- Email: [email protected]
The effect of pre-freeze assisted hatching on post-thaw survival of mouse embryos
using ACS 100 machine. Inhibins A and B were determined by ELISA. The pregnancy rates among patients with serum estradiol concentrations ⬍450 pg/ml were compared with their inhibin A and B concentrations. RESULTS: Eight (8) percent of patients undergoing COH with Antagon had peak estradiol concentrations of ⬍ 450 pg/ml or estradiol/oocyte ⬍130 pg/ml at the time of hCG administration. One-third (33%) of these women became pregnant. All women with peak estradiol ⬍450 pg/ml who became pregnant had inhibin B concentrations ⱖ700 pg/ml and/or inhibin A concentrations ⱖ200 pg/ml. Individuals who had inhibin B/# oocytes between 50 –75 pg/ml had a pregnancy rate of 50%. Women with inhibin A concentrations ⱖ200 had odds ratio for pregnancy of 3.257 (95% conficence interval 1.447–7.33, P⫽0.02). CONCLUSION: Inhibin A and B concentrations at the time of hCG administration may be a helpful tool to the clinician in determining which patients with estradiol levels ⬍450 pg/ml should have their cycle cancelled. Supported by: Grant from Organon Pharmaceuticals USA, Roseland NJ.
P-530 Correlation between the degree of agglutinated spermatozoa and incidence of positive antisperm antibodies. M. T. Langley, D. E. Marek, A. C. Nackley, K. M. Doody, K. J. Doody. Center for Assisted Reproduction, Bedford, TX. OBJECTIVE: 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. DESIGN: A retrospective analysis of semen analysis and antisperm antibody results occurring between January 1st, 2002 and March 31st, 2004. MATERIALS AND METHODS: 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. RESULTS:
CONCLUSION: 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. Supported by: None.
OBJECTIVE: Mitogen-activated protein kinase (MAPK) pathway is one of the most ubiquotus signaling pathways. This pathway is activated by a variety of cellular stimuli and regulates numerous physiological processes. It has been proposed that p38 MAPK functions in the regulation of cytokine and chemokine expression. Given the role of p38 MAPK in cellular inflammatory responses, we aimed to determine whether p38 MAPK pathway is involved in the production of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in endometrial stromal cells. DESIGN: A prospective in vitro study to assess the effect of p38 MAPK inhibition on IL-8 and MCP-1 expression in endometrial stromal cells. MATERIALS AND METHODS: Endometrial tissues were obtained from women undergoing surgery for benign gynecologic conditions. Endometrial stromal cells were isolated and cultured in Ham’s F-12: DME medium that contained 10% fetal bovine serum, and grown to confluence. Experiments were performed after incubating cells in serum-free, phenol red-free medium for 24 h. Statistical analysis of the data was performed using ANOVA. RESULTS: Cells were treated for 24 h with a specific p38 MAPK inhibitor, SB203580 (10 M) or with vehicle (control), and supernatants were collected. IL-8 and MCP-1 levels in culture media were quantified using a specific ELISA for each chemokine. Treatment of cells with p38 MAPK inhibitor resulted in a 37⫾2% (mean⫾SEM; p⬍0.001) decrease in IL-8 levels and a 29⫾2% (mean⫾SEM; p⫽0.003) decrease in MCP-1 levels. CONCLUSION: Inhibition of p38 MAPK signaling pathway significantly inhibited IL-8 and MCP-1 production in endometrial stromal cells. Therefore, we propose that IL-8 and MCP-1 expression in these cells, is at least partially regulated through p38 MAPK pathway. We speculate that this pathway may be involved in the pathogenesis of endometriosis, a proinflammatory disease where high levels of these chemokines are observed. Supported by: None.
