- Email: [email protected]
Detection of sperm DNA damage by raman microspectroscopy
targeted deletion, as well as overexpression of e2f1, causes severe testicular atrophy, apoptosis, and impaired spermatogenesis. DESIGN: A cohort of 8 NOA (normal karyotypes with no Y-chromosome microdeletions) men and 70 controls were evaluated for chromosomal rearrangements by array comparative genomic hybridization (aCGH). MATERIALS AND METHODS: 70 control and 8 NOA men were evaluated using 385K and 720K NimbleGen-aCGH and the data analyzed using Nexus Copy Number. To confirm copy-number-variations (CNV) detected by aCGH, E2F1-CNV-Taqman assays were performed on the original 8 NOA men and 72 additional NOA men as well as 40 fertile men. Exons 27 of E2F1 were sequenced and analyzed using Surveyor. RESULTS: aCGH results revealed a microduplication at 20q11.22 that encompasses the gene E2F1 in one NOA men that was not found in any of the other aCGH analyzed. CNV-Taqman assays showed 8 of 80 (10%) infertile men had microduplications or microdeletions of E2F1 with none of the additional 40 fertile controls having CNVs in E2F1. Sequence analysis of E2F1 revealed mutations at positions 102(Ala/Thr) and 393(Gly/Ser) of unclear significance. CONCLUSION: We identified genetic and genomic changes of E2F1 among NOA men consisting of possibly benign SNPs, as well as E2F1 microdeletions and microduplications that were statistically significant and correlated with abnormal testicular biopsy. This finding, together with the known spermatogenic deficiencies in transgenic and knockout mouse models of e2f1, suggests that E2F1 could play an important role in human male infertility. To our knowledge this is the first reported association of a CNVof E2F1 in non-cancer patients, emphasizing the range of roles of E2F1 in complex diseases that include infertility. Supported by: NIH grants K12DK0083014, P01HD36289.
P-432 Wednesday, October 19, 2011 IN THE ERA OF IVF WITH ICSI, DO WE NEED TO REFER THE MALE PARTNER TO A REPRODUCTIVE UROLOGIST. B. A. McElyea, H. I. G. Cotton, K. McCarty, M. Acosta-La Greca, N. Bar-Chama, A. B. Copperman. Reproductive Medicine Associates of New York, New York, NY; Department of OBGYN and Reproductive Science, Mount Sinai School of Medicine, New York, NY. OBJECTIVE: When presenting for an infertility consult at RMA of NY, female patients undergo a comprehensive examination. Part of this evaluation includes obtaining an initial semen analysis from their male partners. If an abnormality is identified in the semen analysis (decreased sperm counts, motility, or morphology) then the male patient is often referred to the Urology department for a comprehensive infertility work up as well. The purpose of this study was to determine if the current practice of referring male patients for an infertility workup is appropriate, or even necessary, and to determine with the consultation results any change in the couple’s treatment plan. DESIGN: Retrospective data analysis at a private academic fertility center. MATERIALS AND METHODS: A comprehensive chart review of 100 consecutive males who presented to the Urology practice for evaluation was completed. We analyzed the reason for presentation to the Urologist and the resulting diagnosis. Only patients who were referred by a reproductive endocrinologist or Ob-Gyn specifically for the purpose of an infertility workup were included. Excluded were self-referred patients, patients who presented for issues other than fertility, and patients with pre-existing diagnoses who were referred for treatment. The final sample included 66 male patients. RESULTS: More than half (53%) of the men referred were identified to have a pathology that is easily treated.
Pathology
% of Referrals
Hypogonadal Positive Semen Cultures Varicocele Congenital Absence of Vas Deferens
15.2 9.1 27.3 1.5
CONCLUSION: The findings suggest that referral to Urology for further workup is appropriate, given that more than half of the men with abnormal semen analyses had easily treatable pathologies. Despite the fact that the treatment plan for IVF may not be altered in a majority of the cases, early identification of other treatable disorders may improve overall health in our male patient population.
