- Email: [email protected]
Stability of Human Zonae Pellucidae During Extended Culture In Vitro
All were subjected to history taking, examination, ultrasonography, routine investigations, and finally measurement of vitamin E, beta-carotene, MDA, glutathione peroxidase, and superoxide dismutase. Results: There were statistically significant lower levels of superoxide dismutase, vitamin E, beta-carotene, and glutathione peroxidase (protective antioxidants) in both pregnant and nonpregnant women with history of recurrent abortion than in those with previous normal deliveries. It was found that levels of MDA (indicator of peroxidation) in both pregnant and nonpregnant women with history of recurrent abortion were significantly higher than in the women with previous normal deliveries. Conclusions: There is a beneficial role of antioxidants in maintaining pregnancy and preventing spontaneous abortion. This role is achieved by antagonizing the harmful oxygen free radicals which causes peroxidation. Depletion or deficiency of antioxidant during pregnancy predispose to another abortion. Malonyl dialdehyde is a reliable marker of lipid peroxidation.
bly was equilibrated in an incubator (HeraCell 150) set to 37 C with an environment of 6% CO2 in air. Simultaneously, two 15 mL test tubes with caps loosely in place and containing 3 mL of culture medium were placed within the incubator adjacent to the sensor dish assembly. The medium pH was logged at 5-minute intervals throughout. All samples were at room temp (20 C) when placed in the incubator. After a minimum of 23 hours of equilibration, dishes were removed from the incubator and placed on a 37 C warm plate with the lid removed for 1- and 5-minute intervals. The lids were then replaced and the re-equilibration profiles for each dish were recorded. Results: Equilibrated pH at 23 hours: Sample
pH
Time taken to equilibrate (h)
50 mL well 500 mL well Tube 1 Tube 2
7.34 7.34 7.33 7.35
8.03 9.58 n/a n/a
P-55 Ovarian Reserve Markers Update. T.H. Said, H. Damarawy, A. Warda. University of New Mexico; RMFC, Colorado Springs, CO; Department of Obstetrics and Gynecoogy, Alexandria University, Alexandria Egypt.
Time log for pH of 50 mL and 500 mL medium under oil: 7.7 7.6
pH
7.5 pH 500ul
7.4
pH50ul
7.3 7.2
1 10 0 .2 5 10 .5
9 9. 25 9. 5 9. 75
8
25 8. 5 8. 75
8.
7 7. 25 7. 5 7. 75
6 6. 25 6. 5 6. 75
5
25 5. 5 5. 75
5.
4
25 4. 5 4. 75
4.
3 3. 25 3. 5 3. 75
2 2. 25 2. 5 2. 75
1 25 1. 5 1. 75 1.
0.
0 25 0. 5 0. 75
7.1
Time Log Hrs
Time log for pH of 50 mL under pre-equilibrated oil:
2
3 83 1.
7 66 1.
1. 5
7
1. 33 3
1
16 1.
0. 83 3
0. 5
0. 66 7
0.
16
7
pH
0. 33 3
pH
50µl / 500µl Pre-equilibrated Oil 7.36 7.34 7.32 7.3 7.28 7.26 7.24 7.22 7.2
0
Objective: To compare different markers of ovarian reserve and discuss the merits and the limitation of each of the different methods as an indicator of female fecundity, which is related to the total number of primordial follicles remaining within the ovaries (referred to as ovarian reserve). The goal is to predict a patient’s response to ovulation induction agents and her prognosis for pregnancy. Materials and Methods: This review article discussed the different methods including demographic variables (age, BMI); static measurement, including FSH, estradiol, inhibin-B, and antimullerian hormone; dynamic measurement, including clomid challenge, gonadotophin-releasing hormone, and exogenous follicle-stimulating hormone ovarian reserve tests; ultrasonographic parameters, including antral follicle counting, mean ovarian volume, and Doppler study of ovarian vessels; and ovarian biopsy. Results: This article discusses the causes of limitation of every marker from being a reliable marker for estimating the ovarian reserve. It provides a basis for advising women aged 35 years who are trying to conceive or to postpone childbearing. Conclusions: Currently, there is no reliable test of ovarian reserve for an individual woman that could accurately predict her remaining reproductive life span. Integration of more than one marker improves the results, and repetition of some markers may be needed. More studies have to be done to improve the accuracy and interpretation of the current ovarian reserve markers to state clear cut-off levels for each marker and to find another markers which could correlate more with the number of ova retrieved and clinical pregnancy rate.