REPRODUCTIVE LABORATORY TECHNOLOGY P-532 A retrospective analysis: Culture and transfer of day 6 human blastocysts increases pregnancy rates in patients over 40 years old. H. Lambert, A. K. Lopez, J. Chesmore, K. Vu. Hawaii Center for Reproductive Medicine and Surgery, Kailua, HI. OBJECTIVE: Historically, patients ⱖ40 years of age treated for infertility with IVF have diminished results due to rising FSH levels, poor recruitment of follicles and low response to ovulation induction. Recovery of oocytes have customarily been few and of poor quality. Due to the recent availability and improvement of sequential culture medium for IVF, we wanted to examine the possibility of taking the resulting embryos from these patients out to day 6 (blastocyst stage) to see if these fully developed embryos would have an impact on pregnancy rates. DESIGN: This was a retrospective analysis. MATERIALS AND METHODS: 29 patients, ⱖ40 years of age treated with IVF and subsequent embryo transfer from November 2002 to September 2003 were entered into the study based on the presence of 6 or more “A” quality embryos on day 2 of embryo development. Patients received either 2 or 3 embryos at transfer. RESULTS: Out of the 29 patients that participated in the study, the average age was 41.8 and the average FSH level was 7.45. All 29 patients went to egg retrieval with an average of 12.4 eggs per patient with an average of 3 of those eggs making it to blastocyst. A positive pregnancy test was observed in 20 patients for a 68.9% pregnancy rate. CONCLUSION: We conclude that in our study of patients ⱖ40 years of age, IVF, the day 2 selection criteria, and the use of sequential culture medium can be used to greatly improve the quality of embryos and pregnancy rates in this patient population. Supported by: None
P-531 Inhibition of p38 MAP kinase decreases the production of MCP-1 and IL-8 in endometrial stromal cells. Y. Seval, H. Cakmak, U. A. Kayisli, A. Arici. Dept. of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT; Dept. of Histology and Embryology, Akdeniz University School of Medicine, Antalya, Turkey.
FERTILITY & STERILITY威
P-533 The effect of pre-freeze assisted hatching on post-thaw survival of mouse embryos. A. Hershlag, H. Feng. North shore University Hospital, Manhasset, NY.
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OBJECTIVE: Assisted hatching (AH) has been proposed as a means to increase the implantation rate in poor prognosis patients. The objective of this study was to determine the effect of assisted hatching on mouse embryos cryopreservation, survival rate and post-thaw development. DESIGN: Prospective laboratory study. MATERIALS AND METHODS: Pronuclear stage zygotes (B6C3 F1 female/ B6D2F1 male mice) were obtained from Conception Technology, Inc. (San Diego, CA). Zygotes were randomly divided into two groups in the microdrop (50l) of Quinn’s Advantage Cleavage Medium (SageBiopharm, NJ) containing 10% SPS (SageBiopharm, NJ). The culture dishes were equilibrated in at 5% CO2 and 37oC for 18 hours before introducing the zygotes, which were subsequently cultured and scored for cleavage at 24, 48, 72 hours. After culture 72 hours, the embryos from group 1 underwent assisted hatching with Acidified Medium (SageBiopharm, NJ). The assisted hatching procedure consisted of blowing the acid Tyrode over the external surface of the zona and then creating a 30m diameter defect in the zona, typically at the 3 o’clock position next to an area of empty perivitelline space. Thereafter, hatched (group 1) and non-hatched (group 2) cleaved embryos (6 – 8cells) were cryopreserved using a slow freezing protocol with 1,2-propanediol and sucrose as cryoprotectants. Cooling was conducted in programmable freezers (Kryo 10 Series, Planar, TS, PA) at a rate of -2oC/min to -7oc, at which point seeding was induced manually. Cooling was then continued at rate of -0.3oC/min to -30oC before plunging and storage in LN2. After storage in LN2 for one month, embryos were thawed rapidly by removing cryo-vials from storage, exposure to air for 30 s and immersion in a water bath at 37°C for 3 minutes. PROH was removed by using thaw kit (Medicult, Denmark). Rehydration was completed by transfer to sucrose-free PBS with 20% SPS for 5 min. Embryos were transferred to culture in Quinn’s Advantage Blastocyst Medium with 10 % SPS (SageBiopharm, NJ). The embryos were then evaluated for the incidence of cavitation at blastocyst stage after culture for 120 hours. RESULTS: Results are presented in the following table. CONCLUSION: Assisted hatching prior to cryopreservation does not hinder post thaw embryo development. Additionally, our study may suggest an increase in survival and blastulation in the hatched group when compared to control. Therefore, breaching the integrity of the zona pellucida and directly exposing the embryo to cryoprotectants not only does not result in damaging the embryos, but also may afford them an advantage as they go through the freeze-thaw turmoil. Theoretically, assisted hatching my open up important routes to convey nutrients from the incubating media. Supported by: None