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Abstracts
P-433 Wednesday, October 19, 2011 TESTICULAR SPERM PREINCUBATION AND ICSI OUTCOME. J. Kocent, Q. V. Neri, D. Monahan, Z. Rosenwaks, G. D. Palermo. The Ronald O. Perelman & Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY. OBJECTIVE: To determine the effect of lengthening the incubation time of testicular biopsy specimens on ICSI outcome. DESIGN: Fresh or cryopreserved testicular specimens incubated overnight were compared to those used the same day of ICSI. MATERIALS AND METHODS: From 2008 to 2011, microTESE were used on the oocyte retrieval day (SD) or incubated overnight at 37 C (ONI). Maternal age, sperm concentration, motility, fertilization rate, bHCG, and clinical pregnancy (CP) (with the presence of at least 1 FHB) were evaluated. RESULTS: A total of 296 used fresh TESE and 44 of those were ONI cycles with a remarkably lower proportion of motile spermatozoa (P¼0.04). Fertilization was 48.6% (231/475) and CP rate 34.1% (15/44), not different from the SD at 51.1% (129/252) and 40.0% (101/252), respectively. A total of 18 thawed TESE cycles were identified of which 12 ONI and 6 SD was cryopreserved the same day. The ONI group yielded a fertilization of 41.5% (37/89) and a CP of 50.0% (6/12) while the SD cryo had a fertilization of 37.8% (14/37) without achieving any pregnancy. To see whether the cryopreservation itself could have affected the testicular sperm’s performance, the corresponding fresh ONI (n ¼ 12) and SD (n ¼ 3) cycles were analyzed. The concentration was higher in the ONI (P¼0.03). A fertilization of 66.2% (86/130) and clinical pregnancy of 50.0% (6/12) was observed in the ONI while the SD were 48.1% (13/27) and 33.3% (1/3), respectively. To assess for optimal incubation conditions, 40 ONI cycles were assessed in which 36 were incubated at 37 C in 5% CO2 yielded a fertilization of 25.3% (20/79). In 4 cycles the incubation at RT yielded 50.0% (6/12) fertilization. CONCLUSION: Preincubation time of testicular spermatozoa does not seem to affect clinical ICSI outcomes. Cryopreservation paradoxically in the same day insemination did not yield any pregnancy. Although length of incubation does not have a negative affect on clinical outcomes, incubation at room temperature seems promising. Supported by: Institutional.
SPERM BIOLOGY P-434 Wednesday, October 19, 2011 DETECTION OF SPERM DNA DAMAGE BY RAMAN MICROSPECTROSCOPY. V. Sanchez, J. Wistuba, F. W€ubbeling, M. Burger, S. Schlatt, C. Mallidis. Centre of Reproductive Medicine and Andrology (CeRA), M€unster, North Rhine-Westfalia, Germany; Institute of Computational and Applied Mathematics, University of M€unster, M€unster, North Rhine-Westfalia, Germany. OBJECTIVE: Appraise ability of Raman microspectroscopy to identify and characterise oxidative sperm nDNA damage, thus ascertain the method’s potential as a selection tool of sperm suitable for ART. DESIGN: Controlled, blinded, prospective study. MATERIALS AND METHODS: After confirmation that samples were WHO normozoospermic, ejaculates from 5 healthy donors were washed in PBS then split into three aliquots. One remained untreated (control), the other two underwent differing degrees of oxidative attack via Fenton’s reaction (Fe2+ catalyst and H2O2). DNA fragmentation was determined by flow cytometric Acridine Orange Test (FLAOT) and Raman microspectroscopy (LabRam Aramis system, Horiba Jobin Yvon). Optimal instrumental settings/range were determined and duplicate 30 second single point spectra obtained from 200 sperm/sample/treatment. In total 3000 spectra were analyzed. Identification and frequency of sperm nDNA damage were assessed by Fourier filtering, Principal Component Analysis (PCA) and computation of local spectral angles relative to sample means. RESULTS: Oxidation caused by 1:10 and 1:5 concentrations of the Fenton reagent resulted in 50% and 100% (respectively) of sperm having nDNA damage (FLAOT DNA fragmentation index). PCA showed spectra from sperm with nDNA fragmentation were clearly distinguishable and significantly different from those with undamaged nDNA. Percentage of sperm identified as having damage correlated with FLAOT determination. Local spectral angle analysis, focussing on differences in main spectral peak due to DNA PO4 framework, found a clear shift from customary position at 1092 cm-1 towards 1042 cm-1, a translocation consistent with alterations
Vol. 96., No. 3, Supplement, September 2011
in the molecule’s vibrational capabilities due to perturbations in the DNA backbone. CONCLUSION: Oxidative nDNA fragmentation is manifest by a distinct Raman spectral profile which can be readily identified and used to select sperm with undamaged DNA. Supported by: CeRA internal funding.
