Objective To describe the multiple benefits of performing laser hatching for trophectoderm (TE) biopsy at the time of biopsy rather than at an earlier embryonic stage. Type of Study Case Report/Opinion Materials and Methods Embryos were cultured in Global Total Culture Media (Cooper) and were not evaluated after the fertilization check until Day 5 of development. Blastocysts (D5/D6) were biopsied using a Hamilton Thorne Zilos laser on an Olympus IX 71 Inverted microscope. Biopsied blastocysts were vitrified on Cryotops using Kitazato Vitrification kits (CA Cryobank). Results Blastocysts were placed in 6ul mHTF drops under oil and were picked up with holding pipette with the inner cell mass (ICM) placed between 7 and 10 o'clock. Blastocysts were collapsed with a 250µm laser pulse at 3 o'clock and biopsy did not occur until the blastocyst was totally collapsed. The laser was increased to 450µm and the biopsy pipette was introduced into the small hole that was made during collapse. With suction on the biopsy pipette, approximately 5-8 trophectoderm cells were pulled toward the biopsy hole, which stretches the cells and several pulses of the laser sheared the cells off into the biopsy pipette. The biopsy sample was located in the drop and the blastocyst was returned to a labeled post-biopsy holding dish. The blastocysts were vitrified after the biopsied cells were loaded into microfuge tubes and frozen. Conclusions When trophectoderm biopsy became the preferred method for biopsy, most IVF labs performed laser hatching on Day 3 of embryo development followed by a Day 5/6 biopsy. Often the ICM hatched out of the hole that was created. In addition, many of the blastocysts had very thick zona pellucidas (ZP) and some had very low TE numbers. By performing laser hatching at the time of biopsy, the ZP is normally very thin and the TE number is what it should be for a Day 5/6 blastocyst. By not breaching the ZP on Day 3 the embryo develops into an expanded blastocyst like the embryos in culture that are not undergoing biopsy. Usually, 6-8 TE cells are removed during the biopsy procedure and the collapsed blastocyst tolerates the procedure without any visble negative effects. The collapsed blastocyst vitrifies very well and expands quickly post-thaw. Disclosures Nothing to disclose Funding None To describe the multiple benefits of performing laser hatching for trophectoderm (TE) biopsy at the time of biopsy rather than at an earlier embryonic stage. Case Report/Opinion Embryos were cultured in Global Total Culture Media (Cooper) and were not evaluated after the fertilization check until Day 5 of development. Blastocysts (D5/D6) were biopsied using a Hamilton Thorne Zilos laser on an Olympus IX 71 Inverted microscope. Biopsied blastocysts were vitrified on Cryotops using Kitazato Vitrification kits (CA Cryobank). Blastocysts were placed in 6ul mHTF drops under oil and were picked up with holding pipette with the inner cell mass (ICM) placed between 7 and 10 o'clock. Blastocysts were collapsed with a 250µm laser pulse at 3 o'clock and biopsy did not occur until the blastocyst was totally collapsed. The laser was increased to 450µm and the biopsy pipette was introduced into the small hole that was made during collapse. With suction on the biopsy pipette, approximately 5-8 trophectoderm cells were pulled toward the biopsy hole, which stretches the cells and several pulses of the laser sheared the cells off into the biopsy pipette. The biopsy sample was located in the drop and the blastocyst was returned to a labeled post-biopsy holding dish. The blastocysts were vitrified after the biopsied cells were loaded into microfuge tubes and frozen. When trophectoderm biopsy became the preferred method for biopsy, most IVF labs performed laser hatching on Day 3 of embryo development followed by a Day 5/6 biopsy. Often the ICM hatched out of the hole that was created. In addition, many of the blastocysts had very thick zona pellucidas (ZP) and some had very low TE numbers. By performing laser hatching at the time of biopsy, the ZP is normally very thin and the TE number is what it should be for a Day 5/6 blastocyst. By not breaching the ZP on Day 3 the embryo develops into an expanded blastocyst like the embryos in culture that are not undergoing biopsy. Usually, 6-8 TE cells are removed during the biopsy procedure and the collapsed blastocyst tolerates the procedure without any visble negative effects. The collapsed blastocyst vitrifies very well and expands quickly post-thaw.

Cell-free DNA (cfDNA), which is present in the blastocoel cavity of embryos, is believed to result from either physiological apoptosis and, or cellular (re)modeling during development. Additionally, cfDNA has been investigated as a potential reference source for preimplantation genetic screening (PGS). This study assessed cfDNA content in day 5 IVF blastocysts to determine if there was a correlation with embryo morphology. Additionally, this study investigated if the physiological source of the cfDNA is apoptotic in nature. Retrospective study in academic assisted reproductive technology programs. Day 5 IVF generated blastocysts were scored according to the Gardner system (modified to generate a numerical value) and cfDNA was collected from laser-induced blastocoel collapsing prior to cryopreservation in 25μL of media. cfDNA was quantified via fluorospectrometry (AccuBlue NextGen dsDNA Quantification Kit; ThermoScientific NanoDrop 3300 Fluorospectrometer) and apoptosis activity was assessed via a caspase-3 fluorescence assay (Enzo Life Sciences Caspase-3 Cellular Assay; Tecan Infinite M1000 fluorescence reader). Data were compared by linear regression (SigmaPlot for Windows v13.0.0.83). A total of 32 embryos were evaluated. Embryo morphology scores ranged from a low of 15 to a high of 27; with a mean of 21.1. cfDNA content (ng/mL) ranged from a low of 32.3 to a high of 315.3; with a mean of 133.6. Caspase-3 activity (AFU detected with 30 μM DEVD-AMC substrate) ranged from undetectable levels to a high of 2,226.6; with a mean of 383.8. The maximum embryo score of 27 had a cfDNA concentration of 315.3 ng/mL and the minimum embryo score of 15 had a cfDNA concentration of 32.3 ng/mL. There was a significant (p<0.01) and positive correlation (cfDNA = -104.753+(11.281*score); R2=0.200) between embryo score and cfDNA. There was a significant (p<0.05) and positive correlation (cfDNA = 115.9+(0.05*caspase-3); R2=0.128) between caspase-3 activity and cfDNA. There was no significant relationship between caspase-3 activity and embryo morphology score. This study provides further evidence that cfDNA is present in blastocoel fluid, can be quantified, and positively correlates with embryonic morphology. There is also evidence that at least a portion of the cfDNA is due to apoptotic activity. Additional studies are warranted to determine other physiological sources of the cfDNA in blastocyst fluid and to determine the relationship of cfDNA content with embryo morphology, ploidy status (PGS) and, or implantation potential.

