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Timing of multinucleation appearance affects further embryo cleavage and subsequent blastocyst formation rate
MATERIALS AND METHODS: Proliferation assays: 100,000 GLC were plated in SFM and incubated 24 h with different agents; EGF (10 ng/ml); LA as GnRH-a (1, 10 and 100 ng/ml) and ANT (10-7 M), as GnRH antagonist; or a combination of ANT with LA, adding LA 100 ng/mL 3 hours after the addition of ANT 10-7M. 24h before harvesting, 1 Ci 3 H-thymidine was added to each well, and DNA synthesis was assessed by 3 H-thymidine incorporation. Apoptosis: % of apoptotic cells was assessed by the acridine orange-ethidium bromide technique in GLC cultures at basal conditions and after exposure to LA 100 ng/mL or ANT 10-7 M, or a combination of ANT with LA, adding LA 3 hours after the addition of ANT. After addition of the acridine orange-ethidium bromide mix, the cells were viewed by a fluorescence microscope and the apoptotic cells were counted as a percentage of the total. One-way ANOVA was used to compare the mean values among the treatments. When the values were significant, ANOVA analysis was followed by Tukey test. Values of p⬍ 0.05 were considered significant. RESULTS: The GnRH-a LA alone had no effect on basal DNA synthesis in GLC cultures. Unexpectedly, ANT alone produced a significant stimulatory effect (350%) of 3H-thymidine incorporation on basal conditions. To further determine the role of LA in cell proliferation, different concentrations of LA were added to EGF (10 ng/ml) stimulated cell cultures (SCCs). In these conditions 3H-thymidine uptake was down-regulated by LA at 10 and 100 ng/ml (LA 1 ng/ml: 7385.3⫾1164.4 cpm/well, P⬎0.05; LA 10 ng/ml: 1798⫾268 cpm/well and LA 100 ng/ml: 1698.6⫾259.3 cpm/well, p⬍0.05 vs. SCC: 7556⫾758 cpm/well), while ANT did not produce any change in DNA synthesis in the presence of EGF. The inhibitory LA effect was completely reversed by the addition of ANT to the culture medium. To assess whether this DNA synthesis inhibition was produced by an increase of the apoptosis of GLC cells, these cells were cultured in the presence of LA (100 ng/ml). This treatment showed a significant increase in the apoptotic levels from 39.6⫾2.5 % at basal conditions to 58.6⫾4% (expressed as a % of apoptotic cells, p⬍0.05). This effect was reversed by ANT: 40.4⫾4 (vs. basal, P⬎0.05), while ANT alone had no effect on basal apoptosis. CONCLUSION: These findings suggest that the direct inhibitory effect of GnRH agonists on ovarian follicular growth described in conventional ART procedures, could be avoided with the use of GnRH antagonists, and besides these molecules could improve follicular growth. Supported by: ANPCYT (BID 1201 OC-AR PICT 99:05– 06384) and Universidad de Buenos Aires (X909).
P-440 Timing of multinucleation appearance affects further embryo cleavage and subsequent blastocyst formation rate. M. Florensa, P. Ga´ miz, G. Calderon, M. J. De Los Santos, J. Remohı´, A. Pellicer. Instituto Valenciano de Infertilidad, Valencia, Spain.
P-439 Direct effect of gonadotropin-releasing hormone agonist and antagonist on the growth, apoptosis and steroidogenesis in human granulosa cells from women undergoing in vitro fertilization. A. M. Vitale, D. Abramovich, M. C. Peluffo, G. Meresman, M. Tesone. Instituto de Biologia y Medicina Experimental, Buenos Aires, Argentina. OBJECTIVE: To evaluate the direct effect of a GnRH-a (leuprolide acetate, LA) and a GnRH-ant (antide, ANT) on the steroidogenesis, growth and programmed cell death (apoptosis) of human granulosa-luteal cells (GLC) obtained from patients undergoing ART. DESIGN: Human GLC were cultured after isolation from follicular fluid aspirated of patients undergoing ART.
