It has been postulated that in some mammalian species male embryos have greater rates of development than do female embryos prior to gonadal differentiation. It has been demonstrated the most progressive embryos produce the highest pregnancy and implantation rates (Langley M, Fertility and Sterility, Sept 2003, Vol. 80, supp 3). Therefore, most ART programs routinely transfer the most progressive embryos whereby resulting in possible sex selection in favor of males. A retrospective analysis of 1127 fresh blastocyst transfers and 235 frozen blastocyst transfers occurring from January 1, 1999 to July 1, 2003. The use of sequential media was utilized for all cycles. Embryos were cultured in from day 0 to day 3 in S1, G1, G1.2 or G1.3 (IVF Science, Vitrolife). Multi-cell embryos were cultured from day 3 to day 6 in S2, G2, G2.2, CCM or G2.3 (IVF Science, Vitrolife). Embryo transfer occurred on day 5 or day 6 depending on blastocyst development. Results:ü To our knowledge this is the largest single clinic report on sex ratios. These findings are not in accordance with similar finding in literature where a trend toward male offspring can be observed following blastocyst transfer. Our results appear no different than the sex ratios observed following in vivo conception in the general population of 105 to 107 males:100 females (Smith GG (2000) Am J Epidemiol 151, 614–619).View Large Image Figure ViewerDownload Hi-res image Download (PPT)

The objective of this study was to compare the incidence of retained embryos when a full column of media is utilized to expel embryos compared with a column of air. A retrospective analysis of excellent quality fresh embryo transfer results from January 1, 2001 through March 31, 2004. A total of 542 patients underwent fresh embryo transfers. There were no mock or practice embryo transfers performed. All embryo transfers were performed on Day 5 or Day 6 using either a Wallace or Cook catheter with excellent quality embryos. From January 1, 2001 to August 7, 2002, embryos were transferred with approximately 15–20 μl of media and expelled with a column of air. Beginning August 8, 2002, embryos were transferred with approximately 15–20 μl of media and expelled with a full column of media. All embryo transfers were performed with an abdominal ultrasound (5 MHz) and full bladder to aid intrauterine placement of the embryo transfer catheter. *, ** P < .05 (vs. Media Column group using Fisher's Exact) Results indicate no significant difference for retained embryos (P= 1.0) and overall pregnancy rate (P= .7139) between transfers performed when a column of air or a column of media was used to expel embryos. Significant difference was noted for clinical pregnancies compared with the media column group. In addition, there was a significant difference in implantation rate when the two groups were compared.

It has been suggested that the presence of agglutinated spermatozoa is suggestive of the existence of an immunological cause of infertility such as the existence of antisperm antibodies. The objective of this study was to determine whether varying degrees of sperm agglutination can be a predictive indicator of positive antisperm antibodies. A retrospective analysis of semen analysis and antisperm antibody results occurring between January 1st, 2002 and March 31st, 2004. A computer assisted semen analysis (CASA) was performed using an IVOS (Hamilton-Thorne) in coordination with every antisperm antibody test performed. Agglutination was evaluated and was given a grade of "Normal", "Slight", "Moderate" or "Heavy" based on degree of motile spermatozoa sticking to each other via head to head, midpiece to midpiece, tail to tail, or mixed (e.g., midpiece to tail). Antisperm antibodies present on the sperm surface were detected by the immunobead test. Presence of greater than 20% binding of Rabbit Anti-Human IgA Immunobeads (Irvine Scientific, 15376) and/or Rabbit Anti-Human IgG Immunobeads (Irvine Scientific, 15375) indicated positive antisperm antibodies. Of 451 analysis, 36 patients tested positive for IgA and/or IgG (7.98%). The majority of patients had either "Normal" or "Slight" agglutination; however, 49 (10.9%) of the patients analyzed had either "Moderate" or "Heavy" agglutination. Of patients with positive antisperm antibodies, no significant difference was observed between patients with "Normal" or "Slight" agglutination 31 of 402 patients (7.7%) and patients with "Moderate" or "Heavy" agglutination 5 of 49 patients (10.2%). Contrary to the WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction that states the presence of agglutination is suggestive of the existence of an immunological cause of infertility, this study demonstrates that degree of agglutination is not a suitable indicator of presence of antisperm antibodies.

The objective of this study was to compare the pregnancy rate resulting from transfers performed with G2.3 (Vitrolife) and Embryo Glue (Vitrolife). A retrospective analysis of fresh embryo transfer results from November 14, 2002 through March 31, 2004. Fresh embryo transfers were performed on a total of 461 patients (Table 1). Within this group, a subset of 228 patients were evaluated in which only excellent quality embryos were utilized for embryo transfer (Table 2). All embryo transfers were scheduled on Day 5 or Day 6 using a Cook echo-genic catheter. No practice embryo transfers were performed prior to actual attempts. In all transfers, a malleable stylet was pre-positioned in the cervical os prior to introduction of the embryo transfer catheter. Prior to transfer, embryos selected were isolated and placed in either G2.3 media or Embryo Glue for 1–2 hours. Embryos were then loaded with approximately 15–20μl of G2.3 media or Embryo Glue and expelled with either a full column of air or media. An abdominal ultrasound (5 MHz) was used to aid the intrauterine placement of the embryo transfer catheter. Patients were requested to present with a full bladder to help with uterine position. ü Results of Table 1 indicate no significant difference in overall pregnancy (P= .9258), clinical pregnancy (P= .9242) or implantation rate (P= .4975) between embryo transfers performed with G2.3 media or Embryo Glue. Evaluation of a subset of patients who received only excellent embryos for transfer (Table 2) was similiar in that no significant difference in overall (P= .4951) or clinical (P= 1.0) pregnancy was noted. In addition, no significant difference in was seen for implantation rate (P= .2827) between the two groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Objective: To evaluate pregnancy and implantation rates following transfer of two blastocyst stage embryos of similar progression on day 5. Design: A retrospective analysis of day 5 embryo transfer occurring between January 1st, 2000 and March 30th, 2003. Materials and Methods: Patients underwent Antagon® (Organon) or Cetrotide® (Serono) antagonist protocols, Lupron® (TAP) agonist protocols, or flare protocols prior to controlled ovarian hyper-stimulation with Follistim® (Organon) or Gonal-F® (Serono). Oocyte retrieval occurred 36–39 hours post Human Chorionic Gonadotropin (hCG) administration. Insemination was performed 38–42 hours post hCG using a two hour co-incubation of sperm with oocytes or sperm injection. Oocytes were individually placed in 50–100μl micro-drops of sequential media (G1.2, G1.3, IVF Science, Vitrolife) under oil (Squibb, Sigma) and cultured overnight. Fertilization was evaluated 18–20 hours after insemination. Fertilized oocytes were placed into fresh 50–100μl micro-drops of sequential media (G1.2, G1.3) and cultured in groups of two under oil to Day 3. Multi-cell embryos were moved to new 50–100μl micro-drops of sequential media (CCM, G2.2, G2.3, IVF Science, Vitrolife) under oil for culture to Day 5 and subsequent blastocyst embryo transfer. Blastocyst were graded using the system of Gardner and Schoolcraft et al. (Fertility and Sterility, 1999). Categories for analysis included two embryos for transfer progressing as follows: 1) early blastocyst, the blastocoel being less than half the volume of the embryo; 2) blastocyst, the blastocoel being greater than half the volume of the embryo; 3) full blastocyst, the blastocoel completely filling the embryo; 4) expanding blastocyst, the blastocoel volume is larger than that of the early embryo and the zona is thinning. Trophectoderm and inner cell mass grades were not evaluated. In all cycles, an abdominal ultrasound (5 MHz) was utilized to assist intrauterine placement of the embryo transfer catheter (Wallace, Cook). A sonogram to determine total gestational sacs and fetal hearts was performed at 4–6 weeks post positive hCG. Results: Tabled 1† P < 0.05 vs. III‡ P < 0.01 vs. I ¶ P < 0.001 vs. I ¶¶ P < 0.0001 vs. I, II † P < 0.05 vs. III ‡ P < 0.01 vs. I ¶ P < 0.001 vs. I ¶¶ P < 0.0001 vs. I, II Conclusion: Rate of blastocyst expansion, regardless of blastocyst inner-cell-mass or trophectoderm grades, can be associated with improved pregnancy and implantation rates. A significant increase in ongoing pregnancy rates can be observed between group 1 (28.3%) and group 4 (60.2%) (Fisher's Exact; P < 0.01). A significant increase in implantation rates was observed between all groups with exception of groups 1 and 2 and groups 2 and 3 where the most dramatic increase in implantation rate was observed between group 4 and groups 1 and 2 (Fisher's Exact; P < 0.0001).