Objective: In order to optimize ART implantation success, embryologists typically select a preimplantation embryo(s) based on the embryos grade at the time of transfer. This embryonic grading is routinely a combination of objective and subjective assessment methods. However, this objective/subjective combination has been only minimally linked with successful implantations. Recently, significantly higher implantation rates have been observed utilizing novel embryo morphometric (embryometric) analyses that examine blastocyst length-to-width ratio or ICM-to-total surface area ratio that approximates phi (θ = 1.618, aka the Golden Ratio). To further explore the potential of embryometric phi analysis in ART, in particular in an elective single embryo transfer (eSET) program, we further evaluated these ratios in positive term pregnancy outcomes.

Preimplantation embryo grading mixes objective and subjective assessment methods in the hopes of increasing implantation potential. However, the combination of cell stage/number and percent fragmentation utilized has been only minimally linked with successful implantation. Recently significantly higher implantation rates have been found using novel embryometric analysis approaches that examine blastocyst ratios, length-to-width across the inner cell mass (ICM) axis (AAB-2014) or ICM-to-total blastocyst areas (ASRM-2014) that closely approximates phi (θ=1.618, aka the Golden Ratio). To further explore the potential of embryometric analysis in ART, this study combined the phi ratios derived from both length-to-width and ICM-to-total areas ratios of blastocysts reported to have either successfully delivered or failed to implant/delivered following elective single embryo transfer (eSET). Retrospective post-eSET embryometric analysis (length-to-width and ICM-to-total area) was calculated for phi of day-5 blastocysts and analyzed with term delivery outcomes. Day-5 blastocyst images were measured for length-to-width (Word) and ICM-to-total area (tpsDIG2; life.bio.sunysb.edu/morph/) and subsequent phi (θ) ratios calculated. Receiver operating curve (ROC) (MedCalc) was used to assess θ ratios with delivery outcomes and determine definitive θ cut-off value. A total of 54 day-5 blastocysts were evaluated. The overall term delivery rate was 55.6%. In the length-to-width dataset, θ1 ratios ranged from a low of 1.3908 (highly expanded) to a high of 2.1089 (minimal expansion), with a mean of 1.8297 (113.1% of the Golden Ratio). In the ICM-total area dataset, θ2 ratios ranged from a low of 1.0585 to a high of 1.3110, with a mean of 1.1173 (69.1% of the Golden Ratio). In a third phi ratio (θ3=θ1+θ2)/θ1), θ3 ratios ranged from a low of 1.5298 to a high of 1.7610, with a mean of 1.6142 (99.8% of the Golden Ratio). Utilizing the combined embryometric algorithm for θ3, ROC demonstrated a definitive phi cut-off value (i.e. criterion) for a viable pregnancy of θ3=1.6114 (which very closely approximates "true" phi), with specificity 63.3 and sensitivity 73.9, P=0.0184). Embryometric analysis of blastocyst stage embryos were found to provide a more objective assessment than traditional morphological approaches. Additionally, those embryometric ratios which approached phi were found to be a more ideal selection choice for eSET. Finally, these results suggest calculating blastocyst area ratios relative to phi can assist in identifying the more ideal blastocyst and assist in predicting eSET pregnancy potentials.

OBJECTIVE: PGS (preimplantation genetic screening) is often used in patients diagnosed with advanced maternal age (AMA). Often these patients have a poor response to ovarian stimulation which results in cancellation of PGS due to low embryo number. A solution for this problem is to freeze all embryos at the zygote stage prior to genetic testing. The patient would subsequently proceed through another IVF cycle and PGS would be performed on both her fresh and frozen-thawed embryos. It is the goal of this study to determine if embryo banking is advantageous or if a patient with a low embryo yield should proceed with testing regardless of embryo number. DESIGN: Retrospective data analysis. MATERIALS AND METHODS: Patients diagnosed with AMA that elected to perform PGS in conjunction with IVF (in-vitro fertilization) between April 2006 and April 2010. Patients were given the option to freeze all embryos if we had less then 8 zygotes and go through another IVF cycle (group 1), or proceed with PGS without banking (group 2). Embryos were biopsied on day 3 and either 9 probe or 12 probe FISH (fluorescence in-situ hybridization) was performed. RESULTS: The results are shown in Table 1.Table 1Banked (group 1)Not Banked (group 2)P ValueNo. Patients2828Avg. Age40.1 ± 2.839.9 ± 2.80.7903No. Biopsied11.1 ± 4.49.6 ± 5.40.2595No. Normal2.4 ± 2.02.2 ± 2.30.7298No. ET1.8 ± 1.21.4 ± 1.30.2368No. No ET4100.1212+hCG (%)12 (42.9%)11 (39.3%)1.0000+FCA (%)9 (32.1%)10 (35.7%)0.5472 Open table in a new tab CONCLUSION: Embryo banking to increase the number of embryos available for PGS does not increase pregnancy rates. Although not significant, the number of biopsied embryos, number normal, and number transferred is slightly higher in group 1. The number of patients that didn't have a transfer, although not significant, was higher in group 2. Embryo banking may increase embryo number for PGS but our data shows that pregnancy rates are not influenced.