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OBJECTIVE: Multinucleation on human embryos occurs on approximately 17% of cases. It has been associated to failure to complete cytokinesis but not karyokinesis contributing to subsequent mitosis arrest and a lower blastocyst formation rate. The aim of this study is to assess whether the timing of multinucleation appearance affects further embryo development to blastocyst stage. DESIGN: Retrospective study. MATERIALS AND METHODS: A total of 3979 embryos from 531 patients undergoing IVF/ICSI cycles between Jannuary, 2002 and December, 2003. Embryos were derived from regular IVF (n ⫽ 1025) or ovum donation (n ⫽ 2954) programs and were grown until blastocyst stage. Embryos were classified into 4 groups: group A, not multinucleated either on day 2 or day 3; group B, only multinucleated on day 3; group C, only multinucleated on day 2; group D, multinucleated on both day 2 and 3. The percentage of arrested embryos on days 3 and blastocyst stage as well as good-quality (GQ) blastocyst formation rate were assessed. All percentages were statistically analyzed by a chi squared test. RESULTS: The incidence of multinucleated embryos was slightly but significantly lower in embryos coming from donors compared to patients either on day 2 (14.7% vs 18.2%) or day 3 (6.5% vs 8.8%) (p ⬍ 0.05). Group A embryos showed the lowest embryo arrest and the highest blastocyst and good quality blastocyst percentage. Group C embryos had a higher development arrest on day 3, and a lower blastocyst formation rate than groups B and A embryos. Group D embryos showed also low embryo cleavage ability regarding both development arrest on day 3 and blastocyst formation when compared to group A and B.
Vol. 82, Suppl. 2, September 2004
a, b and c showed statistical differences (p ⬍ 0.01) among them in the same column. CONCLUSION: Day 2 embryos obtained from donated oocytes were significantly less multinucleated than embryos derived from patients with their own oocytes. That may defend the hypothesis of multinucleation being depending on maternal rather than paternal factors. Signs of multinucleation on day 2 of embryo cleavage may be indicative of earlier defects on the embryo mitosis machinery that will significantly impair embryo cleavage and good quality blastocyst formation. Multinucleated embryos on day 2 and those that remain multinucleated from day 2 to day 3, have similar developmental ability than those multinucleated only on day 2, therefore, they should be avoided for embryo transfer whenever possible. Supported by: None. P-441 Identification of new proteins in human follicular fluid by high resolution 2D electrophoresis as reliable markers for oocyte developmental potential. T. Anahory, S. Hamamah, H. Dechaud, D. Laoudj, A. Fernandez, N. Lamb. Service de Biologie de la Reproduction B, Montpellier, France; Service de Gyne´ vcologie Obste´ trique, Montpellier, France; Institut de Genetique Humaine, Montpellier, France. OBJECTIVE: The molecular basis of human cytoplasmic oocyte maturity remains unclear. The follicle is formed from granulosa cells which produce follicular fluid containing a number of different proteins. In order to increase our knowledge of the molecular relationship between the stage of oocyte maturity and follicular fluid composition, we have analysed the total and specific protein content of each follicular fluid isolated individually from separate human follicles after hyperstimulation. DESIGN: 66 individual human follicular fluid samples were collected from 10 women undergoing in vitro fertilization. Each follicle was aspirated separately and a total of 66 samples were obtained. MATERIALS AND METHODS: Each oocyte was fertilized in isolation and the corresponding follicular fluid processed by high-resolution 2-D PAGE. The comparison and analysis of the gel was realized by PDQUest software. Two patterns of protein expression were observed which could be directly correlated with oocyte fecundity. From these two different expression patterns, 3 proteins varied with particular interest to oocyte