Time Log Hrs (5 min log interval)
Re-equilibration times after removal from the incubator for 1 or 5 minutes:
Sample
Time out of incubator Max pH pH Variation
50 mL well
1 min 5 min 500 mL well 1 min
7.36 7.40 7.37
0.04 0.8 0.01
5 min
7.40
0.4
Re-equilibration period (min) 47 64 n/a (within limits of error) 148
P-56 pH Equilibration Dynamics of Culture Medium Under Oil. T. Steel J. Conaghan. MediCult, Napa, CA; Pacific Fertility Center, San Francisco, CA. Background: Achieving the correct pH in gamete and embryo culture media is an essential element of laboratory quality control during in vitro fertilization. Until recently, limitations in pH measuring technology required that measurements were performed under ambient conditions after a period of medium equilibration within an incubator, typically using a relatively large volume of medium in a tube of a size compatible with immersing a pH electrode fully within the medium. This system introduces variation into the results and does not replicate actual culture conditions. Recently, however, a pH measuring technology has become available that permits direct testing of medium within the incubator in a configuration that closely mimics actual culture conditions. Objective: This study was designed to examine a range of pH equilibration dynamics using a commercially available culture medium within an incubator under standard embryo culture conditions. Materials and Methods: A pHOnline ‘‘fluorescent decay time’’ pH meter (MTG Altdorf; Germany) was used to provide ‘‘real time’’ in situ measurement of medium pH under actual culture conditions. The device uses a standard Nunc four-well dish in which one well is fitted with a pH reactive disk (sensor dish). Medium to be tested (EmbryoAssist; MediCult, Denmark) was prepared as either a single 50 mL microdrop or as 500 mL overlaid with 500 mL of prewashed paraffin oil (MediCult). The oil-medium sensor dish assem-
FERTILITY & STERILITYÒ
Conclusions: 1) When using 50 or 500 mL of medium under oil, a minimum of 10 hours’ equilibration is suggested to stabilize at the target pH under normal culture conditions. This time can be reduced to less than 1 hour if preequilibrated oil and medium are used. 2) Removing culture dishes from the incubator for up to 5 minutes has minimal effect on the pH of the culture medium. Equilibration and re-equilibration times were largely unaffected by the volume of medium in the dish.
P-57 Stability of Human Zonae Pellucidae During Extended Culture In Vitro. V. Ivakhnenko, T. Tan, B. Kolb, D. Tourgeman, J. Wilcox, B. Behr. Huntington Reproductive Center, Pasadena, CA. Background: Literature data on the fate of the zona pellucida or its remnants, including intact zonae pellucidae containing cellular debris, empty zonae, and/or zona fragments and unfertilized oocytes from previous nonfertile reproductive cycles observed in the oviducts, uterus, and peritoneal cavity, is available for at least two mammalian species: mice (A. McLaren, 1970) and the little bulldog bat (John J. Rasweiler IV, 1977, 1979) however, no similar data were found for humans. Objective: To investigate effects of the prolonged exposure to standard IVF culture conditions on human zonae pellucidae.