OBJECTIVE: AMH (anti-mullerian hormone) has been shown to correlate to ovarian reserve and certain cycle parameters. There are multiple studies demonstrating how AMH correlates to different cycle parameters, however data on patient management in regards to their AMH is lacking. Here we present our clinical experience with the use of AMH level in determining the type of ovarian stimulation and starting dose in IVF cycles.DESIGN: Retrospective data analysis.MATERIALS AND METHODS: AMH was drawn on day 3 of the cycle. Patients were subdivided into 4 groups based on their AMH level, group 1 (0.5-1.2 ng/mL), group 2 (1.2-1.9 ng/mL), group 3 (2.0-4.0 ng/mL), and group 4 (>4.0 ng/mL). The stimulation protocol was determined based on AMH levels:, group 1 was a microdose flare protocol, group 2 was a high dose FSH luteal down regulation protocol, group 3 was a standard luteal down regulation protocol, and group 4 was a low dose luteal down regulation protocol.Table 1Group 1 (0.5-1.2)Group 2 (1.3-1.9)Group 3 (2.0-4.0)Group 4 (>4.0)P ValueNo. Patients26254729Avg. Age35.0 ± 4.034.9 ± 4.331.9 ± 3.831.9 ± 4.10.0009AMH (ng/mL)1.0 ± 0.21.7 ± 0.22.8 ± 0.67.4 ± 4.6<0.0001No. cancelations7 (26.9%)3 (12.0%)0 (0%)0 (0%)0.0002No. oocytes7.5 ± 3.910.5 ± 7.414.9 ± 5.417.3 ± 8.3<0.0001No. ET1.8 ± 1.31.8 ± 1.12.0 ± 0.71.8 ± 1.00.7543+hCG (%)9 (36.0%)13 (52.0%)27 (87.4%)17 (58.6%)0.3117+FCA (%)8 (30.8%)12 (48.0%)26 (55.3%)15 (51.7%)0.2345 Open table in a new tab CONCLUSION: AMH is routinely drawn but not much is known on how to utilize the value for an upcoming IVF cycle. Our data shows that if proper cutoffs are determined then high pregnancy rates can be achieved by altering the starting stimulation dose. OBJECTIVE: AMH (anti-mullerian hormone) has been shown to correlate to ovarian reserve and certain cycle parameters. There are multiple studies demonstrating how AMH correlates to different cycle parameters, however data on patient management in regards to their AMH is lacking. Here we present our clinical experience with the use of AMH level in determining the type of ovarian stimulation and starting dose in IVF cycles. DESIGN: Retrospective data analysis. MATERIALS AND METHODS: AMH was drawn on day 3 of the cycle. Patients were subdivided into 4 groups based on their AMH level, group 1 (0.5-1.2 ng/mL), group 2 (1.2-1.9 ng/mL), group 3 (2.0-4.0 ng/mL), and group 4 (>4.0 ng/mL). The stimulation protocol was determined based on AMH levels:, group 1 was a microdose flare protocol, group 2 was a high dose FSH luteal down regulation protocol, group 3 was a standard luteal down regulation protocol, and group 4 was a low dose luteal down regulation protocol. CONCLUSION: AMH is routinely drawn but not much is known on how to utilize the value for an upcoming IVF cycle. Our data shows that if proper cutoffs are determined then high pregnancy rates can be achieved by altering the starting stimulation dose.