fertilization. RESULTS: The analysis revealed two distinct different patterns of protein expression with three proteins showing significant variations in level. The identification of these 3 proteins was realized by internal amino acid sequence analysis or by Maldi-TOF- MS and gave: retinol binding protein, thioredoxin peroxidase 1, and Transthyretin. These proteins are highly implicated in the early development potential during human embryogenesis. CONCLUSION: These results indicate that three proteins can act as reliable markers for oocyte developmental potential and confirmed that high resolution proteomic analysis of human follicular fluid is a useful tool to study cytoplasmic oocyte maturation. Supported by: CNRS and ARC. P-442 GSTM1 polymorphism analysis in patients with mild and severe endometriosis. C. V. De Carvalho, P. D’Amora, E. Schor, E. C. Baracat, M. J. C. Gira˜ o, I. D. G. Da Silva. Molecular Ginecology Laboratory, Department of Gynecology, Federal University of Sa˜ o Paulo, Sa˜ o Paulo, Brazil. OBJECTIVE: Analyze the prevalence of GSTM1 polymorphism in women with mild and severe endometriosis. DESIGN: Case control study. MATERIALS AND METHODS: We have genotyped 93 patients from the same geographical origin of Sa˜ o Paulo. The study group was consisted of patients with surgically and histologically diagnosed endometriosis. Sixty-one patients with endometriosis were divided into different catego-
FERTILITY & STERILITY威
ries: endometriosis stages I-II (n⫽25) and stages III-IV (n⫽36). The control group was composed by 32 postmenopausal women recruited from the Unit of Gynecological Endocrinology at the Federal University of Sa˜ o Paulo. Allele especific PCR was performed in order to detect the 215-bp fragment of the GSTM1 gene in a multiplex reaction along with the endogenous control gene beta-globin (268-bp). Bands were analyzed in a 2% agarose gel stained with Ethide Bromide. RESULTS: According to our results the GSTM1 polymorphism was not associated with endometriosis in present population. Table I shows frequencies of GSTM1 allele in the studied groups:
CONCLUSION: GSTM1 polymorphism is not associated with mild and/or severe endometriosis. However, the present study is still on going, therefore by increasing the number of patients we might have a better analysis regarding this matter. Supported by: FAPESP 03/04533–1 and grants from Centro Universita´ rio Fundac¸ a˜ o Santo Andre´ , Sa˜ o Paulo, Brazil. P-443 Alpha- and gamma-tocopherol in women with polycystic ovary syndrome. A. J. Duleba, J. Dorey. Yale University School of Medicine, New Haven, CT. OBJECTIVE: Polycystic ovary syndrome (PCOS) is associated with a broad range of metabolic and endocrine manifestations including increased oxidative stress, systemic inflammation and insulin resistance. Alpha-tccopherol (AT) and gamma-tocopherol (GT) play an important role in protection from oxidative stress by scavenging reactive oxygen and nitrogen oxide species. Recent studies suggest that the properties and metabolism of GT are distinctly different from AT. Specifically, GT has a particular affinity to the nitrogen oxide species and possesses unique anti-inflammatory properties related to inhibition of cyclooxygenase activity. This study was designed to evaluate the relationship of AT and GT with selected parameters of PCOS. DESIGN: Cross-sectional evaluation of women with PCOS. MATERIALS AND METHODS: PCOS was diagnosed on the basis of oligo- or amenorrhea (⬍⫽6 spontaneous menses per year) and hyperandrogenism. Subjects with diabetes or other endocrinopathies were excluded. All subjects were evaluated within 3–9 days of spontaneous or progestininduced menses. Primary measures included: leves of AT and GT, body mass index (BMI), lipid profile, high-sensitivity C-reactive protein (hsCRP), as well as insulin and glucose in the fasting state and during a 2-hour glucose tolerance test. Insulin sensitivity was estimated by insulin sensitivity index (ISI). Statistical analysis was performed using regression analysis. RESULTS: The study evaluated 38 subjects. The mean age was 25.6⫾1.3 (⫾SEM) years and the mean BMI was 34.3⫾1.7 (⫾SEM). GT and AT correlated positively with total cholesterol and triglycerides; in view of the lipid distribution of GT and AT, subsequent analysis was also carried out evaluating GT per total lipids (GTTL) and AT per total lipids (ATTL). GTTL correlated positively with age (r⫽0.4, p⬍0.01) while ATTL did not (r⫽0.1, p⫽0.5). Furthermore, GTTL correlated positively with BMI (r⫽0.57, p⬍0.001) while ATTL correlated negatively with BMI (r⫽-0.4, p⫽0.016). GTTL correlated positively with the logarithm of hs-CRP (r⫽0.54, p,0.001) while ATTL did not (r⫽-0.37, p⫽0.1). The relationship of tocopherols with insulin sensitivity was also distinctly discordant. GT correlated negatively with ISI (r⫽-0.39, p⫽0.04) while AT correlated borderline positively (r⫽0.26, p⫽0.1). Furthermore, GTTL correlated borderline negatively with ISI (r⫽-0.32, p⫽0.09) while ATTL correlated positively (r⫽0.39, p⫽0.03). CONCLUSION: Gamma-tocopherol differs from alpha-tocopherol with regard to the relationship with obesity, systemic inflammation and insulin sensitivity. Among subjects with PCOS, level of gamma-tocopherol is greater in the presence of obesity, systemic inflammation and insulin resistance. These observations raise the possibility of unique protective-compensatory increases of gamma-tocopherol in subjects with greater cardiovascular risks. Supported by: NIH grant R01 HD 40207.
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P-440 Timing of multinucleation appearance affects further embryo cleavage and subsequent blastocyst formation rate. M. Florensa, P. Ga´ miz, G. Calderon, M. J. De Los Santos, J. Remohı´, A. Pellicer. Instituto Valenciano de Infertilidad, Valencia, Spain.
P-439 Direct effect of gonadotropin-releasing hormone agonist and antagonist on the growth, apoptosis and steroidogenesis in human granulosa cells from women undergoing in vitro fertilization. A. M. Vitale, D. Abramovich, M. C. Peluffo, G. Meresman, M. Tesone. Instituto de Biologia y Medicina Experimental, Buenos Aires, Argentina. OBJECTIVE: To evaluate the direct effect of a GnRH-a (leuprolide acetate, LA) and a GnRH-ant (antide, ANT) on the steroidogenesis, growth and programmed cell death (apoptosis) of human granulosa-luteal cells (GLC) obtained from patients undergoing ART. DESIGN: Human GLC were cultured after isolation from follicular fluid aspirated of patients undergoing ART.
S290
OBJECTIVE: Multinucleation on human embryos occurs on approximately 17% of cases. It has been associated to failure to complete cytokinesis but not karyokinesis contributing to subsequent mitosis arrest and a lower blastocyst formation rate. The aim of this study is to assess whether the timing of multinucleation appearance affects further embryo development to blastocyst stage. DESIGN: Retrospective study. MATERIALS AND METHODS: A total of 3979 embryos from 531 patients undergoing IVF/ICSI cycles between Jannuary, 2002 and December, 2003. Embryos were derived from regular IVF (n ⫽ 1025) or ovum donation (n ⫽ 2954) programs and were grown until blastocyst stage. Embryos were classified into 4 groups: group A, not multinucleated either on day 2 or day 3; group B, only multinucleated on day 3; group C, only multinucleated on day 2; group D, multinucleated on both day 2 and 3. The percentage of arrested embryos on days 3 and blastocyst stage as well as good-quality (GQ) blastocyst formation rate were assessed. All percentages were statistically analyzed by a chi squared test. RESULTS: The incidence of multinucleated embryos was slightly but significantly lower in embryos coming from donors compared to patients either on day 2 (14.7% vs 18.2%) or day 3 (6.5% vs 8.8%) (p ⬍ 0.05). Group A embryos showed the lowest embryo arrest and the highest blastocyst and good quality blastocyst percentage. Group C embryos had a higher development arrest on day 3, and a lower blastocyst formation rate than groups B and A embryos. Group D embryos showed also low embryo cleavage ability regarding both development arrest on day 3 and blastocyst formation when compared to group A and B.