Objective: Reports of high incidence of multiple gestations associated with in-vitro fertilization (IVF) procedures have led many clinics to limit the number of embryos transferred. These data will evaluate pregnancy and implantation rates resulting from elective transfer of one or two blastocyst embryos derived from extended embryo culture. Design: A retrospective analysis of elective single and double embryo transfer results from January 1, 1998 through March 31, 2003. Materials and Methods: Patients underwent Antagon(r) (Organon) or Cetrotide(r) (Serono) antagonist protocols, Lupron(r) (TAP) agonist protocols, or flare protocols prior to controlled ovarian hyper-stimulation with Follistim(r) (Organon) or Gonal-F(r) (Serono). Oocyte retrieval occurred 36–39 hours post Human Chorionic Gonadotropin (hCG) administration. Insemination was performed 38–42 hours post hCG using a two hour co-incubation of sperm with oocytes or sperm injection. Oocytes were individually placed in 50–100μl micro-drops of sequential media (S1, G1.2, G1.3, IVF Science, Vitrolife) under oil (Squibb, Sigma) and cultured overnight. Fertilization was evaluated 18–20 hours after insemination. Fertilized oocytes were placed into fresh 50–100μl micro-drops of sequential media (G1.2, G1.3) and cultured in groups of two under oil to Day 3. Multi-cell embryos were moved to new 50–100μl micro-drops of sequential media (S2, CCM, G2.2, G2.3, IVF Science, Vitrolife) under oil for culture to Day 5 or Day 6 and subsequent blastocyst embryo transfer. Patients were counseled and given the option to transfer one or two embryos irrespective of embryo grading/scoring and have their additional embryos cryopreserved. To assist intrauterine placement of the embryo transfer catheter (Wallace, Cook), an abdominal ultrasound (5 MHz) was utilized. A sonogram to determine total gestational sacs and fetal hearts was performed at 4–6 weeks post positive hCG. Results: Tabled 1 Conclusion: Upon review of results a large number of patients (95.3%) elected to transfer two embryos rather than one. It is remarkable that no significant difference was noted for overall (P=1.002), clinical (P=.1717) and ongoing (P=.4512) pregnancy rates between transfer of one or two embryos. In addition, no significant difference was recorded for implantation rate between the two groups (P=.6523). Similar pregnancy and implantation rates between elective fresh one and two embryo transfer groups warrant serious consideration for single blastocyst transfer in select patient populations and/or when supernumerary embryos are present. Single blastocyst transfer does show a dramatic reduction in multiple gestations.

Objective: Obesity, defined as a body mass index (BMI) >30, has been postulated to be a negative factor in outcome with ART. One possible mechanism in IVF cycles could be a lower number of mature oocytes due to lower circulating levels of hCG prior to egg retrieval. The optimal level of hCG required to maximize oocyte maturation prior to egg retrieval has not been established. The objective of this study was to evaluate prospectively if BMI or weight correlated with pre-egg retrieval serum hCG levels.Design: Prospective evaluation of pre-egg retrieval serum hCG levels and patient’s body weight and body mass index in all patients undergoing IVF.Materials and Methods: This study included eighty patients undergoing IVF who had received 250mcg rec-hCG (Ovidrel) subcutaneously (SQ) for oocyte maturation between 2/19/03 and 4/30/03. Twelve to 14 hours following administration of rec-hCG, serum hCG levels were obtained using the DPC Immulite assay.Results: View Large Image Figure ViewerDownload (PPT)The results were analyzed using a linear regression model. A significant decline in serum hCG levels was observed with increasing BMI (p=0.0034) and body weight (p=.0170).Conclusion: A significant association between increasing BMI or body weight and lower pre-egg retrieval serum hCG levels was observed. A wide range of hCG values was observed in patients, indicating that factors other than BMI or body weight may also play a role in circulating levels of hCG prior to egg retrieval. It is possible that routine use of a higher dose of hCG in patients with increased body mass may lead to improved pregnancy outcome. Objective: Obesity, defined as a body mass index (BMI) >30, has been postulated to be a negative factor in outcome with ART. One possible mechanism in IVF cycles could be a lower number of mature oocytes due to lower circulating levels of hCG prior to egg retrieval. The optimal level of hCG required to maximize oocyte maturation prior to egg retrieval has not been established. The objective of this study was to evaluate prospectively if BMI or weight correlated with pre-egg retrieval serum hCG levels. Design: Prospective evaluation of pre-egg retrieval serum hCG levels and patient’s body weight and body mass index in all patients undergoing IVF. Materials and Methods: This study included eighty patients undergoing IVF who had received 250mcg rec-hCG (Ovidrel) subcutaneously (SQ) for oocyte maturation between 2/19/03 and 4/30/03. Twelve to 14 hours following administration of rec-hCG, serum hCG levels were obtained using the DPC Immulite assay. Results: The results were analyzed using a linear regression model. A significant decline in serum hCG levels was observed with increasing BMI (p=0.0034) and body weight (p=.0170). Conclusion: A significant association between increasing BMI or body weight and lower pre-egg retrieval serum hCG levels was observed. A wide range of hCG values was observed in patients, indicating that factors other than BMI or body weight may also play a role in circulating levels of hCG prior to egg retrieval. It is possible that routine use of a higher dose of hCG in patients with increased body mass may lead to improved pregnancy outcome.