OBJECTIVE: Anti-Mullerian Hormone (AMH) is a glycoprotein dimer composed of two 72kDa monomers linked by disulfide bridges and is a member of the transforming growth factor-β super family. AMH is produced in small amounts by ovarian granulosa cells after birth until menopause, and then becomes undetectable. AMH performs various physiological functions, e.g. controlling antral follicle development. AMH is routinely used clinically to help determine ovarian reserve status and subsequently, gonadotropin stimulation protocols. Whereas, AMH values can help predict the quantity of oocytes obtained during retrieval, little to no information is available regarding AMH levels on embryo formation rates. To determine if circulating anti-mullerian hormone (AMH) levels relate to blastocyst formation rates following ovarian stimulation in donor IVF.DESIGN: Retrospective analysis of donor cycles. AMH levels were measured and correlated with blastocyst (Grade A or B) formation rates during the IVF cycle.MATERIALS AND METHODS: Circulating AMH levels were measured in donors by a commercially available assay. ART cycles were normalized to control for ovarian stimulation protocol (antagonist) and gonadotropin dose, no male factor, number of stimulation days (10-12 days) and day 5 transfers. Data were analyzed by regression analysis.RESULTS: A total of 18 donor ART cycles were included in this study. The overall blastocyst formation rate was 28.93% (80/342). The mean (+s.e.) AMH level in the donors was 3.941 (+0.459) ng/mL. AMH levels ranged from a low of 1.27 ng/mL to a high of 8.25 ng/mL. Regression analysis revealed a statistically significant binomial correlation between circulating AMH levels in oocyte donors and blastocyst formation rates (P<0.05).CONCLUSION: The data demonstrates a significant and positive correlation between circulating AMH levels in oocyte donors and blastocyst formation rates. This is the first report demonstrating a significant relationship between AMH and blastocyst formation rates. OBJECTIVE: Anti-Mullerian Hormone (AMH) is a glycoprotein dimer composed of two 72kDa monomers linked by disulfide bridges and is a member of the transforming growth factor-β super family. AMH is produced in small amounts by ovarian granulosa cells after birth until menopause, and then becomes undetectable. AMH performs various physiological functions, e.g. controlling antral follicle development. AMH is routinely used clinically to help determine ovarian reserve status and subsequently, gonadotropin stimulation protocols. Whereas, AMH values can help predict the quantity of oocytes obtained during retrieval, little to no information is available regarding AMH levels on embryo formation rates. To determine if circulating anti-mullerian hormone (AMH) levels relate to blastocyst formation rates following ovarian stimulation in donor IVF. DESIGN: Retrospective analysis of donor cycles. AMH levels were measured and correlated with blastocyst (Grade A or B) formation rates during the IVF cycle. MATERIALS AND METHODS: Circulating AMH levels were measured in donors by a commercially available assay. ART cycles were normalized to control for ovarian stimulation protocol (antagonist) and gonadotropin dose, no male factor, number of stimulation days (10-12 days) and day 5 transfers. Data were analyzed by regression analysis. RESULTS: A total of 18 donor ART cycles were included in this study. The overall blastocyst formation rate was 28.93% (80/342). The mean (+s.e.) AMH level in the donors was 3.941 (+0.459) ng/mL. AMH levels ranged from a low of 1.27 ng/mL to a high of 8.25 ng/mL. Regression analysis revealed a statistically significant binomial correlation between circulating AMH levels in oocyte donors and blastocyst formation rates (P<0.05). CONCLUSION: The data demonstrates a significant and positive correlation between circulating AMH levels in oocyte donors and blastocyst formation rates. This is the first report demonstrating a significant relationship between AMH and blastocyst formation rates.

OBJECTIVE: Oocyte vitrification requires extensive training to be successful. The most difficult step is the placement of the oocyte on a vessel (ie cryolock) in a small amount of fluid. The small amount of fluid surrounding the oocyte increases the rate of cooling and decreases the chance of ice formation. These vessels are expensive and the placement of the oocytes is hard to master. This is in contrast to the simple loading process of slow freezing which most often makes use of straws or vials. Our objective was to compare oocyte survival following vitrification in either ¼ cc straws or Cryolocks.DESIGN: prospective randomized study.MATERIALS AND METHODS: Day 1 oocytes were vitrified utilizing a cryolock (group 1) or a regular ¼ cc straw (group 2). Oocytes were exposed to equilibration solution for 10 minutes and then moved to a vitrification solution (VS) for 1 min. Oocytes vitrified on the cryolock were plunged directly into liquid nitrogen and capped. Oocytes vitrified in a straw were aspirated into a straw in 50 uL of VS. The straw was sealed and plunged directly into liquid nitrogen. Oocytes were thawed in thawing solution for 1 minute, followed by 3 minutes in diluent, and 8 minutes in Hepes media.RESULTS: A total of 52 day 1 oocytes were included in this study. The average age between group 1 and 2 was not signfiicant, 33.4 ± 2.3 and 32.1 ± 3.2 years, respectively (P=0.1011). For group 1, 17 of 25 (68.0%) oocytes survived vitrification with the cryolock. For group 2, 19 of 27 (70.4%) survived being vitrified in a ¼ cc straw (P=1.0000).CONCLUSION: Vitrification typically requires an expensive and complex device which requires training to master. Here we present results showing that oocyte vitrification can be done in a sealed ¼ cc straw and yield similar results to that of a vitrification vessel. Utilizing a ¼ cc straw will allow an embryologist to vitrify with little to no experience. Further studies are needed to see if fertilization and embryo quality are similar with this new technique. OBJECTIVE: Oocyte vitrification requires extensive training to be successful. The most difficult step is the placement of the oocyte on a vessel (ie cryolock) in a small amount of fluid. The small amount of fluid surrounding the oocyte increases the rate of cooling and decreases the chance of ice formation. These vessels are expensive and the placement of the oocytes is hard to master. This is in contrast to the simple loading process of slow freezing which most often makes use of straws or vials. Our objective was to compare oocyte survival following vitrification in either ¼ cc straws or Cryolocks. DESIGN: prospective randomized study. MATERIALS AND METHODS: Day 1 oocytes were vitrified utilizing a cryolock (group 1) or a regular ¼ cc straw (group 2). Oocytes were exposed to equilibration solution for 10 minutes and then moved to a vitrification solution (VS) for 1 min. Oocytes vitrified on the cryolock were plunged directly into liquid nitrogen and capped. Oocytes vitrified in a straw were aspirated into a straw in 50 uL of VS. The straw was sealed and plunged directly into liquid nitrogen. Oocytes were thawed in thawing solution for 1 minute, followed by 3 minutes in diluent, and 8 minutes in Hepes media. RESULTS: A total of 52 day 1 oocytes were included in this study. The average age between group 1 and 2 was not signfiicant, 33.4 ± 2.3 and 32.1 ± 3.2 years, respectively (P=0.1011). For group 1, 17 of 25 (68.0%) oocytes survived vitrification with the cryolock. For group 2, 19 of 27 (70.4%) survived being vitrified in a ¼ cc straw (P=1.0000). CONCLUSION: Vitrification typically requires an expensive and complex device which requires training to master. Here we present results showing that oocyte vitrification can be done in a sealed ¼ cc straw and yield similar results to that of a vitrification vessel. Utilizing a ¼ cc straw will allow an embryologist to vitrify with little to no experience. Further studies are needed to see if fertilization and embryo quality are similar with this new technique.

