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thawing and function of human sperm: sperm that remain motile also maintain their plasma membrane integrity and hyaluronic acid binding properties
Design: PAF content was measured and correlated with semen analysis parameters (WHO, 1999). Materials/Methods: Human semen was obtained from sexually mature males (n⫽43) and cell counts, percent motilities and morphologies were recorded prior to PAF measurement by radioimmunoassay. Results: The overall mean (⫹ SEM) PAF content was 4.31 (⫹0.97) pM/106 spermatozoa. Linear regression analysis revealed a positive and significant relationship between PAF content in sperm and percent motility (R⫽0.46; p ⬍0.01) as well as percent normal forms (R⫽0.33; p ⬍0.05). Receiver-operator curve analysis revealed that sperm containing 2.5 picomoles/106 sperm exhibited ⬎50% motility with a sensitivity of 0.31, a specificity of 0.36, a positive predictive value of 0.50 and a negative predictive value of 0.20. Receiver-operator curve analysis revealed that sperm containing 3.5 picomoles/106 sperm exhibited ⬎28% normal morphology with a sensitivity of 0.30, a specificity of 0.44, a positive predictive value of 0.47 and a negative predictive value of 0.27. Conclusions: There is a positive, direct correlation between spermatozoan motility, normal morphology and PAF content. Receiver-operator characteristic curve analysis revealed that PAF content alone does not accurately predict spermatozoan motility or normal morphology. Additional studies will elucidate the role of PAF in sperm functionality and the significance PAF plays in human fertility. Supported by: This study was supported by Reproductive Biology Associates, Atlanta, GA, USA.
P-297 Cryopreservation/thawing and function of human sperm: Sperm that remain motile also maintain their plasma membrane integrity and hyaluronic acid binding properties. Ciler Celik-Ozenci, Sevil Cayli, Jill Stronk, Lynne Vigue, Ramazan Demir, Gabor Huszar. The Sperm Phys Lab, Dept of Ob/Gyn, Yale Sch of Medicine, New Haven, CT. Objective: During human spermiogenesis, simultaneously with cytoplasmic extrusion and the major expression of the HspA2 chaperone protein, there is a remodeling of the sperm plasma membrane, which facilitates the formation of zona pellucida- and hyaluronic acid(HA)-binding sites (Huszar et al BOR 1997 and 2000). Mature sperm bind to the zona pellucida and to HA-coated surfaces, whereas immature sperm with cytoplasmic retention exhibit diminished binding. In the present work using the sperm-HA binding assay, we investigated the functional integrity of the sperm plasma membrane after sperm cryopreservation and thawing. This issue is of interest from the points of view of cryobiology and the preservation of sperm function in cryopreserved sperm. Design: Comparison of sperm HA-binding before and after cryopreservation. Materials/Methods: We utilized modified Cell-VUTM double chamber slides. The A chamber was used for determination of motile sperm concentration, whereas the B chamber was coated with HA for the sperm binding assay (Biocoat Inc., PA). HA-binding was scored after placing a drop of semen in the chamber for 10 minutes (equilibrium is reached at 6 –7 minutes). HA binding is expressed as % bound sperm/total motile sperm. In each sample, the fresh semen and their swim-up fractions (in HTF) were studied, and another aliquot of semen and swim-up fractions were cryopreserved in egg yolk-free medium (1:1 ratio, 5% final cc. of glycerol), and were thawed (37oC ⫻ 10 min) after one week. In the fresh samples motile sperm concentration, HA-binding, and degree of maturity reflected by cytoplasmic retention (expressed as CK-B immunocytochemistry darkness factor, DF) were determined. In the thawed fractions motile sperm concentration and HA-binding were studied. Data analysis was carried out with SigmaStat. Results: We studied 30 men (sperm conc: 86 ⫾ 12 ⫻ 106 sperm/ml, and motility: 50.5 ⫾ 2.9%, all data mean ⫾ SEM). By comparing the semen and swim-up sperm, we studied sperm fractions of different maturities, as indicated by the DF (Table). Accordingly, in the swim-up fractions the HA-binding scores were higher. Sperm motility, as expected, was lower in the thawed sperm. However, HA binding scores of thawed sperm were similar in both the semen and the swim-up fractions. Thus, sperm that remain motile also maintain their plasma membrane integrity and HA binding ability. The DF and HA-binding were related (semen: r ⫽ ⫺0.55, p ⫽ 0.001; swim up: r ⫽ ⫺0.47, p ⬍0.01), reflecting the spermiogenetic connection between cytoplasmic extrusion and plasma membrane remodeling.