P-435 Wednesday, October 19, 2011 IMMUNOREACTIVITY TEST OF SPERM AGAINST PLCZ1: EFFECTIVE DIAGNOSTIC TEST FOR MALE INFERTILITY IN ART. S.-Y. Yoon, T. H. Kim, M. K. Kim, H. H. Seuk, D. R. Lee, T. K. Yoon. Fertility Center of CHA Gangnam Medical Center, CHA University, Seoul, Korea. OBJECTIVE: Phospholipase C zeta 1 (PLCZ1), a strong candidate of egg activating sperm factor induces [Ca2+]i-oscillations in mammalian eggs that activate embryo development. Human sperm devoid PLCZ1 fails to induce [Ca2+]-oscillation in egg and lead to failure of egg activation and pregnancy. Here we produced human PLCZ1-specific antibody and evaluated the correlation of immunoreactivity of PLCZ1 in sperm and fertilization rate in ICSI patients. DESIGN: Study for new diagnostic test for male factor. MATERIALS AND METHODS: Two antibodies were raised in rabbits or rats against human PLCZ. After the completion of ICSI, the remaining patients’ sperm were washed in Dulbecco’s PBS (DPBS) and for immunofluorescence or western blotting. Fertilization rate was assessed 16–19 hours after ICSI by PN formation. We examined the immunoreactivity of sperm sample which showed normal fertilization rate (more than 90% after ICSI) for positive control. From western blot, intensity of PLCZ1 bands were quantified relatively compare to positive control by densitometer. From immunofluorescence, equatorial PLCZ1 staining on the sperm head were scored and compared to control sperm sample. The relevance of band intensity or immunofluorescence score and fertilization rate were investigated. RESULTS: We showed that repeated ICSI failure associated with egg activation failure is linked to the immunoreactivity of sperm against PLCZ1. We also presented evidence that PLCZ1-deficient or low expression of PLCZ1 reproduces the ICSI failure or low fertilization rate. CONCLUSION: According to these result, immunoreactivity of sperm against PLCZ1 could be an effective diagnostic test for male infertility in ART. Supported by: This work was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family affairs, Republic of Korea (A084923).
P-436 Wednesday, October 19, 2011 PHOSPHALIPASE C ZETA (PLCz) SPERM ANALYSIS IN PATIENTS WITH FAILED OR LOW FERTILIZATION AFTER ICSI. T. J. Jellerette-Nolan, H. C. Lee, M. Arny, D. Grow, R. Fissore. Reproductive Biology and Infertility Research, Department of Obstetrics and Gynecology, Baystate Medical Center, Sprignfield, MA; Animal Biotechnology and Biomedical Sciences, Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA. OBJECTIVE: To determine PLC zeta status in patients’ sperm that had either one or two failed or low fertilization ICSI cycles. DESIGN: Case series. MATERIALS AND METHODS: Following institutional IRB approval, infertile patients < 43 years of age undergoing Assisted Reproductive Technologies for intracytoplasmic sperm injection (ICSI) were consented for PLC zeta analysis of the husbands’ sperm after one or two failed or low fertilization ICSI cycles; the presence of protein was assessed by Western blot analysis. Monitoring of Ca2+ with fura-2 acetoxymethylester was performed after ICSI with human sperm into mouse eggs. RESULTS: Three couples presenting to Baystate Medical IVF clinic in 2010-2011 had either one or two failed or low fertilization ICSI cycles. Baystate Medical IVF treats 500 cycles per year; these three couples represent 1% of the patient population, which falls inline with national statistics for failed fertilization after ICSI. Couple 1: Female: 39yo, two previous ICSI cycles where 0/6 and 2/8 eggs fertilized, G1P0, husband’s SAN was within normal range; Mot: 47%; [23] Morph: 4.5. Couple 2: Female: 35yo, one previous cycle where 0/6 eggs fertilized, G0P0, Husband’s SAN was within normal range; Mot: 55%; [73] Morph: 5. Couple 3: Fe-
FERTILITY & STERILITYÒ
male: 42yo, one previous cycle 1/13 eggs fertilized. G0P0, husband’s SAN was not in normal range; Mot: 17%; [4.2] Morph: 2.0. The husband’s sperm was analyzed for calcium release and PLC zeta protein concentration; all sperm samples showed deficient calcium release patterns after ICSI into mouse eggs when compare to control. Western blot analysis revealed low PLC zeta content in all three patients’ sperm samples; patient 3< patient 2< patient 1. CONCLUSION: Our preliminary data demonstrate an association between poor ICSI outcome and sperm deficient in PLC zeta interestingly this is not always associated with poor SAN results. Supported by: This work was supported by Baystate Medical Center OB/ GYN dept.