OBJECTIVE: To assess the relationship between the Embryo Morphologic Grade (EMG) of Day 3 (D3) and Day 5 (D5) IVF embryos and live birth rate. DESIGN: A retrospective study from the IVF database collected by SARTCORS. MATERIALS AND METHODS: From June 2006 to December 2007, SART-affiliated IVF clinics were requested to voluntarily report information about the EMG of transferred embryos to the SARTCORS database. The embryo classification system developed by SART assigned embryos to one of three categories: Good, Fair or Poor (G, F, P). We analyzed the EMG of embryos transferred on D3 (6 to ≥8 cell embryos) and D5 (blastocysts). Live birth rates/embryo transfer (LB/ET) were evaluated in a subset of patients who received two embryos of the same grade. LB/ET was examined by age (<35, 35-37, 38-40, and 41-42 YO). RESULTS: 70,293 embryos from 28,186 ETs representing 19.1% of all fresh autologous ETs were morphologically classified by clinics in 46 states, the District of Columbia and Puerto Rico. D3 ETs were 58.7% and D5 were 29.6% of classified embryos. The distribution of embryos by grade was similar for D3 & D5 (D3 = 70%G, 24.2%F & 5.5%P; D5= 77.3%G, 18.6%F & 3.7%P). The LB/ET rate (p<.05) decreased by EMG on D3 & D5 (D3 = 45.3%G, 35.2%F, 21.3%P; D5 = 56.2%G, 42.16%F, 30.8%P). This decrease was observed in each age group and EMG negatively correlated (p<.05) with increasing age. CONCLUSIONS: The EMG data collected by SART correlated with live birth rate. The predictive values generated by these data represent a valuable national standard that could be used by individual SART IVF programs for quality assurance assessment. Future analysis of a larger SART dataset may: 1) increase the accuracy of EMG for predicting pregnancy outcome; 2) lead to the development of a more accurate and universal EMG that could enhance the selection of fewer embryos at ET that would; 3) reduce the potential for multiple pregnancies.

OBJECTIVE: The CASA is able to measure multiple values for sperm motility. Included within these measurements is VAP (average path velocity), VSL (straight line velocity), VCL (curvilinear velocity), ALH (amplitude of lateral head), BCF (beat cross frequency), STR (straightness), and LIN (linearity). The purpose of this study is to determine if sperm parameters, as calculated by CASA, on sperm used for conventional in-vitro fertilization (cIVF), predict fertilization outcome in cIVF cycles. DESIGN: Retrospective comparison of CASA parameters with cIVF. MATERIALS AND METHODS: Sperm CASA parameters were determined at time of insemination. Sperm were isolated from seminal fluid by a gradient and washed in 3 mL of media. After washing the pellet was overlayed with 1 mL of media and placed in an incubator for a swim-up and capacitation. Retrieved oocytes were placed in 1 mL drop of media. After 2-3 hours, a count of roughly 150K of sperm was added to the oocytes. A 10 μl of sperm, from the same sample used for insemination, was added to a slide. This slide was analyzed using the CASA. Data was subjected to a linear regression analysis. RESULTS: A total of 49 semen specimens were analyzed as described above. The average male age was 34.9 ± 4.8 years. The average sperm count at insemination was 8.9 ± 10.0 Mil/mL. The mean cIVF rate (±SEM) was 0.551 (±0.031). Linear regression analysis between the cIVF and VAP (R2 = 0.0322), VSL (R2 = 0.0127), ALH (R2 = 0.0509), VCL (R2 = 0.0610), BCF (R2 = 0.0001), STR (R2 = 0.0298), and LIN (0.0229) revealed no significant relationship (P>0.05). CONCLUSIONS: The CASA is able to determine multiple sperm parameters on a single sample. In our study we found that these parameters are poor predictors of cIVF outcome. There are no parameters, that this study found, that are able to predict cIVF rates.

OBJECTIVE: The aim of this study was to determine if a female's patient's body mass index (BMI) increases the chances of producing aneuploid embryos. DESIGN: A retrospective data analysis involving patients that went through in-vitro fertilization in conjunction with PGD-AS (preimplantation genetic diagnosis for aneuploidy screening) for recurrent pregnancy loss or advanced maternal age. MATERIALS AND METHODS: Patients were divided into three groups based upon the females BMI at time of egg retrieval. Group 1 included those patients with a BMI of <24 (average weight and height), group 2 included patients with a BMI of 25-29 (overweight), and group 3 included patients with a BMI of >29 (obese). Embryos were biopsied and graded on day 3, where "A" was the highest and "D" the lowest quality. One cell was removed and fixed to a slide. The slide was hybridized and underwent fluorescence in-situ hybridization (FISH). Embryos diagnosed as "normal" by "FISH" were transferred on day 5. Statistical analysis was done utilizing the proper statistical test and P<0.05 was considered significant. RESULTS: A total of 586 embryos were biopsied from 53 patients. The average age was 37.5 ± 4.9, 36.4 ± 3.6, and 37.4 ± 3.8 years for groups 1, 2, and 3 respectively (P=0.8479). Embryo quality on day 3 is described in table 1 (P=0.7672).Table 1Embryo Quality on Day 3Group 1Group 2Group 3No. A (%)119 (30.6%)30 (30.9%)35 (35.0%)No. B (%)173 (44.5%)49 (50.5%)45 (45.0%)No. C (%)77 (19.8%)15 (15.5%)15 (15.0%)No. D (%)20 (5.1%)3 (3.1%)5 (5.0%) Open table in a new tab In group 1, a total of 389 embryos from 36 patients were biopsied and 105 were normal (27.0%). In group 2, 97 embryos from 7 patients were biopsied and 32 (33.0%) were normal. In group 3, 100 embryos from 10 patients were biopsied and 29 (29.0%) were normal (P=0.4960). CONCLUSIONS: Certain patient populations (i.e., recurrent pregnancy lost and advanced maternal age) have an increased incidence of chromosomal aneuploidy and consequently, infertility. Obesity has also been associated with infertility. In our study we found no link between the females BMI and the incidence of aneuploidy in the corresponding cycle.