S27
Materials and Methods: Oocytes were retrieved 34–36 hours after an hCG injection following standard controlled ovarian stimulation. Embryos from patients who participated in PGD were cultured in 50 mL drops in Sage cleavage and blastocyst media under oil (Cooper Surgical, Trumbull, CT) at a temperature of 37.0 C and atmosphere of 5% CO2. Embryos with more than 4 blastomeres on day 3 after insemination were biopsied with the assistance of the noncontact 1.48-mm infrared diode Zilos (Hamilton Biosciences Research, Beverly, MA). After hatching, the resulting blastocysts were transfered to the recipients or disposed of according to institutional policies. As a result, a total of 27 empty zonae pellucidae (EZP) were obtained and left for observation in the original culture conditions. EZP were observed at 2to 4-week intervals during a period of 10 to 14 months under 200 magnification light microscopy and photographed. The age of the oldest group of 3 EZP under observation was 14 months from the time of hatching. Results: All 27 (100%) of empty zonae pellucidae remained unchanged throughout 14 months of exposure to the standard IVF culture conditions. Conclusions: The absence of any changes on the light microscopy level during 14 months demonstrated high stability of human zonae pellucidae. As a result the evidence of immunoreactivity of the zona pellucida components, knowledge of the mechanisms, and duration of human zonae pellucidae digestion within the reproductive tract or peritoneal cavity is important in understanding of some of the causes of infertility, such as autoimmune ovarian failure. This study for the first time contributes data on the significant structural stability of human zona pellucida, though only in in vitro conditions. Support: Huntington Reproductive Center. P-58 Application of Custom-Made Electrofusion Pipettes in Mouse Somatic Cell Nuclear Transfer. Y. Shu, R. Rodriguez, S. Kim, B. Behr. IVF Program, Department of Obstetrics and Gynecology and Department of Developmental Biology, Stanford University, Stanford, CA. Introduction: Electrical fusion has become the method of choice for mammalian somatic cell nuclear transfer (SCNT). However, the fusion rate is not satisfactory when the somatic cell and oocyte are disoriented in the electric field and/or when there is little or no cell-to-cell contact. The distance of the two fixed electrodes of the commonly used electrofusion chamber for SCNT is 0.5 cm, which makes it difficult to clearly visualize the contact surface between somatic cell and ooocyte under the dissection microscope, owing to the small size of somatic cell. To circumvent this limitation, two custommade microelectrodes of 100 mm diameter (Midatlantic, NJ) was used to help contact surface between somatic cell and the enucleated oocyte to parallel to the electrode surface. Materials and Methods: Mouse (B6C3F1) superovulation was performed by injection of 5 IU PMSG and 5 IU hCG 48 h apart interperitoneally. Fourteen to seventeen hours after hCG injection, the cumulus-oocyte complexes (COCs) were collected from oviducts and treated in 40 IU/mL hyaluronidase
S28
PCRS Abstracts
to remove cumulus cells. A slit was made on the zona pellucida by using partial zona dissection before enucleation. After incubation for 5 min in 5 mg/mL cytochalasin-B (CB), the enucleation pipette (20 mm OD) was inserted into the oocyte through the slit and the spindle apparatus was aspirated with a minimal volume of cytoplasm. Fibroblast was used as nuclei donor, and one fibroblast was inserted into the perivetilline space of the enucleated oocyte under the zona. The cell-oocyte couplets were then transfered to electrical fusion medium (0.30 mol/L mannitol, 0.1 mmol/L MgCl2, 0.1 mmol/L CaCl2, 0.5 mmol/L HEPES, 1% BSA). For the conventional electrofusion chamber, each couplet was manually aligned with a fine pipette under dissection microscope so that the contact surface between the oocyte and the donor cell was parallel to the electrodes. For custom-made electrofusion pipettes, alignment was performed under 400 magnification of inverted microscope. Cell fusion was induced with 2 electrical pulses of 1.8-2.0 KV/cm for 30 ms using a BTX ECM 2001. Assessment of fusion was assessed 30 min later. Fused oocytes were then incubated in 10 mmol/L SrCl2 and 5 mg CB for activation 2 h after electric stimulation. Embryo development was recorded 4 days after nuclear transfer. Results: Similar fusion rates were obtained by using conventional electrofusion chamber (56.1%, 37/66) and electrofusion pipettes (51.0%, 21/41). No significant difference was observed in terms of cleavage (81.8% [54/66] vs. 85% [20/40]). Similar proportions of fused embryos appeared to develop to morula and blastocyst stages (pending). Conclusions: Although no further beneficial results were obtained in this study, owing to the small sample size of oocytes, preliminary results showed that electrofusion pipette allows for better visualization of membrane contact between somatic cell and oocyte. Further study is needed to verify if the electrofusion pipette increases rates of nuclear fusion and embryonic development after nuclear transfer.