P-437 Wednesday, October 19, 2011 IS OBESITY DELETERIOUS TO MALE FERTILITY POTENTIAL? S. Belloc, J. de Mouzon, I. Lichtblau, M. Cohen-Bacrie, S. Alvarez, P. Cohen-Bacrie. ART Unit, Laboratoire d’Eylau-Unilabs, Paris, France; INSERM, Unite Statistique, Paris, France; ART Unit, Clinique de la Muette, Paris, France. OBJECTIVE: Obesity is a well established risk factor for many diseases (hypertension, diabetes.). In women, obesity is frequently related to ovarian function troubles. In men, obesity has been shown to be associated to a decrease in testosterone levels. However, its impact on semen quality has been scarcely studied, with few publications. We analysed, on an important sample, the relationship between semen parameters and Body Mass Index (BMI), recorded at the time of examination. DESIGN: Retrospective study of an observational cohort of 5874 spermograms in a private ART Unit. MATERIALS AND METHODS: Self-reported men height and weight values have been recorded at each spermogram since October 2010 and until April 15, 2011. In case of several measurements, only the first one was included in the study, allowing to compare 400 obese men (BMI > 30 Kg/ m2) and 3415 normal weight men (18-25 Kg/m2). The following parameters were analyzed: semen volume, sperm concentration, total sperm count, motility, vitality and normal morphology (WHO criteria). Men’s age was also entered. Statistical analysis was performed using variance, chi-square, and multivariate logistic models. P< 0.05 was considered statistically significant. RESULTS: The rate of azoospermia increased from 1.0% to 3.8% between normal and obese men (P<0.01). Semen volume decreased from 3.3 1.6 to 3.0 1.6 ml (P<0.001), concentration from 60.9 61.9 to 48.8 53.8 millions/ml (P<0.001), and total sperm count from 184 194 to 135 157 million sperm (P<0.001). A similar decrease was found for overall motility (39.8 16.6 vs. 36.8 14.7 %) and progressive motility (36.8 16.7 vs. 33.5 15.0 % P<0.001). Logistic models including men’s age (strongly related to BMI and sperm concentration) confirmed the results of monovariate analysis. CONCLUSION: Sperm production is clearly altered in obese men quantitatively (sperm count) and qualitatively (motility). Male obesity needs to be taken into account in infertility diagnosis.
P-438 Wednesday, October 19, 2011 ASSESSMENT OF THE SPERM CENTROSOME. Q. V. Neri, V. Scala, Z. Rosenwaks, G. D. Palermo. The Ronald O. Perelman & Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY. OBJECTIVE: Different assays are available to screen the male gamete but only sperm morphology, including high magnification techniques, has been used to ascertain indirect information on the centrosome without actually assessing for its presence and integrity. DESIGN: We carried out an assay to screen for presence and integrity of the sperm centrosome in infertile and fertile men. MATERIALS AND METHODS: The centrin immunofluorescence localization at the edges of the proximal centriole allowed the angle measurement of the centriolar axis against the axonemal axis passing through the basal body. The centrosome was considered intact when 2 signals were observed. The angle generated between the proximal centriole and the flagellum was measured in control and study groups. An attempt to obtain sperm centrioles devoid of nuclear DNA was performed by mechanical dissection of spermatozoa.
S235
P-432 Wednesday, October 19, 2011 IN THE ERA OF IVF WITH ICSI, DO WE NEED TO REFER THE MALE PARTNER TO A REPRODUCTIVE UROLOGIST. B. A. McElyea, H. I. G. Cotton, K. McCarty, M. Acosta-La Greca, N. Bar-Chama, A. B. Copperman. Reproductive Medicine Associates of New York, New York, NY; Department of OBGYN and Reproductive Science, Mount Sinai School of Medicine, New York, NY. OBJECTIVE: When presenting for an infertility consult at RMA of NY, female patients undergo a comprehensive examination. Part of this evaluation includes obtaining an initial semen analysis from their male partners. If an abnormality is identified in the semen analysis (decreased sperm counts, motility, or morphology) then the male patient is often referred to the Urology department for a comprehensive infertility work up as well. The purpose of this study was to determine if the current practice of referring male patients for an infertility workup is appropriate, or even necessary, and to determine with the consultation results any change in the couple’s treatment plan. DESIGN: Retrospective data analysis at a private academic fertility center. MATERIALS AND METHODS: A comprehensive chart review of 100 consecutive males who presented to the Urology practice for evaluation was completed. We analyzed the reason for presentation to the Urologist and the resulting diagnosis. Only patients who were referred by a reproductive endocrinologist or Ob-Gyn specifically for the purpose of an infertility workup were included. Excluded were self-referred patients, patients who presented for issues other than fertility, and patients with pre-existing diagnoses who were referred for treatment. The final sample included 66 male patients. RESULTS: More than half (53%) of the men referred were identified to have a pathology that is easily treated.
