Oocyte survival and fertilization rates were similar between groups, 78.6% (44/56) and 70.5% (31/44), and 80.7% (46/57) and 69.6% (32/46) for Fabri and Boldt protocols, respectively. The percentage of embryos >6 cell were 7% (2/31) and 38% (12/32) for Fabri and Boldt, respectively (p<0.01). Conclusions: Although these data are preliminary and the study is still ongoing, both protocols appear to yield cryo-survival rates similar to those of 2PN's and fertilization rates by ICSI equal that achieved from the fresh oocytes. However the Boldt group had significantly better D3 embryo development. Future studies are planned to assess the implantation potential of the embryos derived from oocyte cryopreservation and whether the differences in D3 embryo development translate into differences in pregnancy.

Background: Cryopreservation of pronuclear embryos is common practice in ART clinics due to the high survivability compared to cleavage stage embryos or blastocysts. However, there is little data guiding the clinical and embryology team as to the prognosis and affect of ICSI on parameters of outcome. Consideration needs to be given to micromanipulation of the oocyte prior to fertilization and if this may have any subsequent effect. Objective: This study was undertaken to determine whether the method of fertilization has a significant impact on survival and/or clinical pregnancy rates of cryopreserved human pronuclear (2PN) stage embryos. Design: A retrospective analysis of cryosurvival and clinical pregnancy rates after thawing of 2PN stage embryos. Material and Methods: A total of 279 human 2PN embryos were cryopreserved using a Freeze Control (CL-863) freezing unit (Biogenics, Napa Ca) and the standard Testart/Lasalle 1.5M PrOH/0.1M Suc freeze/thaw procedure, with the modification of starting the freeze at −6C. On thawing, embryos were cultured in vitro in cleavage medium (Sage) with 10% Serum Substitute Supplement (Irvine Scientific). Embryo transfer was performed at 40 to 48 hours from time of thaw into a recipient uterus after standard estradiol/progesterone preparation. Significant differences were assigned at p<0.05. Results: From 2001 to 2003, 91% of all frozen 2PN embryos survived and were transferred in 53 cycles producing a clinic pregnancy rate of 54.7% in various age women. The clinic pregnancy rate of patients who had vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) was similar (56.5% and 53.3%, respectively) (P>0.05). However, 56.6% of these cycles were ICSI, and the survival of frozen 2PN from these cycles (88%) was lower than the respective survival of frozen 2PN embryos from standard IVF (95.5%; P<0.05). Conclusion: 2PN stage embryo cryosurvival may be negatively affected by ICSI, possibly caused by disruption of the zona pellucida and vitelline membrane before cryopreservation, and/or because ICSI promotes fertilization of some compromised eggs (producing compromised 2PN embryos) that would not have fertilized by conventional IVF. However, the clinical outcomes of cryopreserved pronuclear embryos from standard IVF and ICSI are not significantly different.

Some concerns have been raised regarding the potential negative effects of GnRH antagonists on ovum quality. This study seeks to compare the outcomes in ovum donor cycles when donors used traditional down-regulation protocols, compared to using newer antagonist protocols. Retrospective case-control study by chart review. Ninety-seven women underwent an ovum donor cycle between 2002–2003. Sixty-six donors were treated with leuprolide acetate, while taking oral contraceptives(OC), and then commenced gonadotropin therapy (225–300U) after stopping the pills. Thirty-one donors used similar doses of gonadotropins 5 days after stopping OC and added one of the two brands of GnRH antagonists when 14mm follicles were seen on sonogram. Recipients used OC and leuprolide, when appropriate, and intramuscular estradiol valerate and combinations of progesterone in oil and vaginal suppositories to create mock cycles synchronized to the donor cycles. Outcome parameters of eggs received, embryos transferred, pregnancy rates, and implantation rates were compared in the two groups. Student's t-test and chi square were used for statistical analysis. Down-regulation cycles produced a mean of 13.9 eggs with 2.3 embryos transferred per cycle, compared to 12.4 eggs and 2.5 embryos in the antagonist group. These were statistically similar. The ongoing pregnancy rate in the leuprolide group was 30/66 (45%) vs. 17/31 (55%) in the antagonist group. In the down-regulation group, 41/157 (26%) of the embryos implanted, while the recipients in the antagonist group experienced a 22/65 (34%) implantation rate. These differences were not statistically significant. Ovum donor cycles using traditional down-regulation protocols and GnRH antagonist protocols for the donors produce similar results. Concerns over the potential detrimental effects of agonists on egg quality are not supported by our results. The increased convenience and patient acceptance of the antagonists make these drugs the preferred method of preventing LH surges in the donors.