Objective: To compare the pregnancy outcomes following transfer of two blastocyst stage embryos of similar grade, progression and transfer day.Design: A retrospective analysis of blastocyst embryo transfer occurring between January 1st, 2000 and March 31st, 2003.Materials and Methods: Blastocyst for transfer on day 5 or 6 were graded using the system of Gardner and Schoolcraft et al. (Fertility and Sterility, 1999). Categories for analysis were grouped according to grade of blastocyst inner-cell-mass and trophectoderm cells where a combination of "AB" was considered excellent/good and a combination of "BC" was considered fair/poor. Category I included transfer of two good/fair quality blastocyst embryos with a grade of "BC", "CB, or "CC" sorted by progression and transfer day. Category II included transfer of two excellent/good quality blastocyst embryos with a grade of "AA", "AB", "BA", or "BB" sorted by progression and transfer day. Each subcategory was broken down by degree of blastocyst expansion and transfer day as follows: A) day 5 early blastocyst and blastocyst, B) day 5 full blastocyst and expanding blastocyst, and C) day 6 blastocyst transfers.Results: Tabled 1 Tabled 1† P < 0.05 vs. I-C, II-C ‡ P < 0.01 vs. I-A, I-C, II-C P < 0.05 vs. II-AConclusion: A trend toward increased pregnancy and implantation rates were observed when transferring two rapidly progressing blastocyst embryos versus transferring two slower blastocyst embryos in both groups. In addition, the transfer of excellent/good quality blastocyst embryos (category II) showed higher pregnancy and implantation rates when compared to transferring fair/poor quality blastocyst embryos (category I). A significant increase in pregnancy rates were observed when day 5 good/excellent quality progressive blastocyst embryos (II-B) were transferred compared to day 6 fair/poor quality blastocyst embryos (I-C) (Fisher Exact; P < 0.05) and day 6 good/excellent quality, blastocyst embryos (II-C) (Fisher Exact; P < 0.05). Significant improvements in implantation rates were also observed when comparing day 5 good/excellent quality progressive blastocyst embryos (II-B) with all groups except day 5 fair/poor quality progressive embryos where improvements were observed but not significant. Although pregnancy and implantation rates are improved when better quality, more progressive embryos are transferred, successful pregnancies are not uncommon when transferring embryos of retarded development and/or fair to poor quality. Objective: To compare the pregnancy outcomes following transfer of two blastocyst stage embryos of similar grade, progression and transfer day. Design: A retrospective analysis of blastocyst embryo transfer occurring between January 1st, 2000 and March 31st, 2003. Materials and Methods: Blastocyst for transfer on day 5 or 6 were graded using the system of Gardner and Schoolcraft et al. (Fertility and Sterility, 1999). Categories for analysis were grouped according to grade of blastocyst inner-cell-mass and trophectoderm cells where a combination of "AB" was considered excellent/good and a combination of "BC" was considered fair/poor. Category I included transfer of two good/fair quality blastocyst embryos with a grade of "BC", "CB, or "CC" sorted by progression and transfer day. Category II included transfer of two excellent/good quality blastocyst embryos with a grade of "AA", "AB", "BA", or "BB" sorted by progression and transfer day. Each subcategory was broken down by degree of blastocyst expansion and transfer day as follows: A) day 5 early blastocyst and blastocyst, B) day 5 full blastocyst and expanding blastocyst, and C) day 6 blastocyst transfers. Results: Tabled 1 Tabled 1† P < 0.05 vs. I-C, II-C ‡ P < 0.01 vs. I-A, I-C, II-C P < 0.05 vs. II-A † P < 0.05 vs. I-C, II-C ‡ P < 0.01 vs. I-A, I-C, II-C P < 0.05 vs. II-A Conclusion: A trend toward increased pregnancy and implantation rates were observed when transferring two rapidly progressing blastocyst embryos versus transferring two slower blastocyst embryos in both groups. In addition, the transfer of excellent/good quality blastocyst embryos (category II) showed higher pregnancy and implantation rates when compared to transferring fair/poor quality blastocyst embryos (category I). A significant increase in pregnancy rates were observed when day 5 good/excellent quality progressive blastocyst embryos (II-B) were transferred compared to day 6 fair/poor quality blastocyst embryos (I-C) (Fisher Exact; P < 0.05) and day 6 good/excellent quality, blastocyst embryos (II-C) (Fisher Exact; P < 0.05). Significant improvements in implantation rates were also observed when comparing day 5 good/excellent quality progressive blastocyst embryos (II-B) with all groups except day 5 fair/poor quality progressive embryos where improvements were observed but not significant. Although pregnancy and implantation rates are improved when better quality, more progressive embryos are transferred, successful pregnancies are not uncommon when transferring embryos of retarded development and/or fair to poor quality.

Objective: Quality assurance (QA) programs in clinical testing laboratories typically employ analysis of quality control (QC) samples the results of which are interpreted through the use of Levy-Jennings graphs. Through the use of such analysis, sub-optimal function of the testing mechanism can be quickly identified and bring about prompt and appropriate corrective action. Quality assurance (QA) programs in the IVF laboratory have traditionally not employed Levy-Jennings type analysis because of the lack of commercial availability of appropriate QC materials (human eggs or embryos). Design: A modification of Levy-Jennings type analysis was developed in attempt to improve identification of laboratory issues that may not be easily identified through the use of other standard IVF QC processes (e.g. monitoring of equipment function, mouse embryo testing etc.). Materials and Methods: A retrospective evaluation (12 prior months) of fertilization and embryo development in all patients was used to develop “expected values”. The number of eggs fertilized for each consecutive 50 eggs obtained in the laboratory was examined. Similarly, the number of blastocysts produced per 50 consecutive 2PN zygotes was also assessed. The mean value and standard deviation were calculated. A frequency histogram of each parameter was plotted to evaluate for symmetry and probable Gaussian distribution. At present, an incident report and search for corrective action is triggered for two consecutive values of poor fertilization or poor blastulation defined as greater than one standard deviation from the mean. Results: Expected values were established using the mean and SD (standard deviation) of 2001 IVF rates of fertilization and blastulation for both traditional insemination and sperm injection procedures (see Table). Initially, comparison of 2002 fertilization and blastulation rates using the 2SD defined limits produced no consecutive incidents. Given this, our defined limits have been narrowed to 1SD resulting in a hightened awareness of trends or shifts toward lowered rates of fertilization and/or blastulation. Tabled 1 Conclusion: This implementation of parametric statistical analysis appears to be a novel approach to QA an IVF laboratory. A drawback to this type of analysis is that the lack of appropriate QC materials introduces potential variability related to individual patient type. This may cause increased incidence of “false rejection”. Increasing the sample size will decrease false rejection, however, may delay identification of a laboratory problem. Further refinement of sample size and development of rules analogous to “Westgard rejection rules” (commonly employed by clinical testing laboratories) may improve the value of this type of analysis.

Objective: GnRH analogs are widely used and generally considered to be an important part of IVF stimulation protocols. The primary rationale for using these drugs is to prevent the onset of premature LH surge. Studies using GnRH agonists have demonstrated reduced cycle cancellation compared to stimulations utilizing gonadotropins alone. Additionally, most studies have also demonstrated an additional benefit of increased egg numbers per retrieval in patients treated with GnRH agonists in a “long protocol”. This second benefit has been attributed to ovarian suppression and “synchronization of the follicle cohort” prior to exposure to exogenous gonadotropins. This benefit is not present in women treated with conventional GnRH antagonist protocols or flare protocols using GnRH agonists. In these protocols, ovarian stimulation with FSH is initiated either after onset of menses or days after discontinuation of oral contraceptives. Although GnRH antagonist protocols are highly advantageous in certain respects (fewer injections, shorter treatment duration), in contrast to GnRH long protocols, they have not been demonstrated to increase egg numbers per retrieval compared to gonadotropins alone or “short protocol” GnRH agonist use. Design: Retrospective analysis of IVF patients undergoing treatment from October 1st, 2002 to March 31st, 2003. Materials and Methods: Beginning January 1, 2003, the clinic’s standard IVF stimulation protocol (down regulation with Lupron®, TAP) was changed to a modified antagonist protocol. The basic elements of the protocol include the following: 1) oral contraceptive pretreatment for 1–3 weeks beginning with the onset of menses, 2) documentation of ovarian suppression by measurement of serum estradiol while on oral contraceptives, 3) administration of a 3mg dose of Cetrotide® (Serono) on the day following the last oral contraceptive pill, and 4) gonadotropin stimulation with recombinant FSH and micro-dose recombinant hCG (2.5 mcg / day) beginning 4 days following the dose of Cetrotide. Finally, Cetrotide 3mg was administered again when lead follicles exceeded 12 mm in mean diameter. Results: Tabled 1 Conclusion: The use of a GnRH antagonist prior to the initiation of stimulation with gonadotropins yields stimulation parameters nearly identical to stimulation with a traditional GnRH antagonist long protocol. Both protocols allow for ovarian suppression which has been proposed to increase egg numbers at retrieval via synchronization of the follicle cohort. Both protocols effectively prevent the occurrence of a spontaneous LH surge. The GnRH antagonist protocol does give a significant advantage in decreasing substantially the number of injections per cycle as well as the total length of the treatment cycle. Further study is needed to confirm that this modified “double use” of GnRH antagonist does improve egg production as compared to traditional antagonist protocols.