Vol. 89, Suppl 2, April 2008
bly was equilibrated in an incubator (HeraCell 150) set to 37 C with an environment of 6% CO2 in air. Simultaneously, two 15 mL test tubes with caps loosely in place and containing 3 mL of culture medium were placed within the incubator adjacent to the sensor dish assembly. The medium pH was logged at 5-minute intervals throughout. All samples were at room temp (20 C) when placed in the incubator. After a minimum of 23 hours of equilibration, dishes were removed from the incubator and placed on a 37 C warm plate with the lid removed for 1- and 5-minute intervals. The lids were then replaced and the re-equilibration profiles for each dish were recorded. Results: Equilibrated pH at 23 hours: Sample
pH
Time taken to equilibrate (h)
50 mL well 500 mL well Tube 1 Tube 2
7.34 7.34 7.33 7.35
8.03 9.58 n/a n/a
P-55 Ovarian Reserve Markers Update. T.H. Said, H. Damarawy, A. Warda. University of New Mexico; RMFC, Colorado Springs, CO; Department of Obstetrics and Gynecoogy, Alexandria University, Alexandria Egypt.
Time log for pH of 50 mL and 500 mL medium under oil: 7.7 7.6
pH
7.5 pH 500ul
7.4
pH50ul
7.3 7.2
1 10 0 .2 5 10 .5
9 9. 25 9. 5 9. 75
8
25 8. 5 8. 75
8.
7 7. 25 7. 5 7. 75
6 6. 25 6. 5 6. 75
5
25 5. 5 5. 75
5.
4
25 4. 5 4. 75
4.
3 3. 25 3. 5 3. 75
2 2. 25 2. 5 2. 75
1 25 1. 5 1. 75 1.
0.
0 25 0. 5 0. 75
7.1
Time Log Hrs
Time log for pH of 50 mL under pre-equilibrated oil:
2
3 83 1.
7 66 1.
1. 5
7
1. 33 3
1
16 1.
0. 83 3
0. 5
0. 66 7
0.
16
7
pH
0. 33 3
pH
50µl / 500µl Pre-equilibrated Oil 7.36 7.34 7.32 7.3 7.28 7.26 7.24 7.22 7.2
0
Objective: To compare different markers of ovarian reserve and discuss the merits and the limitation of each of the different methods as an indicator of female fecundity, which is related to the total number of primordial follicles remaining within the ovaries (referred to as ovarian reserve). The goal is to predict a patient’s response to ovulation induction agents and her prognosis for pregnancy. Materials and Methods: This review article discussed the different methods including demographic variables (age, BMI); static measurement, including FSH, estradiol, inhibin-B, and antimullerian hormone; dynamic measurement, including clomid challenge, gonadotophin-releasing hormone, and exogenous follicle-stimulating hormone ovarian reserve tests; ultrasonographic parameters, including antral follicle counting, mean ovarian volume, and Doppler study of ovarian vessels; and ovarian biopsy. Results: This article discusses the causes of limitation of every marker from being a reliable marker for estimating the ovarian reserve. It provides a basis for advising women aged 35 years who are trying to conceive or to postpone childbearing. Conclusions: Currently, there is no reliable test of ovarian reserve for an individual woman that could accurately predict her remaining reproductive life span. Integration of more than one marker improves the results, and repetition of some markers may be needed. More studies have to be done to improve the accuracy and interpretation of the current ovarian reserve markers to state clear cut-off levels for each marker and to find another markers which could correlate more with the number of ova retrieved and clinical pregnancy rate.