FERTILITY & STERILITY威
DF initial
Motility (%) initial
Binding (%) initial
Motility (%) thawed
Binding (%) thawed
Semen sperm 52.8 ⫾ 4.1 50.5 ⫾ 2.4 87.3 ⫾ 2.4 27 ⫾ 2.4 86.1 ⫾ 3.5 P value P ⫽ 0.002 P ⬍ 0.001 P ⫽ 0.005 P ⬍ 0.001 P ⫽ 0.15 Swim-up sperm 34.6 ⫾ 3.9 78.5 ⫾ 3.0 93.1 ⫾ 2.0 19.5 ⫾ 2.0 88.3 ⫾ 3.2
Conclusions: The sperm HA-binding scores were unchanged between the fresh and thawed samples, whether in semen or in the swim-up fractions. This indicates that those spermatozoa that retain motility also retain their functional integrity in HA (and presumably zona pellucida) binding properties. HA binding provides an objective measure of sperm function in cryopreserved/thawed samples, and also allows testing of sperm for reproductive toxicity at a remote laboratory after shipping. Supported by: (Supp. HD-19505, HD-32902 and OH-04061).
P-298 A novel microfluidic device for separating motile sperm from nonmotile sperm via inter-streamline crossing. Timothy G. Schuster, Brenda Cho, Laura M. Keller, Dana A. Ohl, Shuichi Takayama, Gary D. Smith. Univ of Michigan, Ann Arbor, MI. Objective: To assess if motile sperm could be separated from nonmotile sperm and non-gamete cells using a novel microfluidic device. Design: Laboratory study. Materials/Methods: A microfluidic device was designed with 2 parallel laminar flow channels where nonmotile sperm and debris would flow along their initial streamlines and exit one outlet whereas motile sperm had an opportunity to swim into a parallel stream and exit a separate outlet. Overnight sperm motility of density gradient prepared sperm samples (N⫽5) with and without exposure to polydimethylsiloxane (PDMS) used to construct the device was assessed. Ten density gradient prepared samples containing 5 million motile sperm/ml were placed in thin inlet channels while processing media was placed in wider parallel channels. Sperm (N⫽200) in the inflow and parallel stream outflow channels were evaluated for motility. Sperm samples (N⫽10) were also placed in wider channels to assess if switching channels affected sorting ability. Lastly, unprocessed semen samples (N⫽10) were placed in wider channels and sperm motility and strict Kruger morphology were assessed from sorted outlets. Results: Compared to initial sperm motility (75% ⫹/⫺ 5.6%; Mean ⫹/⫺ S.E.), overnight sperm motility was significantly lower for samples with and without exposure to PDMS (64% ⫹/⫺ 3.9% and 69% ⫹/⫺ 4.6%; p ⬍0.05, respectively). No difference was observed between overnight motilities (P ⬎0.05). Sperm motility for samples placed in thin inlet channels increased from 74% ⫹/⫺ 2.6% to 96% ⫹/⫺ 0.9% (p ⬍0.05). Placing sperm in wider channels increased motility from 58% ⫹/⫺ 3.9% to 98% ⫹/⫺ 0.6% (p ⬍0.05). Unprocessed sperm motility increased from 44% ⫹/⫺ 4.5% to 98% ⫹/⫺ 0.4% (p ⬍0.05) and morphology increased from 10% ⫹/⫺ 1.05% to 22% ⫹/⫺ 3.3% (p ⬍0.05) following microfluidic inter-streamline crossing isolation. Conclusions: We have demonstrated inter-streamline crossing can be used for isolating human sperm. This microfluidic device provides a novel method for isolating motile, morphologically normal sperm from semen samples without centrifugation. This technology may prove useful in isolating motile sperm from oligozoospermic samples, even with high amounts of nonmotile gamete and/or non-gamete cell contamination. Supported by: None.