Introduction: There is a void in clinical training of embryo transfer during Reproductive Endocrinology fellowships. Due to the inherent nature of these programs, experience is often limited to observation. However, developing a protocol utilizing mock transfers with the assistance of a transvaginal ultrasound may prevent compromising pregnancy rates immediately post fellowship.Purpose: To compare pregnancy rates post fellowship to those of an experienced operator subsequent to initiating a meticulous protocol.Methods: After a short period of observation, Dr A rehearsed embryo transfers with a Softpass catheter and transvaginal ultrasound during intrauterine inseminations. After competency was established with inseminations, the technique was applied to embryo transfer. A mock transfer was performed (by Doctor A) at the time of egg retrieval and then again just prior to transfer on day 3 or day 5. Doctor B, who has 15 years of experience, performed mock transfers during the stimulation phase with a Jones Catheter. After several transfers were supervised (by Doctor B) and pregnancies were established, this protocol was adhered to during the first 95 fresh and frozen transfers during practice. Pregnancy rates were then compared. Statistical analyses were performed using the Chi-Square test.Results: Table 1Fresh ≤ 37Fresh ≥ 38FrozenDoctor A57.4% (35/61)42.0% (8/19)53.3% (8/15)Doctor B54.5% (36/66)36.7% (18/49)60.0% (36/60)∗ P > 0.05Table 1 is a summary comparing the first 95 transfers post fellowship, which include fresh non donor and frozen including donor and non-donor cycles from December 2002 through October 2003. Open table in a new tab Discussion: According to this case control investigation there is no significant difference in pregnancy rates when a rigorous and conscientious embryo transfer protocol is implemented post fellowship. The use of echo tip catheters and ultrasound are essential in establishing the location of embryo transfer placement. Whereas the transabdominal ultrasound may be limited in specific clinical scenarios, the transvaginal ultrasound is persistently effective in obese women and women with a retroverted uterus. Moreover, it is of interest to note that mock transfers performed close to the actual day of embryo transfer does not appear to disturb the endometrium and affect pregnancy rates when compared to mock transfers performed considerably earlier in the treatment cycle.Conclusion: Despite the lack of training of embryo transfers during fellowship, a meticulous protocol utilizing mock transfers and transvaginal ultrasound can prevent impaired pregnancy rates during the first year in practice. Introduction: There is a void in clinical training of embryo transfer during Reproductive Endocrinology fellowships. Due to the inherent nature of these programs, experience is often limited to observation. However, developing a protocol utilizing mock transfers with the assistance of a transvaginal ultrasound may prevent compromising pregnancy rates immediately post fellowship. Purpose: To compare pregnancy rates post fellowship to those of an experienced operator subsequent to initiating a meticulous protocol. Methods: After a short period of observation, Dr A rehearsed embryo transfers with a Softpass catheter and transvaginal ultrasound during intrauterine inseminations. After competency was established with inseminations, the technique was applied to embryo transfer. A mock transfer was performed (by Doctor A) at the time of egg retrieval and then again just prior to transfer on day 3 or day 5. Doctor B, who has 15 years of experience, performed mock transfers during the stimulation phase with a Jones Catheter. After several transfers were supervised (by Doctor B) and pregnancies were established, this protocol was adhered to during the first 95 fresh and frozen transfers during practice. Pregnancy rates were then compared. Statistical analyses were performed using the Chi-Square test. Results: ∗ P > 0.05 Table 1 is a summary comparing the first 95 transfers post fellowship, which include fresh non donor and frozen including donor and non-donor cycles from December 2002 through October 2003. Discussion: According to this case control investigation there is no significant difference in pregnancy rates when a rigorous and conscientious embryo transfer protocol is implemented post fellowship. The use of echo tip catheters and ultrasound are essential in establishing the location of embryo transfer placement. Whereas the transabdominal ultrasound may be limited in specific clinical scenarios, the transvaginal ultrasound is persistently effective in obese women and women with a retroverted uterus. Moreover, it is of interest to note that mock transfers performed close to the actual day of embryo transfer does not appear to disturb the endometrium and affect pregnancy rates when compared to mock transfers performed considerably earlier in the treatment cycle. Conclusion: Despite the lack of training of embryo transfers during fellowship, a meticulous protocol utilizing mock transfers and transvaginal ultrasound can prevent impaired pregnancy rates during the first year in practice.