Vol. 82, Suppl. 2, September 2004
a, b and c showed statistical differences (p ⬍ 0.01) among them in the same column. CONCLUSION: Day 2 embryos obtained from donated oocytes were significantly less multinucleated than embryos derived from patients with their own oocytes. That may defend the hypothesis of multinucleation being depending on maternal rather than paternal factors. Signs of multinucleation on day 2 of embryo cleavage may be indicative of earlier defects on the embryo mitosis machinery that will significantly impair embryo cleavage and good quality blastocyst formation. Multinucleated embryos on day 2 and those that remain multinucleated from day 2 to day 3, have similar developmental ability than those multinucleated only on day 2, therefore, they should be avoided for embryo transfer whenever possible. Supported by: None. P-441 Identification of new proteins in human follicular fluid by high resolution 2D electrophoresis as reliable markers for oocyte developmental potential. T. Anahory, S. Hamamah, H. Dechaud, D. Laoudj, A. Fernandez, N. Lamb. Service de Biologie de la Reproduction B, Montpellier, France; Service de Gyne´ vcologie Obste´ trique, Montpellier, France; Institut de Genetique Humaine, Montpellier, France. OBJECTIVE: The molecular basis of human cytoplasmic oocyte maturity remains unclear. The follicle is formed from granulosa cells which produce follicular fluid containing a number of different proteins. In order to increase our knowledge of the molecular relationship between the stage of oocyte maturity and follicular fluid composition, we have analysed the total and specific protein content of each follicular fluid isolated individually from separate human follicles after hyperstimulation. DESIGN: 66 individual human follicular fluid samples were collected from 10 women undergoing in vitro fertilization. Each follicle was aspirated separately and a total of 66 samples were obtained. MATERIALS AND METHODS: Each oocyte was fertilized in isolation and the corresponding follicular fluid processed by high-resolution 2-D PAGE. The comparison and analysis of the gel was realized by PDQUest software. Two patterns of protein expression were observed which could be directly correlated with oocyte fecundity. From these two different expression patterns, 3 proteins varied with particular interest to oocyte fertilization. RESULTS: The analysis revealed two distinct different patterns of protein expression with three proteins showing significant variations in level. The identification of these 3 proteins was realized by internal amino acid sequence analysis or by Maldi-TOF- MS and gave: retinol binding protein, thioredoxin peroxidase 1, and Transthyretin. These proteins are highly implicated in the early development potential during human embryogenesis. CONCLUSION: These results indicate that three proteins can act as reliable markers for oocyte developmental potential and confirmed that high resolution proteomic analysis of human follicular fluid is a useful tool to study cytoplasmic oocyte maturation. Supported by: CNRS and ARC. P-442 GSTM1 polymorphism analysis in patients with mild and severe endometriosis. C. V. De Carvalho, P. D’Amora, E. Schor, E. C. Baracat, M. J. C. Gira˜ o, I. D. G. Da Silva. Molecular Ginecology Laboratory, Department of Gynecology, Federal University of Sa˜ o Paulo, Sa˜ o Paulo, Brazil. OBJECTIVE: Analyze the prevalence of GSTM1 polymorphism in women with mild and severe endometriosis. DESIGN: Case control study. MATERIALS AND METHODS: We have genotyped 93 patients from the same geographical origin of Sa˜ o Paulo. The study group was consisted of patients with surgically and histologically diagnosed endometriosis. Sixty-one patients with endometriosis were divided into different catego-
FERTILITY & STERILITY威