Objective: Recent reports regarding the incidence of biochemical pregnancy and miscarriage rates associated with in-vitro fertilization (IVF) and frozen embryo transfer (FET) procedures have prompted clinics to evaluate the effect of sequential embryo culture on fresh and frozen embryo quality as well as transfer and freezing techniques.Design: A retrospective analysis of embryo transfer results from January 1, 1998 through April 15, 2003.Materials and Methods: IVF patients underwent Antagon(r) (Organon) or Cetrotide(r) (Serono) antagonist protocols, Lupron(r) (TAP) agonist protocols, or flare protocols prior to controlled ovarian hyper-stimulation with Follistim(r) (Organon) or Gonal-F(r) (Serono). Oocyte retrieval and insemination occurred 36–39 and 38–42 hours respectively post Human Chorionic Gonadotropin (hCG) administration. Oocytes were individually placed in 50μl drops of sequential media (S1, G1.2, G1.3, IVF Science, Vitrolife) under oil (Squibb, Sigma) and cultured overnight. Fertilization was evaluated 18–20 hours after insemination. Fertilized oocytes were placed into fresh 50μl drops of sequential media (G1.2, G1.3) and cultured in groups of two under oil to Day 3. Multi-cell embryos were moved to new 50μl drops of sequential media (S2, CCM, G2.2, G2.3, IVF Science, Vitrolife) under oil for culture to Day 5 or Day 6 and subsequent morula or blastocyst embryo transfer. For FET, priority was given to thaw excellent then good quality embryos for culture in 50μl drops of sequential media (S2, CCM, G2.2, G2.3) under oil approximately four hours prior to transfer. Blastocyst embryos of fair, good and excellent quality were generally frozen on Day 5 and 6. All embryos were frozen and thawed using modified glycerol protocols previously described.Results: Tabled 1∗, ∗∗ P < .01; † P < .001 (vs. frozen age group using Fisher's Exact) Tabled 1Conclusion: Review of biochemical pregnancy per positive hCG between fresh and frozen cycles indicate significant difference for age groups <35 Years (P=.0074), 38–40 Years (P=.0039) and >41 Years (P=.0006). No significant difference was noted for age group 35–37 Years. In addition, evaluation of miscarriage per positive hCG between fresh and frozen cycles indicate no significant difference between age groups. The marked increase in biochemical pregnancy with frozen blastocyst embryos may be attributable either to embryo selection for transfer prior to cryopreservation or to the effects of cryopreservation on the embryo. These findings warrant review of pre-freeze and pre-transfer embryo quality. Objective: Recent reports regarding the incidence of biochemical pregnancy and miscarriage rates associated with in-vitro fertilization (IVF) and frozen embryo transfer (FET) procedures have prompted clinics to evaluate the effect of sequential embryo culture on fresh and frozen embryo quality as well as transfer and freezing techniques. Design: A retrospective analysis of embryo transfer results from January 1, 1998 through April 15, 2003. Materials and Methods: IVF patients underwent Antagon(r) (Organon) or Cetrotide(r) (Serono) antagonist protocols, Lupron(r) (TAP) agonist protocols, or flare protocols prior to controlled ovarian hyper-stimulation with Follistim(r) (Organon) or Gonal-F(r) (Serono). Oocyte retrieval and insemination occurred 36–39 and 38–42 hours respectively post Human Chorionic Gonadotropin (hCG) administration. Oocytes were individually placed in 50μl drops of sequential media (S1, G1.2, G1.3, IVF Science, Vitrolife) under oil (Squibb, Sigma) and cultured overnight. Fertilization was evaluated 18–20 hours after insemination. Fertilized oocytes were placed into fresh 50μl drops of sequential media (G1.2, G1.3) and cultured in groups of two under oil to Day 3. Multi-cell embryos were moved to new 50μl drops of sequential media (S2, CCM, G2.2, G2.3, IVF Science, Vitrolife) under oil for culture to Day 5 or Day 6 and subsequent morula or blastocyst embryo transfer. For FET, priority was given to thaw excellent then good quality embryos for culture in 50μl drops of sequential media (S2, CCM, G2.2, G2.3) under oil approximately four hours prior to transfer. Blastocyst embryos of fair, good and excellent quality were generally frozen on Day 5 and 6. All embryos were frozen and thawed using modified glycerol protocols previously described. Results: Tabled 1∗, ∗∗ P < .01; † P < .001 (vs. frozen age group using Fisher's Exact) Tabled 1 ∗, ∗∗ P < .01; † P < .001 (vs. frozen age group using Fisher's Exact) Conclusion: Review of biochemical pregnancy per positive hCG between fresh and frozen cycles indicate significant difference for age groups <35 Years (P=.0074), 38–40 Years (P=.0039) and >41 Years (P=.0006). No significant difference was noted for age group 35–37 Years. In addition, evaluation of miscarriage per positive hCG between fresh and frozen cycles indicate no significant difference between age groups. The marked increase in biochemical pregnancy with frozen blastocyst embryos may be attributable either to embryo selection for transfer prior to cryopreservation or to the effects of cryopreservation on the embryo. These findings warrant review of pre-freeze and pre-transfer embryo quality.