P-534 The effect of temperature and the duration of cryopreservation on human sperm chromatin. S. Pal, A. Varghese, A. Agarwal, A. K. Bhattacharyya. University of Calcutta, Calcutta, India; Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: The present study was carried out to assess the effect of different freezing temperatures and duration of freezing on human sperm chromatin integrity. DESIGN: Prospective study. MATERIALS AND METHODS: Semen samples were obtained from 93 randomly selected men attending the University Clinic, specimens from 5 proven fertile volunteers served as the controls. After preliminary semen analysis, each sample was divided in five aliquots and mixed with commercially available cryopreservation medium (Tardigrade, IMV, France). One aliquot (A) was used for the pre-freeze study of chromatin integrity, two other aliquots (B1, B2) were frozen in a mechanical freezer at -80°C and the last two aliquots (C1, C2) were frozen in liquid nitrogen (-196°C). The
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duration of cryopreservation for aliquots B1 and C1 was 1 week, and 3 months for aliquots B2 and C2. Chromatin cryo-injury was examined under fluorescent microscope using acridine orange. RESULTS: The mean percentage of spermatozoa with intact chromatin in pre-freeze samples (aliquot A) was 90.1 ⫾ 5.9%, it showed no difference from that of whole semen without a cryopreservative. After 1 week of freezing, chromatin integrity reduced to 76.5 ⫾ 7.7% and 81.6 ⫾ 8.1% in aliquots B1 & C1, respectively. It dropped to 69.6 ⫾ 8.6% and 76.3 ⫾ 7.1% for aliquots B2 & C2, respectively, upon prolonged freezing for 3 months. CONCLUSION: The process of freeze-thawing has an adverse effect on human sperm chromatin. The loss of chromatin integrity is more prominent in case of mechanical freezing at -80°C than with liquid nitrogen, both at 1 week as well as after prolonged preservation for three months. Therefore, cryopreservation in liquid nitrogen should be recommended for freezing of semen specimens used for assisted reproduction. Supported by: Indian Council of Medical Research.
P-535 Reductions in volatile organic compounds, aldehydes, and particulate air contaminants in an IVF laboratory by centralized and stand-alone air filtration systems. M. Forman, V. Polanski, P. Horvath, A. Gilligan, D. Rieger. Albany IVF, Fertility and Gynecology, Albany, NY; Alpha Environmental, Jersey City, NJ; LifeGlobal, Guelph, ON, Canada. OBJECTIVE: To determine the effects of a dedicated central air-handling system and a stand-alone air filtration system on volatile organic compounds (VOC), aldehydes, and particulate concentrations in an IVF laboratory. DESIGN: Prospective, descriptive measurements of air quality. MATERIALS AND METHODS: The IVF laboratory at Albany IVF is supplied by a dedicated central air-handling system which includes HEPA filtration, and high-activity charcoal and potassium permanganate filters. The hallway outside of the laboratory is supplied by an unfiltered air system. The central air-handling system in the laboratory was turned off and the air vents and doors to the laboratory were sealed with plastic sheeting. Baseline air samples were then taken in the hallway and the laboratory over 4 hours for the determination of VOC, aldehydes and particulates. A freestanding air-filtration system (Coda Tower, IVFonline/genX) was installed in the laboratory for 24 hours, and the air in the laboratory was again sampled. Air samples were analyzed for VOC by cryoconcentrated gas chromatography/mass spectrometry, and for aldehydes by high-performance liquid chromatography. The size and number of particulates were determined by dispersion of laser light. RESULTS: A total of 28 VOC and 6 aldehydes, were detected in the baseline samples taken from the hallway and the laboratory. Of these, 24 were at lower concentration in the laboratory, 9 were at higher concentration in the laboratory, and one was at the same concentration in the hallway and laboratory. Total baseline particulate concentrations were 2.8 times higher in the laboratory than in the hallway. After 24 hours of operation of the Coda Tower, the concentrations of 14 VOC and 6 aldehydes in the laboratory decreased (range 13 to 100%), the concentrations of 4 were unchanged, and the concentrations of 3 (acetonitrile, dichlorodifluoromethane (CFC 12), and n-heptane) increased. The concentrations of six size classes of particulates in the laboratory decreased (range 37 to 98%) during the 24 hours of operation of the Coda Tower. CONCLUSION: The results indicate that a dedicated central air-handling system can reduce the concentrations of VOC, aldehydes, and particulates in an IVF laboratory. However, the Coda Tower produced further decreases in the concentrations of VOC, aldehydes, and particulates. The increase in the concentration of acetonitrile in the laboratory was probably due to offgassing from the plastic sheeting used to seal the air vents and doors. These results suggest that a stand-alone air treatment system can improve air quality over that produced by a dedicated central air-handling system. Supported by: Financial support was provided by IVFonline/genX International, Guilford, CT.