OBJECTIVE: Few studies have been conducted as to the effects of the freezing procedure on aneuploidy rates as determined by preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). The aim of this study is to determine if the cryopreservation of zygotes affects aneuploidy rates in subsequent embryos. DESIGN: This study was retrospective and observational. MATERIALS AND METHODS: Eighteen patients underwent a fresh IVF cycle, had all embryos frozen at the 2PN stage, and then proceeded through a second fresh cycle. PGD-AS, due to recurrent pregnancy loss or advanced maternal age, was performed on both the fresh and thawed frozen embryos. Embryos were divided into 2 groups: Group 1 consisted of the embryos derived from the fresh cycle and group 2 consisted of embryos derived from the frozen/thaw cycle. Day 3 fresh and frozen embryos underwent embryo biopsy, 9 probe FISH, and transferred on day 5. Embryos were graded on day 3 where "A" quality was considered excellent and "D" quality was considered poor. Statistical analysis was done utilizing either the unpaired t-test or chi-square. P<0.05 was considered significant. RESULTS: Ages between the group 1 and group 2 were not significant, 36.5±2.7 and 37.1±2.9 respectively (P=0.5249). Embryo quality between group 1 and group 2 is represented in table 1 (P=0.0701).Table 1Day 3 Embryo QualityEmbryo QualityGroup 1Group 2No. A (%)72 (49.7%)32 (34.0%)No. B (%)47 (32.4%)35 (37.2%)No. C (%)16 (11.0%)19 (20.2%)No. D (%)10 (6.9%)8 (8.5%) Open table in a new tab Of the 94 embryos from group 2 that underwent PGD-AS, 17 (22.1%) were chromosomally normal (P=0.1199). For group 1, a total of 145 embryos developed to day 3 and underwent PGD-AS. A total of 40 (27.6%) and 17 (22.1%) embryos were chromosomally normal from group 1 and group 2 (P=0.1199). CONCLUSIONS: The cryopreservation of embryos is a common and necessary practice in IVF. The effects of cryopreservation on chromosome segregation are unknown. Although not significant, data shows a trend towards an increase in chromosomal aneuploidy in frozen/thawed zygotes. However, further studies need to be conducted involving sibling oocytes before any conclusions can be drawn.

OBJECTIVE: To determine if the 12 probe panel for FISH (Fluorescent in-situ hybridization) detects more chromosome abnormalities then the 9 probe panel.DESIGN: A retrospective study involving 42 patients attending a fertility clinic between January 2005 and April 2008, who had preimplantation genetic diagnosis for aneuploidy (PGD-AS), due to recurrent pregnancy loss or advanced maternal age, with a day 5 embryo transfer.MATERIALS AND METHODS: Patients' embryos were subdivided into two groups. In group 1 embryos were screened using a 9 probe FISH, while in Group 2 the embryos were screened using a 12 probe FISH panel. Group 2 was further divided into those embryos which would have been successfully screened out using the 9 probe FISH panel and those whose abnormalities would only have been detected using 12 probe FISH. Embryos were graded as A - D on day 3 based on cell number and quality where “A” was best quality. Embryos were biopsied on Day 3 and the cells were fixed and analyzed by FISH with either 9 probes (X,Y,13,15,16,17,18,21,22) or 12 probes (X,Y,8,13,14,15,16,17,18,20,21,22). P<0.05 was considered significant.Table 1Embryo Quality on Day 3Group 1Group 2No. A (%)54 (46.6%)150 (39.6%)No. B (%)35 (30.1%)129 (34.0%)No. C (%)25 (21.6%)92 (24.3%)No. D (%)2 (1.7%)8 (2.1%) Open table in a new tab Chromosomal analysis by FISH showed that 69.8% (n=116) and 74.7% (n = 379) of embryos were abnormal in groups 1 and 2 respectively (P=0.3009). When group 2 was further analyzed to determine which embryos would have had chromosomal anomalies detected in the 9 probe FISH panel a total of 71.2 % (n=379) were considered abnormal (P=0.4463) showing a difference of 3.5% in abnormality detection rate in the 9 and 12 chromosome FISH panels.CONCLUSIONS: The screening of an additional 3 chromosomes using the 12 panel FISH provided detection for 3.5% of chromosomal abnormalities not already identified using the 9 panel FISH. The 12 probe panel provided no statistical significant advantage over the 9 probe. The 9 probe still remains a viable alternative with PGD-AS. OBJECTIVE: To determine if the 12 probe panel for FISH (Fluorescent in-situ hybridization) detects more chromosome abnormalities then the 9 probe panel. DESIGN: A retrospective study involving 42 patients attending a fertility clinic between January 2005 and April 2008, who had preimplantation genetic diagnosis for aneuploidy (PGD-AS), due to recurrent pregnancy loss or advanced maternal age, with a day 5 embryo transfer. MATERIALS AND METHODS: Patients' embryos were subdivided into two groups. In group 1 embryos were screened using a 9 probe FISH, while in Group 2 the embryos were screened using a 12 probe FISH panel. Group 2 was further divided into those embryos which would have been successfully screened out using the 9 probe FISH panel and those whose abnormalities would only have been detected using 12 probe FISH. Embryos were graded as A - D on day 3 based on cell number and quality where “A” was best quality. Embryos were biopsied on Day 3 and the cells were fixed and analyzed by FISH with either 9 probes (X,Y,13,15,16,17,18,21,22) or 12 probes (X,Y,8,13,14,15,16,17,18,20,21,22). P<0.05 was considered significant. Chromosomal analysis by FISH showed that 69.8% (n=116) and 74.7% (n = 379) of embryos were abnormal in groups 1 and 2 respectively (P=0.3009). When group 2 was further analyzed to determine which embryos would have had chromosomal anomalies detected in the 9 probe FISH panel a total of 71.2 % (n=379) were considered abnormal (P=0.4463) showing a difference of 3.5% in abnormality detection rate in the 9 and 12 chromosome FISH panels. CONCLUSIONS: The screening of an additional 3 chromosomes using the 12 panel FISH provided detection for 3.5% of chromosomal abnormalities not already identified using the 9 panel FISH. The 12 probe panel provided no statistical significant advantage over the 9 probe. The 9 probe still remains a viable alternative with PGD-AS.