Objective: Reports from several In-Vitro Fertilization (IVF) clinics have documented that pronuclear (PN) and nucleolar orientation may be important to blatulation success. This study was undertaken to compare the incidence of blastulation, transfer and cryopreservation within identified groups of fertilized oocytes.Design: A retrospective analysis of pronuclear development from June 6, 2002 through March 31, 2003.Materials and Methods: Patients underwent Antagon® (Organon) or Cetrotide® (Serono) antagonist protocols, down regulation with the gonadotropin releasing hormone analog, Lupron® (TAP) or flare protocols prior to controlled ovarian hyper-stimulation with Follistim® (Organon) or Gonal-F® (Serono). Oocyte retrieval occurred 36–39 hours post Human Chorionic Gonadotropin (hCG) administration. Insemination was performed 38–42 hours post hCG using a two hour co-incubation of sperm with oocytes or intracytoplasmic sperm injection (ICSI). Oocytes were individually placed in 50 μl micro-drops of sequential media (G1.2, G1.3, IVF Science) under oil (Squibb, Sigma) and cultured overnight. Fertilization was evaluated 18–20 hours after insemination and assigned a grade based on pronuclear orientation and nucleoli orientation. Grading was assigned as follows: 1)TD: PN orientated together in extreme proximity to each other with nucleolar evenly dispersed. 2)TC: PN orientated together in extreme proximity to each other with nucleolar concentrated. 3)TU: PN orientated together in extreme proximity to each other with nucleolar unevenly dispersed. 4)SD: PN orientated separate from each other with nucleolar evenly dispersed. 5)SC: PN orientated separate from each other with nucleolar concentrated. 6) SU: PN orientated separate from each other with nucleolar unevenly dispersed. Fertilized oocytes of the same grade were placed into fresh 50μl micro-drops of sequential media (G1.2, G1.3) and generally cultured in groups of two under oil to Day 3. Multi-cell embryos were moved to new 50μl micro-drops of sequential media (G2.2, G2.3, IVF Science) under oil for culture to Day 5 or Day 6 at which point blastulation success was recorded.Results: Tabled 1∗ P < .01 (vs. TU using Chi-Square); † P < .0001 (vs. TD using Fisher Exact); ‡ P = .01 (vs. SD using Fisher Exact)Conclusion: Analysis of 2389 normally fertilized oocytes resulted in 1427 blastocyst embryos at a rate of 59.7%. Upon evaluation of pronuclear grades, TD(58.0%), TC(70.5%) and TU(57.5%) oocyte groups resulted in a higher percentage of blastocyst embryos compared to the SD(37.5%) group. Limited observations were recorded for the SD, SC and SU groups. Statistical review of these data suggest that fertilized oocytes exhibiting two pronuclei orientated together in extreme proximity to each other and with nucleolar concentrated (TC oocyte group) yields significantly higher blastocyst embryos compared with the TD(Pμ.0001),TU(P = .0018) and SD(P = .01) oocyte groups. Objective: Reports from several In-Vitro Fertilization (IVF) clinics have documented that pronuclear (PN) and nucleolar orientation may be important to blatulation success. This study was undertaken to compare the incidence of blastulation, transfer and cryopreservation within identified groups of fertilized oocytes. Design: A retrospective analysis of pronuclear development from June 6, 2002 through March 31, 2003. Materials and Methods: Patients underwent Antagon® (Organon) or Cetrotide® (Serono) antagonist protocols, down regulation with the gonadotropin releasing hormone analog, Lupron® (TAP) or flare protocols prior to controlled ovarian hyper-stimulation with Follistim® (Organon) or Gonal-F® (Serono). Oocyte retrieval occurred 36–39 hours post Human Chorionic Gonadotropin (hCG) administration. Insemination was performed 38–42 hours post hCG using a two hour co-incubation of sperm with oocytes or intracytoplasmic sperm injection (ICSI). Oocytes were individually placed in 50 μl micro-drops of sequential media (G1.2, G1.3, IVF Science) under oil (Squibb, Sigma) and cultured overnight. Fertilization was evaluated 18–20 hours after insemination and assigned a grade based on pronuclear orientation and nucleoli orientation. Grading was assigned as follows: 1)TD: PN orientated together in extreme proximity to each other with nucleolar evenly dispersed. 2)TC: PN orientated together in extreme proximity to each other with nucleolar concentrated. 3)TU: PN orientated together in extreme proximity to each other with nucleolar unevenly dispersed. 4)SD: PN orientated separate from each other with nucleolar evenly dispersed. 5)SC: PN orientated separate from each other with nucleolar concentrated. 6) SU: PN orientated separate from each other with nucleolar unevenly dispersed. Fertilized oocytes of the same grade were placed into fresh 50μl micro-drops of sequential media (G1.2, G1.3) and generally cultured in groups of two under oil to Day 3. Multi-cell embryos were moved to new 50μl micro-drops of sequential media (G2.2, G2.3, IVF Science) under oil for culture to Day 5 or Day 6 at which point blastulation success was recorded. Results: Tabled 1∗ P < .01 (vs. TU using Chi-Square); † P < .0001 (vs. TD using Fisher Exact); ‡ P = .01 (vs. SD using Fisher Exact) ∗ P < .01 (vs. TU using Chi-Square); † P < .0001 (vs. TD using Fisher Exact); ‡ P = .01 (vs. SD using Fisher Exact) Conclusion: Analysis of 2389 normally fertilized oocytes resulted in 1427 blastocyst embryos at a rate of 59.7%. Upon evaluation of pronuclear grades, TD(58.0%), TC(70.5%) and TU(57.5%) oocyte groups resulted in a higher percentage of blastocyst embryos compared to the SD(37.5%) group. Limited observations were recorded for the SD, SC and SU groups. Statistical review of these data suggest that fertilized oocytes exhibiting two pronuclei orientated together in extreme proximity to each other and with nucleolar concentrated (TC oocyte group) yields significantly higher blastocyst embryos compared with the TD(Pμ.0001),TU(P = .0018) and SD(P = .01) oocyte groups.

Objective: Obesity, defined as a BMI greater than 30, is a growing epidemic in our nation. Obesity is well known to increase the risk of medical disorders such as diabetes and hypertension. This study was designed to evaluate the impact of obesity and morbid obesity (BMI >35) on outcome with IVF. Design: A retrospective analysis of non-donor, IVF patients, less than age 40, undergoing IVF or IVF/ICSI from March 1st, 2000 to March 31st, 2003. Materials and Methods: Patients underwent Antagon(r) (Organon) or Cetrotide(r) (Serono) antagonist protocols, Lupron(r) (TAP) agonist protocols, or flare protocols prior to controlled ovarian hyper-stimulation with Follistim(r) (Organon) or Gonal-F(r) (Serono). Oocyte retrieval occurred 36–39 hours post Human Chorionic Gonadotropin (hCG) administration. Insemination was performed 38–42 hours post hCG using a two hour co-incubation of sperm with oocytes or sperm injection. Fertilization was evaluated 18–20 hours after insemination. Fertilized oocytes were placed into fresh micro-drops of sequential media (G1.2, G1.3) and cultured in groups of two under oil to Day 3. Multi-cell embryos were moved to new micro-drops of sequential media (CCM, G2.2, G2.3, Vitrolife) under oil for culture to Day 5 or Day 6 and subsequent blastocyst embryo transfer. Abdominal ultrasound (5 MHz) was utilized to assist intrauterine placement of the embryo transfer catheter (Wallace, Cook). Results: There was no significant difference in the mean age of the patients in the four groups (range: 33.1 to 33.7 years). Nor was there a difference in the number of patients undergoing ICSI versus IVF. Total gonadotropin consumption was likewise similar between the groups, ranging from 37.4 to 41 amps. There was a trend towards a lower peak estradiol level in the >35 BMI group; however, this was not statistically significant. The fertilization rate was similar between all groups (62.6 to 67.3%). The average number of embryos transferred was similar in all groups (1.9 to 2.0). The ongoing pregnancy rate and implantation rate was significantly lower (Fisher Exact) in the >35 BMI group compared to <25 and 25–29.9 BMI groups. Tabled 1† P < 0.05 vs. BMI >35‡ P < 0.01 vs. BMI >35P < 0.001 vs. BMI >35 † P < 0.05 vs. BMI >35 ‡ P < 0.01 vs. BMI >35 P < 0.001 vs. BMI >35 Conclusion: Patients undergoing IVF or IVF/ICSI with a BMI > 35, have a significantly decreased implantation rate and ongoing pregnancy rate in comparison to obese (BMI 30 -34.9), overweight (BMI 25–25.9) or normal weight (BMI < 25) patients. There was a trend towards decreasing implantation rate with overweight (BMI 25–29.9) and obese patients but this was not statistically significant. Possible etiologies for this decrease in pregnancy rate may include: greater volume of distribution resulting in lower serum levels of FSH/decreased stimulation of the ovaries, lower hCG levels for oocyte maturation prior to retrieval or possible endometrial effects. Counseling patients regarding the impact of weight loss prior to an IVF cycle should become a routine part of the physician/patient discussion.