Time Log Hrs (5 min log interval)
Re-equilibration times after removal from the incubator for 1 or 5 minutes:
Sample
Time out of incubator Max pH pH Variation
50 mL well
1 min 5 min 500 mL well 1 min
7.36 7.40 7.37
0.04 0.8 0.01
5 min
7.40
0.4
Re-equilibration period (min) 47 64 n/a (within limits of error) 148
P-56 pH Equilibration Dynamics of Culture Medium Under Oil. T. Steel J. Conaghan. MediCult, Napa, CA; Pacific Fertility Center, San Francisco, CA. Background: Achieving the correct pH in gamete and embryo culture media is an essential element of laboratory quality control during in vitro fertilization. Until recently, limitations in pH measuring technology required that measurements were performed under ambient conditions after a period of medium equilibration within an incubator, typically using a relatively large volume of medium in a tube of a size compatible with immersing a pH electrode fully within the medium. This system introduces variation into the results and does not replicate actual culture conditions. Recently, however, a pH measuring technology has become available that permits direct testing of medium within the incubator in a configuration that closely mimics actual culture conditions. Objective: This study was designed to examine a range of pH equilibration dynamics using a commercially available culture medium within an incubator under standard embryo culture conditions. Materials and Methods: A pHOnline ‘‘fluorescent decay time’’ pH meter (MTG Altdorf; Germany) was used to provide ‘‘real time’’ in situ measurement of medium pH under actual culture conditions. The device uses a standard Nunc four-well dish in which one well is fitted with a pH reactive disk (sensor dish). Medium to be tested (EmbryoAssist; MediCult, Denmark) was prepared as either a single 50 mL microdrop or as 500 mL overlaid with 500 mL of prewashed paraffin oil (MediCult). The oil-medium sensor dish assem-
FERTILITY & STERILITYÒ
Conclusions: 1) When using 50 or 500 mL of medium under oil, a minimum of 10 hours’ equilibration is suggested to stabilize at the target pH under normal culture conditions. This time can be reduced to less than 1 hour if preequilibrated oil and medium are used. 2) Removing culture dishes from the incubator for up to 5 minutes has minimal effect on the pH of the culture medium. Equilibration and re-equilibration times were largely unaffected by the volume of medium in the dish.
P-57 Stability of Human Zonae Pellucidae During Extended Culture In Vitro. V. Ivakhnenko, T. Tan, B. Kolb, D. Tourgeman, J. Wilcox, B. Behr. Huntington Reproductive Center, Pasadena, CA. Background: Literature data on the fate of the zona pellucida or its remnants, including intact zonae pellucidae containing cellular debris, empty zonae, and/or zona fragments and unfertilized oocytes from previous nonfertile reproductive cycles observed in the oviducts, uterus, and peritoneal cavity, is available for at least two mammalian species: mice (A. McLaren, 1970) and the little bulldog bat (John J. Rasweiler IV, 1977, 1979) however, no similar data were found for humans. Objective: To investigate effects of the prolonged exposure to standard IVF culture conditions on human zonae pellucidae.