OBJECTIVE:To study the response of human ovarian xenografts to transplantation into different sites and in different host conditions.DESIGN:Controlled experiment.SETTING:Academic research laboratory.PATIENT(S):Donated ovarian tissue from two young women.INTERVENTION(S):Human ovarian cortical pieces were transplanted either under the kidney capsule or to the subcutaneous space of intact or castrated male nonobese diabetic (NOD) severe combined immune-deficient (SCID) mice. Grafts were recovered after euthanasia.MAIN OUTCOME MEASURE(S):Microscopic examination of histologic sections to determine proportions of growing follicles, and serum estradiol concentration measurements.RESULT(S):Six months after transplantation, ovarian grafts transplanted under the kidney capsule of intact male mice had significantly higher proportions of growing follicles compared with those recovered from the castrated/kidney capsule and intact/subcutaneous groups. However, no difference was detected between the intact/kidney capsule and the castrated/subcutaneous groups. Mean estradiol concentrations in serum were nonsignificantly increased in mice with ovarian grafts compared with those in mice without a graft.CONCLUSION(S):Follicular development in xenotransplanted human ovarian tissue is influenced by the site of transplantation and the condition of the host.

Ovarian cortex cryopreservation and xenotransplantation into immunodeficient mice represents a potential means for female germplasm conservation and an immediate model for investigation of folliculogenesis. The objectives of this study were to: (1) assess follicle survival after cryopreservation and transplantation of cat ovarian tissue into non-obese diabetic severely combined immunodeficient (NOD SCID) mice; and (2) evaluate the effects of gonadotropin treatments on follicular development in the transplanted tissue. Slices from the cat ovarian cortex were frozen and after thawing, transplanted under each kidney capsule of castrated male NOD SCID mice (eight xenografts in four mice). Sixty-two days after surgery, mice were randomly assigned (two per group) to gonadotropin-treated (eCG and hCG 88 h later) or control (saline-treated) groups. Twenty-four hours after the last injection, ovarian tissue was recovered and processed for histology. Fresh ovarian tissue from the same original source was similarly processed. Follicles were counted, measured, and classified as primordial, primary, secondary, or antral. Immunoreactive proliferating cell nuclear antigen (PCNA) stain was used to assess follicle viability. Microscopic examination revealed no evidence of necrosis or fibrosis. The grafts were well-vascularized, with follicles at all stages of development. Numbers of follicles in the transplanted tissue were markedly reduced compared to fresh tissue, with approximately 10% of follicles surviving freezing and transplantation procedures. Growing follicles positive for PCNA were found in all xenografts. Gonadotropin treatment did not alter the proportion of resting to growing follicles or mean follicle diameter by comparison with controls from untreated mice. By contrast, luteinization, but not ovulation, of antral follicles was observed only in grafts from treated mice. In summary, frozen-thawed cat ovarian cortex tissue not only survived xenotransplantation, it also contained follicles able to grow to antral stages. Exogenous gonadotropin treatment in this model resulted in luteinization of antral follicles but enhancement of follicular growth and ovulation did not occur.

BACKGROUND:A definitive need exists to identify a biomarker of embryonic viability. Platelet-activating factor (PAF) production by human embryos is related to pregnancy potential. METHODS:Conditioned embryo culture media were obtained following conventional IVF on day 3, with PAF levels and pregnancy outcomes correlated. RESULTS:Overall pregnancy rate was 68% (17/25) with a mean of 84.1 (+/- 8.5) pmol/l/embryo PAF level. PAF levels ranged from a 216.4 pmol/l/embryo (pregnant) to a 3.7 pmol/l/embryo (not pregnant). There was a significant difference (P < 0.05) in PAF content between pregnant (92.1 +/- 9.5 pmol/l/embryo) and non-pregnant groups (52.5 +/- 16.6 pmol/l/embryo). Patients were categorized into three groups based upon PAF levels: low (< or= 5 pmol/l/embryo); medium (51-100 pmol/l/embryo) and high (>100 pmol/l/embryo). The low (60%) group had a significantly (P < 0.05) lower pregnancy rate than either the medium (85%) or high (89%) groups. A receiver-operator characteristic curve predicted a cut-off limit of 45 pmol/l/embryo for PAF content in human embryo conditioned culture media. CONCLUSIONS:The data demonstrate a correlation between PAF levels in human embryo conditioned culture media and pregnancy outcome. Additionally, as embryonic PAF levels increase so does the corresponding pregnancy rate. Therefore, PAF may be used as an indicator of embryo viability and for predicting pregnancy outcome.