ries: endometriosis stages I-II (n⫽25) and stages III-IV (n⫽36). The control group was composed by 32 postmenopausal women recruited from the Unit of Gynecological Endocrinology at the Federal University of Sa˜ o Paulo. Allele especific PCR was performed in order to detect the 215-bp fragment of the GSTM1 gene in a multiplex reaction along with the endogenous control gene beta-globin (268-bp). Bands were analyzed in a 2% agarose gel stained with Ethide Bromide. RESULTS: According to our results the GSTM1 polymorphism was not associated with endometriosis in present population. Table I shows frequencies of GSTM1 allele in the studied groups:
CONCLUSION: GSTM1 polymorphism is not associated with mild and/or severe endometriosis. However, the present study is still on going, therefore by increasing the number of patients we might have a better analysis regarding this matter. Supported by: FAPESP 03/04533–1 and grants from Centro Universita´ rio Fundac¸ a˜ o Santo Andre´ , Sa˜ o Paulo, Brazil. P-443 Alpha- and gamma-tocopherol in women with polycystic ovary syndrome. A. J. Duleba, J. Dorey. Yale University School of Medicine, New Haven, CT. OBJECTIVE: Polycystic ovary syndrome (PCOS) is associated with a broad range of metabolic and endocrine manifestations including increased oxidative stress, systemic inflammation and insulin resistance. Alpha-tccopherol (AT) and gamma-tocopherol (GT) play an important role in protection from oxidative stress by scavenging reactive oxygen and nitrogen oxide species. Recent studies suggest that the properties and metabolism of GT are distinctly different from AT. Specifically, GT has a particular affinity to the nitrogen oxide species and possesses unique anti-inflammatory properties related to inhibition of cyclooxygenase activity. This study was designed to evaluate the relationship of AT and GT with selected parameters of PCOS. DESIGN: Cross-sectional evaluation of women with PCOS. MATERIALS AND METHODS: PCOS was diagnosed on the basis of oligo- or amenorrhea (⬍⫽6 spontaneous menses per year) and hyperandrogenism. Subjects with diabetes or other endocrinopathies were excluded. All subjects were evaluated within 3–9 days of spontaneous or progestininduced menses. Primary measures included: leves of AT and GT, body mass index (BMI), lipid profile, high-sensitivity C-reactive protein (hsCRP), as well as insulin and glucose in the fasting state and during a 2-hour glucose tolerance test. Insulin sensitivity was estimated by insulin sensitivity index (ISI). Statistical analysis was performed using regression analysis. RESULTS: The study evaluated 38 subjects. The mean age was 25.6⫾1.3 (⫾SEM) years and the mean BMI was 34.3⫾1.7 (⫾SEM). GT and AT correlated positively with total cholesterol and triglycerides; in view of the lipid distribution of GT and AT, subsequent analysis was also carried out evaluating GT per total lipids (GTTL) and AT per total lipids (ATTL). GTTL correlated positively with age (r⫽0.4, p⬍0.01) while ATTL did not (r⫽0.1, p⫽0.5). Furthermore, GTTL correlated positively with BMI (r⫽0.57, p⬍0.001) while ATTL correlated negatively with BMI (r⫽-0.4, p⫽0.016). GTTL correlated positively with the logarithm of hs-CRP (r⫽0.54, p,0.001) while ATTL did not (r⫽-0.37, p⫽0.1). The relationship of tocopherols with insulin sensitivity was also distinctly discordant. GT correlated negatively with ISI (r⫽-0.39, p⫽0.04) while AT correlated borderline positively (r⫽0.26, p⫽0.1). Furthermore, GTTL correlated borderline negatively with ISI (r⫽-0.32, p⫽0.09) while ATTL correlated positively (r⫽0.39, p⫽0.03). CONCLUSION: Gamma-tocopherol differs from alpha-tocopherol with regard to the relationship with obesity, systemic inflammation and insulin sensitivity. Among subjects with PCOS, level of gamma-tocopherol is greater in the presence of obesity, systemic inflammation and insulin resistance. These observations raise the possibility of unique protective-compensatory increases of gamma-tocopherol in subjects with greater cardiovascular risks. Supported by: NIH grant R01 HD 40207.
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