S27
Materials and Methods: Oocytes were retrieved 34–36 hours after an hCG injection following standard controlled ovarian stimulation. Embryos from patients who participated in PGD were cultured in 50 mL drops in Sage cleavage and blastocyst media under oil (Cooper Surgical, Trumbull, CT) at a temperature of 37.0 C and atmosphere of 5% CO2. Embryos with more than 4 blastomeres on day 3 after insemination were biopsied with the assistance of the noncontact 1.48-mm infrared diode Zilos (Hamilton Biosciences Research, Beverly, MA). After hatching, the resulting blastocysts were transfered to the recipients or disposed of according to institutional policies. As a result, a total of 27 empty zonae pellucidae (EZP) were obtained and left for observation in the original culture conditions. EZP were observed at 2to 4-week intervals during a period of 10 to 14 months under 200 magnification light microscopy and photographed. The age of the oldest group of 3 EZP under observation was 14 months from the time of hatching. Results: All 27 (100%) of empty zonae pellucidae remained unchanged throughout 14 months of exposure to the standard IVF culture conditions. Conclusions: The absence of any changes on the light microscopy level during 14 months demonstrated high stability of human zonae pellucidae. As a result the evidence of immunoreactivity of the zona pellucida components, knowledge of the mechanisms, and duration of human zonae pellucidae digestion within the reproductive tract or peritoneal cavity is important in understanding of some of the causes of infertility, such as autoimmune ovarian failure. This study for the first time contributes data on the significant structural stability of human zona pellucida, though only in in vitro conditions. Support: Huntington Reproductive Center. P-58 Application of Custom-Made Electrofusion Pipettes in Mouse Somatic Cell Nuclear Transfer. Y. Shu, R. Rodriguez, S. Kim, B. Behr. IVF Program, Department of Obstetrics and Gynecology and Department of Developmental Biology, Stanford University, Stanford, CA. Introduction: Electrical fusion has become the method of choice for mammalian somatic cell nuclear transfer (SCNT). However, the fusion rate is not satisfactory when the somatic cell and oocyte are disoriented in the electric field and/or when there is little or no cell-to-cell contact. The distance of the two fixed electrodes of the commonly used electrofusion chamber for SCNT is 0.5 cm, which makes it difficult to clearly visualize the contact surface between somatic cell and ooocyte under the dissection microscope, owing to the small size of somatic cell. To circumvent this limitation, two custommade microelectrodes of 100 mm diameter (Midatlantic, NJ) was used to help contact surface between somatic cell and the enucleated oocyte to parallel to the electrode surface. Materials and Methods: Mouse (B6C3F1) superovulation was performed by injection of 5 IU PMSG and 5 IU hCG 48 h apart interperitoneally. Fourteen to seventeen hours after hCG injection, the cumulus-oocyte complexes (COCs) were collected from oviducts and treated in 40 IU/mL hyaluronidase
S28
PCRS Abstracts
to remove cumulus cells. A slit was made on the zona pellucida by using partial zona dissection before enucleation. After incubation for 5 min in 5 mg/mL cytochalasin-B (CB), the enucleation pipette (20 mm OD) was inserted into the oocyte through the slit and the spindle apparatus was aspirated with a minimal volume of cytoplasm. Fibroblast was used as nuclei donor, and one fibroblast was inserted into the perivetilline space of the enucleated oocyte under the zona. The cell-oocyte couplets were then transfered to electrical fusion medium (0.30 mol/L mannitol, 0.1 mmol/L MgCl2, 0.1 mmol/L CaCl2, 0.5 mmol/L HEPES, 1% BSA). For the conventional electrofusion chamber, each couplet was manually aligned with a fine pipette under dissection microscope so that the contact surface between the oocyte and the donor cell was parallel to the electrodes. For custom-made electrofusion pipettes, alignment was performed under 400 magnification of inverted microscope. Cell fusion was induced with 2 electrical pulses of 1.8-2.0 KV/cm for 30 ms using a BTX ECM 2001. Assessment of fusion was assessed 30 min later. Fused oocytes were then incubated in 10 mmol/L SrCl2 and 5 mg CB for activation 2 h after electric stimulation. Embryo development was recorded 4 days after nuclear transfer. Results: Similar fusion rates were obtained by using conventional electrofusion chamber (56.1%, 37/66) and electrofusion pipettes (51.0%, 21/41). No significant difference was observed in terms of cleavage (81.8% [54/66] vs. 85% [20/40]). Similar proportions of fused embryos appeared to develop to morula and blastocyst stages (pending). Conclusions: Although no further beneficial results were obtained in this study, owing to the small sample size of oocytes, preliminary results showed that electrofusion pipette allows for better visualization of membrane contact between somatic cell and oocyte. Further study is needed to verify if the electrofusion pipette increases rates of nuclear fusion and embryonic development after nuclear transfer.
Vol. 89, Suppl 2, April 2008