P-536 Antioxidant effect of pentoxifylline in reducing oxidative stress induced embryotoxicity. X. Zhang, R. K. Sharma, A. Agarwal, T. Falcone. Cleveland Clinic Foundation, Cleveland, OH.
Vol. 82, Suppl. 2, September 2004
P-530 Correlation between the degree of agglutinated spermatozoa and incidence of positive antisperm antibodies. M. T. Langley, D. E. Marek, A. C. Nackley, K. M. Doody, K. J. Doody. Center for Assisted Reproduction, Bedford, TX. OBJECTIVE: 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. DESIGN: A retrospective analysis of semen analysis and antisperm antibody results occurring between January 1st, 2002 and March 31st, 2004. MATERIALS AND METHODS: 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. RESULTS:
CONCLUSION: 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. Supported by: None.
OBJECTIVE: Mitogen-activated protein kinase (MAPK) pathway is one of the most ubiquotus signaling pathways. This pathway is activated by a variety of cellular stimuli and regulates numerous physiological processes. It has been proposed that p38 MAPK functions in the regulation of cytokine and chemokine expression. Given the role of p38 MAPK in cellular inflammatory responses, we aimed to determine whether p38 MAPK pathway is involved in the production of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in endometrial stromal cells. DESIGN: A prospective in vitro study to assess the effect of p38 MAPK inhibition on IL-8 and MCP-1 expression in endometrial stromal cells. MATERIALS AND METHODS: Endometrial tissues were obtained from women undergoing surgery for benign gynecologic conditions. Endometrial stromal cells were isolated and cultured in Ham’s F-12: DME medium that contained 10% fetal bovine serum, and grown to confluence. Experiments were performed after incubating cells in serum-free, phenol red-free medium for 24 h. Statistical analysis of the data was performed using ANOVA. RESULTS: Cells were treated for 24 h with a specific p38 MAPK inhibitor, SB203580 (10 M) or with vehicle (control), and supernatants were collected. IL-8 and MCP-1 levels in culture media were quantified using a specific ELISA for each chemokine. Treatment of cells with p38 MAPK inhibitor resulted in a 37⫾2% (mean⫾SEM; p⬍0.001) decrease in IL-8 levels and a 29⫾2% (mean⫾SEM; p⫽0.003) decrease in MCP-1 levels. CONCLUSION: Inhibition of p38 MAPK signaling pathway significantly inhibited IL-8 and MCP-1 production in endometrial stromal cells. Therefore, we propose that IL-8 and MCP-1 expression in these cells, is at least partially regulated through p38 MAPK pathway. We speculate that this pathway may be involved in the pathogenesis of endometriosis, a proinflammatory disease where high levels of these chemokines are observed. Supported by: None.
REPRODUCTIVE LABORATORY TECHNOLOGY P-532 A retrospective analysis: Culture and transfer of day 6 human blastocysts increases pregnancy rates in patients over 40 years old. H. Lambert, A. K. Lopez, J. Chesmore, K. Vu. Hawaii Center for Reproductive Medicine and Surgery, Kailua, HI. OBJECTIVE: Historically, patients ⱖ40 years of age treated for infertility with IVF have diminished results due to rising FSH levels, poor recruitment of follicles and low response to ovulation induction. Recovery of oocytes have customarily been few and of poor quality. Due to the recent availability and improvement of sequential culture medium for IVF, we wanted to examine the possibility of taking the resulting embryos from these patients out to day 6 (blastocyst stage) to see if these fully developed embryos would have an impact on pregnancy rates. DESIGN: This was a retrospective analysis. MATERIALS AND METHODS: 29 patients, ⱖ40 years of age treated with IVF and subsequent embryo transfer from November 2002 to September 2003 were entered into the study based on the presence of 6 or more “A” quality embryos on day 2 of embryo development. Patients received either 2 or 3 embryos at transfer. RESULTS: Out of the 29 patients that participated in the study, the average age was 41.8 and the average FSH level was 7.45. All 29 patients went to egg retrieval with an average of 12.4 eggs per patient with an average of 3 of those eggs making it to blastocyst. A positive pregnancy test was observed in 20 patients for a 68.9% pregnancy rate. CONCLUSION: We conclude that in our study of patients ⱖ40 years of age, IVF, the day 2 selection criteria, and the use of sequential culture medium can be used to greatly improve the quality of embryos and pregnancy rates in this patient population. Supported by: None
P-531 Inhibition of p38 MAP kinase decreases the production of MCP-1 and IL-8 in endometrial stromal cells. Y. Seval, H. Cakmak, U. A. Kayisli, A. Arici. Dept. of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT; Dept. of Histology and Embryology, Akdeniz University School of Medicine, Antalya, Turkey.