P-299 Fertilization and preimplantation development of oocytes injected with spermatozoa of hormone-sensitive lipase deficient mice. Akihisa Fujimoto, Toshihiro Fujiwara, Yutaka Osuga, Tetsu Yano, Osamu Tsutsumi, Yuji Taketani. Dept of Ob and Gyn, Univ of Tokyo, Tokyo, Japan. Objective: Hormone-sensitive lipase (HSL) is a multifunctional enzyme that mediates hydrolysis of cholesterol esters and triacylglycerol in adipose tissues, adrenals, ovaries and testes. In the previous reports, HSL deficient male mice were shown to be infertile because of severe oligozoospermia, elucidating HSL plays an important role in mouse spermatogenesis. How-
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P-297 Cryopreservation/thawing and function of human sperm: Sperm that remain motile also maintain their plasma membrane integrity and hyaluronic acid binding properties. Ciler Celik-Ozenci, Sevil Cayli, Jill Stronk, Lynne Vigue, Ramazan Demir, Gabor Huszar. The Sperm Phys Lab, Dept of Ob/Gyn, Yale Sch of Medicine, New Haven, CT. Objective: During human spermiogenesis, simultaneously with cytoplasmic extrusion and the major expression of the HspA2 chaperone protein, there is a remodeling of the sperm plasma membrane, which facilitates the formation of zona pellucida- and hyaluronic acid(HA)-binding sites (Huszar et al BOR 1997 and 2000). Mature sperm bind to the zona pellucida and to HA-coated surfaces, whereas immature sperm with cytoplasmic retention exhibit diminished binding. In the present work using the sperm-HA binding assay, we investigated the functional integrity of the sperm plasma membrane after sperm cryopreservation and thawing. This issue is of interest from the points of view of cryobiology and the preservation of sperm function in cryopreserved sperm. Design: Comparison of sperm HA-binding before and after cryopreservation. Materials/Methods: We utilized modified Cell-VUTM double chamber slides. The A chamber was used for determination of motile sperm concentration, whereas the B chamber was coated with HA for the sperm binding assay (Biocoat Inc., PA). HA-binding was scored after placing a drop of semen in the chamber for 10 minutes (equilibrium is reached at 6 –7 minutes). HA binding is expressed as % bound sperm/total motile sperm. In each sample, the fresh semen and their swim-up fractions (in HTF) were studied, and another aliquot of semen and swim-up fractions were cryopreserved in egg yolk-free medium (1:1 ratio, 5% final cc. of glycerol), and were thawed (37oC ⫻ 10 min) after one week. In the fresh samples motile sperm concentration, HA-binding, and degree of maturity reflected by cytoplasmic retention (expressed as CK-B immunocytochemistry darkness factor, DF) were determined. In the thawed fractions motile sperm concentration and HA-binding were studied. Data analysis was carried out with SigmaStat. Results: We studied 30 men (sperm conc: 86 ⫾ 12 ⫻ 106 sperm/ml, and motility: 50.5 ⫾ 2.9%, all data mean ⫾ SEM). By comparing the semen and swim-up sperm, we studied sperm fractions of different maturities, as indicated by the DF (Table). Accordingly, in the swim-up fractions the HA-binding scores were higher. Sperm motility, as expected, was lower in the thawed sperm. However, HA binding scores of thawed sperm were similar in both the semen and the swim-up fractions. Thus, sperm that remain motile also maintain their plasma membrane integrity and HA binding ability. The DF and HA-binding were related (semen: r ⫽ ⫺0.55, p ⫽ 0.001; swim up: r ⫽ ⫺0.47, p ⬍0.01), reflecting the spermiogenetic connection between cytoplasmic extrusion and plasma membrane remodeling.