Objective: Previous reports indicating potential toxic effects of disposable materials and plasticware used with in-vitro fertilization (IVF) procedures have led many clinics to examine and improve quality control practices in the laboratory. This study will evaluate two different brands and types of syringe used in oocyte aspiration procedures. Design: A retrospective analysis of fertilzation and embryo transfer results from August 1, 2002 through September 30, 2002. Materials and Methods: During this trial period, patients underwent Lupron(r) (TAP) agonist protocols, or flare protocols prior to controlled ovarian hyper-stimulation with Follistim(r) (Organon) or Gonal-F(r) (Serono). Oocyte retrieval occurred 36–39 hours post Human Chorionic Gonadotropin (hCG) administration. To facillitate oocyte aspiration, a single lumen needle was used in conjunction with either a Terumo(r) (latex free, thermal plastic synthetic elastomer gasket type) or Airtight(r) (no gasket type) 10cc syringe. Both syringe types passed quality control with mouse embryos prior to use. Insemination was performed 38–42 hours post hCG using a two hour co-incubation of sperm with oocytes or sperm injection. Oocytes were individually placed in 50–100μl micro-drops of sequential media (G1.2, IVF Science, Vitrolife) under oil (Squibb) and cultured overnight. Fertilization was evaluated 18–20 hours after insemination. Fertilized oocytes were placed into fresh 50–100μl micro-drops of sequential media (G1.2) and cultured in groups of two under oil to Day 3. Multi-cell embryos were moved to new 50–100μl micro-drops of sequential media (CCM, G2.2, IVF Science, Vitrolife) under oil for culture to Day 5 or Day 6 and subsequent blastocyst embryo transfer. In all cycles, an abdominal ultrasound (5 MHz) was utilized to assist intrauterine placement of the embryo transfer catheter (Cook). A sonogram to determine total gestational sacs and fetal hearts was performed at 4–6 weeks post positive hCG. Results: Tabled 1 Conclusion: Statistical review (Fisher Exact) of data indicate no significant difference between syringe type for fertilization (P=1.000), clinical pregnancy (P=.7891) and blastulation rates (P=1.000). Although a trend was seen for implantation rate, this was not significant (P=.1961). Evaluation of excellent embryo quality per fertilized oocyte indicated no significant difference (P=.3134) between the Airtight (15.8%) and Terumo (12.1%) groups.

Objective: Culture micro-drop temperature measurements differ substantially from heated stage temperature set points. Extensive monitoring of micro-drop temperature showed while stage temperature set points were 37°C, a significantly lower culture drop temperature was observed. Reasons for this discrepancy may be due to the insulting properties of the culture dish or radiation of heat from the culture dish to the cooler ambient air of the IVF Laboratory and/or the objective lens opening of the microscope. Temperature adjustments were made on each stage by increasing the set points to approximately 41°C and 43°C degrees on each microscope respectively raising the micro-drop temperature to 37°C. This abstract evaluated blastulation rates, fertilization rates, and number of embryos remaining for cryopreservation for IVF and ICSI cases three months before and after temperature adjustment. Design: A retrospective analysis of IVF and ICSI cases performed between October 1, 2001 and April 12, 2002. Materials/Methods: Temperature monitoring and adjustments of all IVF equipment was performed with a GMH 3230 Thermometer (Greisinger) utilizing both micro-submergible and surface temperature probes before the start of all 2002 IVF cases. The heated stage (Research Instruments) temperature setting was adjusted for both IVF inverted microscopes (Nikon) while monitoring culture media micro-drop temperature. Oocytes underwent ICSI with maximum exposure time outside of incubator, on the heated stage being approximately five to seven minutes. Embryos were evaluated at 24 hour intervals from Day 2 to Day 5 or Day 6, with subsequent embryo transfer and freezing at the blastocyst stage. Results: Please refer to table. TableResults:IVF: Before Temperature AdjustmentIVF: After Temperature AdjustmentICSI: Before Temperature AdjustmentICSI: After Temperature Adjustmentn49536765Fertilization Rate:63.23%63.61%70.72%73.12%†P < 0.01 (Fisher's Exact)Blastulation Rate:62.22%69.68%46.88%58.86%Average Number of Embryos Frozen:3.53.31.72.9†P < 0.01 (Fisher's Exact)Percent of 2PN Frozen:38.46%38.06%22.85%35.62%†P < 0.01 (Fisher's Exact)† P < 0.01 (Fisher's Exact) Open table in a new tab Conclusions: It was determined that while each heated stage set point was 37°C, the micro-drop in a culture dish sitting on the heated stage over a period of five to seven minutes temperature was 4°C and 6°C cooler on each inverted microscope stage respectively. While no change in fertilization rate, blastulation rate, or number of embryos frozen was observed in the traditional insemination group, possibly due to the fact of limited exposure outside of the incubator, a significant improvement was observed in blastulation rate and number of embryos frozen in the ICSI group after temperature adjustment. It is hypothesized that increasing the stage temperature to maintain micro-drop temperature close to 37°C decreased incidence of spindle apparatus disruption due to temperature fluctuation, therefore decreasing embryonic aneuploidy and thus increasing blastocyst formation in the ICSI group. Supported by: N/A.