FERTILITY & STERILITY威
P-533 The effect of pre-freeze assisted hatching on post-thaw survival of mouse embryos. A. Hershlag, H. Feng. North shore University Hospital, Manhasset, NY.
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OBJECTIVE: Assisted hatching (AH) has been proposed as a means to increase the implantation rate in poor prognosis patients. The objective of this study was to determine the effect of assisted hatching on mouse embryos cryopreservation, survival rate and post-thaw development. DESIGN: Prospective laboratory study. MATERIALS AND METHODS: Pronuclear stage zygotes (B6C3 F1 female/ B6D2F1 male mice) were obtained from Conception Technology, Inc. (San Diego, CA). Zygotes were randomly divided into two groups in the microdrop (50l) of Quinn’s Advantage Cleavage Medium (SageBiopharm, NJ) containing 10% SPS (SageBiopharm, NJ). The culture dishes were equilibrated in at 5% CO2 and 37oC for 18 hours before introducing the zygotes, which were subsequently cultured and scored for cleavage at 24, 48, 72 hours. After culture 72 hours, the embryos from group 1 underwent assisted hatching with Acidified Medium (SageBiopharm, NJ). The assisted hatching procedure consisted of blowing the acid Tyrode over the external surface of the zona and then creating a 30m diameter defect in the zona, typically at the 3 o’clock position next to an area of empty perivitelline space. Thereafter, hatched (group 1) and non-hatched (group 2) cleaved embryos (6 – 8cells) were cryopreserved using a slow freezing protocol with 1,2-propanediol and sucrose as cryoprotectants. Cooling was conducted in programmable freezers (Kryo 10 Series, Planar, TS, PA) at a rate of -2oC/min to -7oc, at which point seeding was induced manually. Cooling was then continued at rate of -0.3oC/min to -30oC before plunging and storage in LN2. After storage in LN2 for one month, embryos were thawed rapidly by removing cryo-vials from storage, exposure to air for 30 s and immersion in a water bath at 37°C for 3 minutes. PROH was removed by using thaw kit (Medicult, Denmark). Rehydration was completed by transfer to sucrose-free PBS with 20% SPS for 5 min. Embryos were transferred to culture in Quinn’s Advantage Blastocyst Medium with 10 % SPS (SageBiopharm, NJ). The embryos were then evaluated for the incidence of cavitation at blastocyst stage after culture for 120 hours. RESULTS: Results are presented in the following table. CONCLUSION: Assisted hatching prior to cryopreservation does not hinder post thaw embryo development. Additionally, our study may suggest an increase in survival and blastulation in the hatched group when compared to control. Therefore, breaching the integrity of the zona pellucida and directly exposing the embryo to cryoprotectants not only does not result in damaging the embryos, but also may afford them an advantage as they go through the freeze-thaw turmoil. Theoretically, assisted hatching my open up important routes to convey nutrients from the incubating media. Supported by: None
P-534 The effect of temperature and the duration of cryopreservation on human sperm chromatin. S. Pal, A. Varghese, A. Agarwal, A. K. Bhattacharyya. University of Calcutta, Calcutta, India; Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: The present study was carried out to assess the effect of different freezing temperatures and duration of freezing on human sperm chromatin integrity. DESIGN: Prospective study. MATERIALS AND METHODS: Semen samples were obtained from 93 randomly selected men attending the University Clinic, specimens from 5 proven fertile volunteers served as the controls. After preliminary semen analysis, each sample was divided in five aliquots and mixed with commercially available cryopreservation medium (Tardigrade, IMV, France). One aliquot (A) was used for the pre-freeze study of chromatin integrity, two other aliquots (B1, B2) were frozen in a mechanical freezer at -80°C and the last two aliquots (C1, C2) were frozen in liquid nitrogen (-196°C). The
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duration of cryopreservation for aliquots B1 and C1 was 1 week, and 3 months for aliquots B2 and C2. Chromatin cryo-injury was examined under fluorescent microscope using acridine orange. RESULTS: The mean percentage of spermatozoa with intact chromatin in pre-freeze samples (aliquot A) was 90.1 ⫾ 5.9%, it showed no difference from that of whole semen without a cryopreservative. After 1 week of freezing, chromatin integrity reduced to 76.5 ⫾ 7.7% and 81.6 ⫾ 8.1% in aliquots B1 & C1, respectively. It dropped to 69.6 ⫾ 8.6% and 76.3 ⫾ 7.1% for aliquots B2 & C2, respectively, upon prolonged freezing for 3 months. CONCLUSION: The process of freeze-thawing has an adverse effect on human sperm chromatin. The loss of chromatin integrity is more prominent in case of mechanical freezing at -80°C than with liquid nitrogen, both at 1 week as well as after prolonged preservation for three months. Therefore, cryopreservation in liquid nitrogen should be recommended for freezing of semen specimens used for assisted reproduction. Supported by: Indian Council of Medical Research.