ObjectiveCryopreservation of human embryos at pronuclear or cleavage stage has commonly been performed using the standard Testart-Lassalle method. However, not much has been done to compare the actual viability and pregnancy rates when starting temperature for freezing is lower than room temperature as in the standard method.DesignRetrospective analysis of pregnancy and cryo-survival rates of Day 3 embryos with two different methods at different starting temperatures over a two-year period in a clinical IVF program.Materials and methodsThree hundred and eighty-two patients undergoing IVF treatments who had supernumerary Day 3 embryos had cryopreservation. Exclusion criteria were women >39 years old or day 3 FSH level ≥20 IU/ml. Day 3 embryos with more than 4 cells and less than 20% fragmentation were cryopreserved in 0.25 cc straws. In Group I, embryos were placed into the freezing machine at -6oC, held for 2 minutes and seeded, then held for a further 10 minutes, followed by cooling at 0.5oC/min to -30oC and plunged into liquid nitrogen. In Group II (Testart-Lassalle method), embryos were cooled at 2oC/min from room temperature to -6oC and continued the same way as in Group I followed by cooling at 0.3oC/min to -35oC and plunged into LN2. Both groups of embryos were thawed using the same procedure.ResultsGroup I yielded better survival rate and pregnancy rate than Group II (p<0.005). See table.Conclusion ObjectiveCryopreservation of human embryos at pronuclear or cleavage stage has commonly been performed using the standard Testart-Lassalle method. However, not much has been done to compare the actual viability and pregnancy rates when starting temperature for freezing is lower than room temperature as in the standard method. Cryopreservation of human embryos at pronuclear or cleavage stage has commonly been performed using the standard Testart-Lassalle method. However, not much has been done to compare the actual viability and pregnancy rates when starting temperature for freezing is lower than room temperature as in the standard method. DesignRetrospective analysis of pregnancy and cryo-survival rates of Day 3 embryos with two different methods at different starting temperatures over a two-year period in a clinical IVF program. Retrospective analysis of pregnancy and cryo-survival rates of Day 3 embryos with two different methods at different starting temperatures over a two-year period in a clinical IVF program. Materials and methodsThree hundred and eighty-two patients undergoing IVF treatments who had supernumerary Day 3 embryos had cryopreservation. Exclusion criteria were women >39 years old or day 3 FSH level ≥20 IU/ml. Day 3 embryos with more than 4 cells and less than 20% fragmentation were cryopreserved in 0.25 cc straws. In Group I, embryos were placed into the freezing machine at -6oC, held for 2 minutes and seeded, then held for a further 10 minutes, followed by cooling at 0.5oC/min to -30oC and plunged into liquid nitrogen. In Group II (Testart-Lassalle method), embryos were cooled at 2oC/min from room temperature to -6oC and continued the same way as in Group I followed by cooling at 0.3oC/min to -35oC and plunged into LN2. Both groups of embryos were thawed using the same procedure. Three hundred and eighty-two patients undergoing IVF treatments who had supernumerary Day 3 embryos had cryopreservation. Exclusion criteria were women >39 years old or day 3 FSH level ≥20 IU/ml. Day 3 embryos with more than 4 cells and less than 20% fragmentation were cryopreserved in 0.25 cc straws. In Group I, embryos were placed into the freezing machine at -6oC, held for 2 minutes and seeded, then held for a further 10 minutes, followed by cooling at 0.5oC/min to -30oC and plunged into liquid nitrogen. In Group II (Testart-Lassalle method), embryos were cooled at 2oC/min from room temperature to -6oC and continued the same way as in Group I followed by cooling at 0.3oC/min to -35oC and plunged into LN2. Both groups of embryos were thawed using the same procedure. ResultsGroup I yielded better survival rate and pregnancy rate than Group II (p<0.005). See table. Group I yielded better survival rate and pregnancy rate than Group II (p<0.005). See table. Conclusion