Objective: Stimulation protocols often utilize oral contraceptive pre-treatment prior to initiation of GnRH agonists or antagonists to facilitate scheduling of the IVF cycle. In some cases, this leads to profound suppression of endogenous gonadotropins. Prior investigation has demonstrated that initiation of FSH prior to resolution of this suppression is associated with delayed follicular growth and increased consumption of gonadotropins. It has been proposed that LH deficiency is responsible for this delayed response. Many IVF programs currently employ 'mixed' stimulation protocols using HMG together with FSH preparations in attempt to overcome this problem. Despite this approach, some patients may continue to have a suboptimal response. This is likely due to the short half-life of the LH component of hMG preparations. Recombinant HCG has recently become available. This drug may serve as a source of LH activity with a significantly longer half life and duration of action than the LH component of HMG. This study was undertaken to evaluate the potential benefit of using a daily low dose of recombinant hCG as an adjunct to recombinant FSH treatment for ovulation induction in IVF patients.Design: Prospective trial of egg donor patients undergoing stimulation with recombinant FSH following pre-treatment with oral contraceptives.Materials/Methods: 82 donor cycles were conducted following pretreatment with oral contraceptives. Blood was obtained on the day gonadotropins (recombinant FSH) were initiated for later analysis of LH. After the first 52 consecutive donors, the next 30 were treated with microdose recombinant hCG (Ovidrel) 2.5 ug daily beginning the first day of stimulation. This was continued until follicles reached maturity maintaining serum hCG levels in the 1 to 3 mIU/ml range.Results: Please refer to Table. TableResultsTotal Donors With OvidrilTotal Donors Without OvidrilLH <0.5 With OvidrilLH <0.5 Without OvidrilLH 0.5 With OvidrilLH 0.5 Without Ovidriln305216141438Average Number of Ampules:29.729.333.434.82527.3Average Peak E2:3092.9†P<0.011594.53361.8†P<0.011383.22785.7†P<0.011672Average Number Ova (All Cases):19.8†P<0.0113.621.9†P<0.01131713.9Average Number IVF Ova (non-atretic ova)/IVF 2PN:18.8†P<0.01/ 12.213.5/ 11.121†P<0.01/ 14.815/ 10.417.3‡P<0.05/ 10.413.1/ 8.9Average Number ICSI Ova (MII Ova)/ICSI 2PN:15.9‡P<0.05/ 11.410.2/ 7.916.6‡P<0.05/ 12.48/614/8.311.4/8.9Fertilization: IVF/ ICSI/ Overall64.8%/ 71.7%/ 67.2%68.4%/ 77.5%/ 71.3%70.2%/ 75.2%/ 72.6%69.5%/ 75.0%/ 71.4%60.0%/ 59.5%/ 59.9%62.1%/ 79.5%‡P<0.05/ 71.7%Blastulation: IVF/ ICSI/ Overall70.7%/ 57.7%/ 65.9%64.2%/ 69.6%/ 66.1%81.4%/ 67.9%/ 74.8%48.0%/ 66.7%/ 54.8%63.2%/ 56.0%/ 61.9%69.5%/ 66.1%/ 68.3%Average Number Embryos Frozen per Cycle:5.53.66.6‡P<0.052.74.13.8† P<0.01‡ P<0.05 Open table in a new tab Conclusions: Low dose recombinant hCG during FSH stimulation for IVF increases the ovarian response with a significantly increased peak estradiol, egg number and embryos available for cryopreservation. This increase was most apparent in donors with initially low LH levels (<0.5 mIU/ml), but also seen in donors exhibiting less suppression.Supported by: N/A. Objective: Stimulation protocols often utilize oral contraceptive pre-treatment prior to initiation of GnRH agonists or antagonists to facilitate scheduling of the IVF cycle. In some cases, this leads to profound suppression of endogenous gonadotropins. Prior investigation has demonstrated that initiation of FSH prior to resolution of this suppression is associated with delayed follicular growth and increased consumption of gonadotropins. It has been proposed that LH deficiency is responsible for this delayed response. Many IVF programs currently employ 'mixed' stimulation protocols using HMG together with FSH preparations in attempt to overcome this problem. Despite this approach, some patients may continue to have a suboptimal response. This is likely due to the short half-life of the LH component of hMG preparations. Recombinant HCG has recently become available. This drug may serve as a source of LH activity with a significantly longer half life and duration of action than the LH component of HMG. This study was undertaken to evaluate the potential benefit of using a daily low dose of recombinant hCG as an adjunct to recombinant FSH treatment for ovulation induction in IVF patients. Design: Prospective trial of egg donor patients undergoing stimulation with recombinant FSH following pre-treatment with oral contraceptives. Materials/Methods: 82 donor cycles were conducted following pretreatment with oral contraceptives. Blood was obtained on the day gonadotropins (recombinant FSH) were initiated for later analysis of LH. After the first 52 consecutive donors, the next 30 were treated with microdose recombinant hCG (Ovidrel) 2.5 ug daily beginning the first day of stimulation. This was continued until follicles reached maturity maintaining serum hCG levels in the 1 to 3 mIU/ml range. Results: Please refer to Table. Conclusions: Low dose recombinant hCG during FSH stimulation for IVF increases the ovarian response with a significantly increased peak estradiol, egg number and embryos available for cryopreservation. This increase was most apparent in donors with initially low LH levels (<0.5 mIU/ml), but also seen in donors exhibiting less suppression. Supported by: N/A.

Objective: Frozen embryo transfer of one single embryo has been reported to result in a reduction of multiple gestations with consistent implantation and pregnancy rates. The purpose of this study is to evaluate pregnancy and implantation rates resulting from transfer of one blastocyst embryo derived from frozen cycles.Design: A retrospective analysis of frozen embryo transfer results from January 1, 1998 through March 31, 2002.Materials/Methods: Frozen embryos thawed for transfer were placed into 100l micro-drops of sequential media (S-2, G-2.2 or CCM; IVF Science) under oil and cultured in 5% CO2 and air at 37°C approximately four hours prior to transfer. Embryos were frozen on Day 5, 6 or 7. All embryos were frozen and thawed using modified glycerol protocols previously reported. In preparation for embryo transfer, estrogen and progesterone supplementation was administered to all patients. An abdominal ultrasound (5 MHz) was utilized to assist intrauterine placement of the embryo transfer catheter (Wallace; Cook). A sonogram to determine total gestational sacs and fetal hearts was performed at 4–6 weeks post positive hCG. If cardiac activity was not documented at the initial sonogram, a follow-up sonogram was performed 1 week later.Results: Please refer to the table. TableSingle Frozen Embryo Transfer ResultsElective Frozen Embryo TransferNon-Elective Frozen Embryo TransferTotal Cycles:1348Mean Patient Age:35.635.9Pregnancies (+hCG)/Ongoing:5/216/7Biochemical/Miscarriages:2/15/4Pregnancy Rate (+hCG):38.5% NS33.3%Clinical Pregnancy Rate:23.1% NS22.9%Ongoing Pregnancy Rate:15.4% NS14.6%Clinical Sacs (1/2/3):3/0/011/0/0Implantation Rate:23.1% NS22.9%Not Significant (NS) vs. non-elective frozen embryo transfers Open table in a new tab Conclusions: Of the 61 single frozen embryo transfers, 13 (21.3%) patients requested to transfer one embryo and have their additional embryos remain cryopreserved. The remaining patients utilizing frozen embryos were forced to transfer only one embryo due to the limited availability of supernumerary embryos for transfer. The selection of patients for single embryo transfer was generally based on the non-availability of additional embryos rather than patient choice. Similiar pregnancy and implantation rates are evident. No multiple gestations were noted.Supported by: Not Applicable. Objective: Frozen embryo transfer of one single embryo has been reported to result in a reduction of multiple gestations with consistent implantation and pregnancy rates. The purpose of this study is to evaluate pregnancy and implantation rates resulting from transfer of one blastocyst embryo derived from frozen cycles. Design: A retrospective analysis of frozen embryo transfer results from January 1, 1998 through March 31, 2002. Materials/Methods: Frozen embryos thawed for transfer were placed into 100l micro-drops of sequential media (S-2, G-2.2 or CCM; IVF Science) under oil and cultured in 5% CO2 and air at 37°C approximately four hours prior to transfer. Embryos were frozen on Day 5, 6 or 7. All embryos were frozen and thawed using modified glycerol protocols previously reported. In preparation for embryo transfer, estrogen and progesterone supplementation was administered to all patients. An abdominal ultrasound (5 MHz) was utilized to assist intrauterine placement of the embryo transfer catheter (Wallace; Cook). A sonogram to determine total gestational sacs and fetal hearts was performed at 4–6 weeks post positive hCG. If cardiac activity was not documented at the initial sonogram, a follow-up sonogram was performed 1 week later. Results: Please refer to the table. Not Significant (NS) vs. non-elective frozen embryo transfers Conclusions: Of the 61 single frozen embryo transfers, 13 (21.3%) patients requested to transfer one embryo and have their additional embryos remain cryopreserved. The remaining patients utilizing frozen embryos were forced to transfer only one embryo due to the limited availability of supernumerary embryos for transfer. The selection of patients for single embryo transfer was generally based on the non-availability of additional embryos rather than patient choice. Similiar pregnancy and implantation rates are evident. No multiple gestations were noted. Supported by: Not Applicable.