P-535 Reductions in volatile organic compounds, aldehydes, and particulate air contaminants in an IVF laboratory by centralized and stand-alone air filtration systems. M. Forman, V. Polanski, P. Horvath, A. Gilligan, D. Rieger. Albany IVF, Fertility and Gynecology, Albany, NY; Alpha Environmental, Jersey City, NJ; LifeGlobal, Guelph, ON, Canada. OBJECTIVE: To determine the effects of a dedicated central air-handling system and a stand-alone air filtration system on volatile organic compounds (VOC), aldehydes, and particulate concentrations in an IVF laboratory. DESIGN: Prospective, descriptive measurements of air quality. MATERIALS AND METHODS: The IVF laboratory at Albany IVF is supplied by a dedicated central air-handling system which includes HEPA filtration, and high-activity charcoal and potassium permanganate filters. The hallway outside of the laboratory is supplied by an unfiltered air system. The central air-handling system in the laboratory was turned off and the air vents and doors to the laboratory were sealed with plastic sheeting. Baseline air samples were then taken in the hallway and the laboratory over 4 hours for the determination of VOC, aldehydes and particulates. A freestanding air-filtration system (Coda Tower, IVFonline/genX) was installed in the laboratory for 24 hours, and the air in the laboratory was again sampled. Air samples were analyzed for VOC by cryoconcentrated gas chromatography/mass spectrometry, and for aldehydes by high-performance liquid chromatography. The size and number of particulates were determined by dispersion of laser light. RESULTS: A total of 28 VOC and 6 aldehydes, were detected in the baseline samples taken from the hallway and the laboratory. Of these, 24 were at lower concentration in the laboratory, 9 were at higher concentration in the laboratory, and one was at the same concentration in the hallway and laboratory. Total baseline particulate concentrations were 2.8 times higher in the laboratory than in the hallway. After 24 hours of operation of the Coda Tower, the concentrations of 14 VOC and 6 aldehydes in the laboratory decreased (range 13 to 100%), the concentrations of 4 were unchanged, and the concentrations of 3 (acetonitrile, dichlorodifluoromethane (CFC 12), and n-heptane) increased. The concentrations of six size classes of particulates in the laboratory decreased (range 37 to 98%) during the 24 hours of operation of the Coda Tower. CONCLUSION: The results indicate that a dedicated central air-handling system can reduce the concentrations of VOC, aldehydes, and particulates in an IVF laboratory. However, the Coda Tower produced further decreases in the concentrations of VOC, aldehydes, and particulates. The increase in the concentration of acetonitrile in the laboratory was probably due to offgassing from the plastic sheeting used to seal the air vents and doors. These results suggest that a stand-alone air treatment system can improve air quality over that produced by a dedicated central air-handling system. Supported by: Financial support was provided by IVFonline/genX International, Guilford, CT.
P-536 Antioxidant effect of pentoxifylline in reducing oxidative stress induced embryotoxicity. X. Zhang, R. K. Sharma, A. Agarwal, T. Falcone. Cleveland Clinic Foundation, Cleveland, OH.
Vol. 82, Suppl. 2, September 2004