Objective: Historically, embryo transfers following embryo biopsy and preimplantation diagnosis (PGD) were performed on day 3 or 4. With the development of sequential media and blastocyst transfer, patients undergoing PGD now have the option of a day 4 or day 5 transfer. The objective of this study was to compare the pregnancy and implantation rates between patients who received a day 4 or day 5 embryo transfer following PGD for aneuploidy Design: Retrospective Analysis Materials/Methods: Embryo biopsy was performed in Ca2+/Mg2+ -free medium on day 3 and a diagnosis was determined using fluorescent in situ hybridization (FISH) for chromosomes X,Y,13,18 and 21. On the morning of day 4 all biopsied embryos were transferred into blastocyst culture medium. Patients who did not want their embryos cultured until day 5 had an embryo transfer on day 4. Embryos which were not transferred remained in culture until day 6. Results: Blastocyst transfer was offered to 50 patients post preimplantation genetic diagnosis. Twenty-two patients chose to have a day 5 transfer, while the remaining 40 requested to have a day 4 transfer. In the blastocyst transfer group 56% (13/23) of the patients had a positive pregnancy test of which 52% (12/23) are ongoing. The average age in this group was 38 and the implantation rate was 27% (16/59). Patients who received a day 4 embryo transfer had a 33.3%% (9/27) pregnancy rate of which 26% (7/27) are ongoing. The average age in this group was 39 and the implantation rate was 12% (8/66). Conclusions: The removal of a cell or cells from a day 3 embryo for the purpose of PGD does not preclude the subsequent in vitro development of that embryo to the blastocyst stage. The transfer of blastocysts instead of day 4 embryos post PGD may allow the embryo to better withstand the trauma of embryo transfer procedure. In this study blastocysts replaced to the uterus had a higher implantation rate than embryos transferred on day 4. Culture to the blastocyst stage may allow for selection of embryos for ET when multiple chromosomally normal embryos are available, or allow extra time for performing the genetic diagnosis or rechecking results. Many smaller IVF centers cannot justify the cost of equipment needed for PGD, though a micromanipulator is available. Cells removed from embryos can be shipped to a reference laboratory for analysis, allowing 48 hours for reporting results before embryo transfer.

Objective: Human stem cells derived from embryos provide potential cell based therapies for repair of degenerating or damaged tissue. However, there are many ethical concerns surrounding the use of human embryos for stem cell research. Parthenogenetic activation has been studied extensively in invertebrates, amphibians, and mice. Activation occurred in these models following an increased intracellular calcium transient. Inhibitors of protein kinases have also been used sequentially with calcium ionophores to suppress the second meiotic reductional division allowing production of diploid parthenones. The aim of this study was to assess parthenogenetic activation and blastocyst development of human donor oocytes using calcium ionophore and protein kinase inhibitors. Design: Prospective, experimental study utilizing donor oocytes. Materials/Methods: Consented oocyte donors underwent follicular stimulation with rFSH following treatment with either a GnRH agonist or antagonist. Oocyte retrieval occurred 36 hours after HCG (10,000 IU) injection and the cumulus cells were removed with hyaluronidase(80 IU/ml) one hour later. Metaphase II oocytes were treated with 5 uM calcium ionophore A23187 for 5 minutes at 33 degrees Celcius followed by a 3 hour incubation in 1 mM 6 dimethyl -aminopurine (6-DMAP) at 37 degrees. Activated oocytes with one pronucleus were cultured in IVC-1 media for 72 hours and were then moved to Gen-X Blastocyst media for 48 hours. Blastocysts which displayed a large blastocoel cavity with the clear presence of an ICM were hatched with acidified tyrodes and treated with anti-human trophoectoderm antibody followed by guinea pig complement. Treated cells were moved to 4-well dishes which were previously coated with mitotically inactivated murine embryonic feeder cells (ATCC) in Dulbeco's modified Eagle's medium supplented with 20% FBS. Attachment of the inner cell mass cells was evaluated after 24 hours. Results: Seventy-six oocytes were retrieved from five donors. 89% (68/88) were mature, metaphase II oocytes. 59% (40/68) activated following treatment with calcium ionophore and DMAP. 37% (15/40) of the parthenotes progressed to the blastocyst stage and underwent assisted hatching (see photo). Following immunosurgery, 60% (9/15) of the treated blastocysts attached to the feeder cells. Cell masses remained attatched to the feeder cells for 3–7 days before their growth arrested. Conclusions: Activation of human donor oocytes with calcium ionophore and protein kinase inhibitors is an effective method for production of parthenotes displaying the morphological characteristics of in vitro produced blastocysts. Using current methodologies for culture of embryo derived stem cells, blastocysts which underwent immunosurgery attached to feeder cells and remained attached for up to seven days. Genetic analysis of the the parthenotes to determine ploidy is currently underway. This method, as demonstrated in the monkey and mouse, is a very promising approach for the production of stem cell lines and may help to minimimize the ethical concerns surrounding embryonic stem cell research. Supported by: Stemron Corporation; Gaithersburg, MD.