Objective: The objective of this study was to demonstrate the ability to successfully freeze and thaw embryos twice resulting in successful pregnancy.Design: A retrospective analysis of frozen embryo transfers following two freeze-thaw cycles.Materials/Methods: Frozen multi-cell Day 3 embryos selected for culture to Day 6 (D6) were thawed and placed into 50μl micro-drops of G2.2 or CCM media under oil and cultured three days prior to transfer. All embryos were cultured in 5% CO2 and air at 37°C. Blastocyst (BL) and multi-cell (MC) embryos were frozen and thawed using modified glycerol and propanediol protocols (Testart et al. 1986, Tucker et al. 1997, Menezo et al. 1992, Menezo et al 1996). Supernumerary frozen-thawed embryos were cryopreserved at the blastocyst stage for a second time and subsequently thawed following identical protocols as mentioned above. In preparation for embryo transfer, estrogen and progesterone supplementation was administered to all patients. Transfers were performed with an abdominal ultrasound (5 MHz) to aid intrauterine placement of the embryo transfer catheter (Wallace).Results: Please refer to table. TableResults:PatientNumber of MC Embryos1st Freeze/Thaw2nd Freeze/ThawOutcomeEmbryo Stage: Pre- FreezeEmbryo Stage: Post- ThawEmbryo Culture (hours)Embryo Stage: Re- FreezeEmbryo Stage: Post ThawEmbryo Trans- ferred: (Yes/ No)137C/6C/8C5C/4C/7C72 hrs (D6)3BB/3BB/4BBAtretic/3BB/4BBN/Y/YFull-term Delivery; Male216C6C72 hrs (D6)4BC4BCYBio- chemical Pregnancy336C/7C/8C5C/7C/8C72 hrs (D6)4BC/4BC/4BBAtretic/Atretic/4CCN/N/NN/A427C/8C7C/8C72 hrs (D6)4BB/3BBAtretic/AtreticN/NN/A538C/6C/6C7C/6C/6C72 hrs (D6)3AA/1BC/1CC3BB/Atretic/1CCY/N/YFull-term Delivery; Female Open table in a new tab Conclusions: This data set consisted of 5 patients where a total of 39 MC embryos were initially frozen. Following the first thaw, a total of 9 (23.1%) embryos were transferred, 12 (25.6%) supernumerary embryos were refrozen at the blastocyst stage (D6) and the remaining 18 (46.2%) showed no evident signs of survival and were discarded. Of the 12 embryos refrozen, 5 (41.7%) embryos survived the second thaw and were transferred. Overall blastulation rate from frozen MC embryos was 53.9%. These results demonstrate the ability to refreeze blastocyst stage embryos derived from extended embryo culture of previously frozen day 3 embryos for subsequent thaw and transfer. Refreezing blastocyst stage embryos is of great advantage to the patient and clinician where surplus or supernumerary embryos exist. The alternative to refreeze embryos is of greater potential benefit than transferring excessive number of embryos or discarding viable zygotes.Supported by: N/A. Objective: The objective of this study was to demonstrate the ability to successfully freeze and thaw embryos twice resulting in successful pregnancy. Design: A retrospective analysis of frozen embryo transfers following two freeze-thaw cycles. Materials/Methods: Frozen multi-cell Day 3 embryos selected for culture to Day 6 (D6) were thawed and placed into 50μl micro-drops of G2.2 or CCM media under oil and cultured three days prior to transfer. All embryos were cultured in 5% CO2 and air at 37°C. Blastocyst (BL) and multi-cell (MC) embryos were frozen and thawed using modified glycerol and propanediol protocols (Testart et al. 1986, Tucker et al. 1997, Menezo et al. 1992, Menezo et al 1996). Supernumerary frozen-thawed embryos were cryopreserved at the blastocyst stage for a second time and subsequently thawed following identical protocols as mentioned above. In preparation for embryo transfer, estrogen and progesterone supplementation was administered to all patients. Transfers were performed with an abdominal ultrasound (5 MHz) to aid intrauterine placement of the embryo transfer catheter (Wallace). Results: Please refer to table. Conclusions: This data set consisted of 5 patients where a total of 39 MC embryos were initially frozen. Following the first thaw, a total of 9 (23.1%) embryos were transferred, 12 (25.6%) supernumerary embryos were refrozen at the blastocyst stage (D6) and the remaining 18 (46.2%) showed no evident signs of survival and were discarded. Of the 12 embryos refrozen, 5 (41.7%) embryos survived the second thaw and were transferred. Overall blastulation rate from frozen MC embryos was 53.9%. These results demonstrate the ability to refreeze blastocyst stage embryos derived from extended embryo culture of previously frozen day 3 embryos for subsequent thaw and transfer. Refreezing blastocyst stage embryos is of great advantage to the patient and clinician where surplus or supernumerary embryos exist. The alternative to refreeze embryos is of greater potential benefit than transferring excessive number of embryos or discarding viable zygotes. Supported by: N/A.

Objective: Previous reports have suggested that extended embryo 2culture can be used to increase the implantation rate in unselected In-Vitro Fertilization (IVF) patients. This study was undertaken to see if application of extended embryo culture to all IVF patients combined with a policy of transfer of no more than two embryos in all but the worst prognosis patients could reduce the risk of high order multiple gestation. Design: Analysis of IVF outcome of all IVF cases from January 1, 2000 through December 31, 2000. Materials/Methods: Extended embryo culture in sequential media (G-1.2, G-2.2 or CCM, IVF Science) was applied to all patients undergoing IVF. A maximum of two embryos was transferred in all patients unless multiple poor prognosis factors (maternal age >38, elevated FSH, extremely poor embryo quality or multiple prior failed cycles) were present. In those cases with more than two or more poor prognosis factor, a maximum of three embryos were transferred. Results: Please refer to table. Blastocyst embryo transfer results. Tabled 11 Embryo transferred2 Embryos transferred3 Embryos transferredTotal transferredMean Patient Age:34.334.837.134.9Total Cycles/Embryos Transferred:40/40337/67429/87406/801Pregnancies (+hCG)/ Ongoing:15/6209/15915/10239/175Biochemical/Miscarriages:8/134/162/344/20Clinical/Ongoing Rate:17.5%/15.0%51.9%/46.9%44.8%/34.5%48.0%/42.9%Clinical Sacs (1/2/3):7/0/0104/69/16/6/1117/75/2Fetal Hearts (0/1/2/3/4):0/6/1/0/014/103/52/4/11/7/5/0/015/116/58/4/1Implantation Rate:17.5%25.8%14.9%24.2% Open table in a new tab Conclusions: Extended embryo culture combined with transfer of only two embryos (or three in patients with extreme poor prognosis) can reduce, but not eliminate high order multiple gestation pregnancies. Although no poor prognosis patients with three embryos transferred conceived with greater than a twin pregnancy, a small number of two embryos transfers resulted in high order multiple gestations secondary to monozygotic embryo splitting. Despite this phenomenon, this strategy resulted in a good pregnancy rate with a much lower high order multiple gestation rate than seen in programs using conventional culture techniques in conjunction with a strategy of transferring more embryos (SART data comparison).