FERTILITY & STERILITY威
DF initial
Motility (%) initial
Binding (%) initial
Motility (%) thawed
Binding (%) thawed
Semen sperm 52.8 ⫾ 4.1 50.5 ⫾ 2.4 87.3 ⫾ 2.4 27 ⫾ 2.4 86.1 ⫾ 3.5 P value P ⫽ 0.002 P ⬍ 0.001 P ⫽ 0.005 P ⬍ 0.001 P ⫽ 0.15 Swim-up sperm 34.6 ⫾ 3.9 78.5 ⫾ 3.0 93.1 ⫾ 2.0 19.5 ⫾ 2.0 88.3 ⫾ 3.2
Conclusions: The sperm HA-binding scores were unchanged between the fresh and thawed samples, whether in semen or in the swim-up fractions. This indicates that those spermatozoa that retain motility also retain their functional integrity in HA (and presumably zona pellucida) binding properties. HA binding provides an objective measure of sperm function in cryopreserved/thawed samples, and also allows testing of sperm for reproductive toxicity at a remote laboratory after shipping. Supported by: (Supp. HD-19505, HD-32902 and OH-04061).
P-298 A novel microfluidic device for separating motile sperm from nonmotile sperm via inter-streamline crossing. Timothy G. Schuster, Brenda Cho, Laura M. Keller, Dana A. Ohl, Shuichi Takayama, Gary D. Smith. Univ of Michigan, Ann Arbor, MI. Objective: To assess if motile sperm could be separated from nonmotile sperm and non-gamete cells using a novel microfluidic device. Design: Laboratory study. Materials/Methods: A microfluidic device was designed with 2 parallel laminar flow channels where nonmotile sperm and debris would flow along their initial streamlines and exit one outlet whereas motile sperm had an opportunity to swim into a parallel stream and exit a separate outlet. Overnight sperm motility of density gradient prepared sperm samples (N⫽5) with and without exposure to polydimethylsiloxane (PDMS) used to construct the device was assessed. Ten density gradient prepared samples containing 5 million motile sperm/ml were placed in thin inlet channels while processing media was placed in wider parallel channels. Sperm (N⫽200) in the inflow and parallel stream outflow channels were evaluated for motility. Sperm samples (N⫽10) were also placed in wider channels to assess if switching channels affected sorting ability. Lastly, unprocessed semen samples (N⫽10) were placed in wider channels and sperm motility and strict Kruger morphology were assessed from sorted outlets. Results: Compared to initial sperm motility (75% ⫹/⫺ 5.6%; Mean ⫹/⫺ S.E.), overnight sperm motility was significantly lower for samples with and without exposure to PDMS (64% ⫹/⫺ 3.9% and 69% ⫹/⫺ 4.6%; p ⬍0.05, respectively). No difference was observed between overnight motilities (P ⬎0.05). Sperm motility for samples placed in thin inlet channels increased from 74% ⫹/⫺ 2.6% to 96% ⫹/⫺ 0.9% (p ⬍0.05). Placing sperm in wider channels increased motility from 58% ⫹/⫺ 3.9% to 98% ⫹/⫺ 0.6% (p ⬍0.05). Unprocessed sperm motility increased from 44% ⫹/⫺ 4.5% to 98% ⫹/⫺ 0.4% (p ⬍0.05) and morphology increased from 10% ⫹/⫺ 1.05% to 22% ⫹/⫺ 3.3% (p ⬍0.05) following microfluidic inter-streamline crossing isolation. Conclusions: We have demonstrated inter-streamline crossing can be used for isolating human sperm. This microfluidic device provides a novel method for isolating motile, morphologically normal sperm from semen samples without centrifugation. This technology may prove useful in isolating motile sperm from oligozoospermic samples, even with high amounts of nonmotile gamete and/or non-gamete cell contamination. Supported by: None.
P-299 Fertilization and preimplantation development of oocytes injected with spermatozoa of hormone-sensitive lipase deficient mice. Akihisa Fujimoto, Toshihiro Fujiwara, Yutaka Osuga, Tetsu Yano, Osamu Tsutsumi, Yuji Taketani. Dept of Ob and Gyn, Univ of Tokyo, Tokyo, Japan. Objective: Hormone-sensitive lipase (HSL) is a multifunctional enzyme that mediates hydrolysis of cholesterol esters and triacylglycerol in adipose tissues, adrenals, ovaries and testes. In the previous reports, HSL deficient male mice were shown to be infertile because of severe oligozoospermia, elucidating HSL plays an important role in mouse spermatogenesis. How-
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