Pathology
% of Referrals
Hypogonadal Positive Semen Cultures Varicocele Congenital Absence of Vas Deferens
15.2 9.1 27.3 1.5
CONCLUSION: The findings suggest that referral to Urology for further workup is appropriate, given that more than half of the men with abnormal semen analyses had easily treatable pathologies. Despite the fact that the treatment plan for IVF may not be altered in a majority of the cases, early identification of other treatable disorders may improve overall health in our male patient population.
S234
Abstracts
P-433 Wednesday, October 19, 2011 TESTICULAR SPERM PREINCUBATION AND ICSI OUTCOME. J. Kocent, Q. V. Neri, D. Monahan, Z. Rosenwaks, G. D. Palermo. The Ronald O. Perelman & Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY. OBJECTIVE: To determine the effect of lengthening the incubation time of testicular biopsy specimens on ICSI outcome. DESIGN: Fresh or cryopreserved testicular specimens incubated overnight were compared to those used the same day of ICSI. MATERIALS AND METHODS: From 2008 to 2011, microTESE were used on the oocyte retrieval day (SD) or incubated overnight at 37 C (ONI). Maternal age, sperm concentration, motility, fertilization rate, bHCG, and clinical pregnancy (CP) (with the presence of at least 1 FHB) were evaluated. RESULTS: A total of 296 used fresh TESE and 44 of those were ONI cycles with a remarkably lower proportion of motile spermatozoa (P¼0.04). Fertilization was 48.6% (231/475) and CP rate 34.1% (15/44), not different from the SD at 51.1% (129/252) and 40.0% (101/252), respectively. A total of 18 thawed TESE cycles were identified of which 12 ONI and 6 SD was cryopreserved the same day. The ONI group yielded a fertilization of 41.5% (37/89) and a CP of 50.0% (6/12) while the SD cryo had a fertilization of 37.8% (14/37) without achieving any pregnancy. To see whether the cryopreservation itself could have affected the testicular sperm’s performance, the corresponding fresh ONI (n ¼ 12) and SD (n ¼ 3) cycles were analyzed. The concentration was higher in the ONI (P¼0.03). A fertilization of 66.2% (86/130) and clinical pregnancy of 50.0% (6/12) was observed in the ONI while the SD were 48.1% (13/27) and 33.3% (1/3), respectively. To assess for optimal incubation conditions, 40 ONI cycles were assessed in which 36 were incubated at 37 C in 5% CO2 yielded a fertilization of 25.3% (20/79). In 4 cycles the incubation at RT yielded 50.0% (6/12) fertilization. CONCLUSION: Preincubation time of testicular spermatozoa does not seem to affect clinical ICSI outcomes. Cryopreservation paradoxically in the same day insemination did not yield any pregnancy. Although length of incubation does not have a negative affect on clinical outcomes, incubation at room temperature seems promising. Supported by: Institutional.
SPERM BIOLOGY P-434 Wednesday, October 19, 2011 DETECTION OF SPERM DNA DAMAGE BY RAMAN MICROSPECTROSCOPY. V. Sanchez, J. Wistuba, F. W€ubbeling, M. Burger, S. Schlatt, C. Mallidis. Centre of Reproductive Medicine and Andrology (CeRA), M€unster, North Rhine-Westfalia, Germany; Institute of Computational and Applied Mathematics, University of M€unster, M€unster, North Rhine-Westfalia, Germany. OBJECTIVE: Appraise ability of Raman microspectroscopy to identify and characterise oxidative sperm nDNA damage, thus ascertain the method’s potential as a selection tool of sperm suitable for ART. DESIGN: Controlled, blinded, prospective study. MATERIALS AND METHODS: After confirmation that samples were WHO normozoospermic, ejaculates from 5 healthy donors were washed in PBS then split into three aliquots. One remained untreated (control), the other two underwent differing degrees of oxidative attack via Fenton’s reaction (Fe2+ catalyst and H2O2). DNA fragmentation was determined by flow cytometric Acridine Orange Test (FLAOT) and Raman microspectroscopy (LabRam Aramis system, Horiba Jobin Yvon). Optimal instrumental settings/range were determined and duplicate 30 second single point spectra obtained from 200 sperm/sample/treatment. In total 3000 spectra were analyzed. Identification and frequency of sperm nDNA damage were assessed by Fourier filtering, Principal Component Analysis (PCA) and computation of local spectral angles relative to sample means. RESULTS: Oxidation caused by 1:10 and 1:5 concentrations of the Fenton reagent resulted in 50% and 100% (respectively) of sperm having nDNA damage (FLAOT DNA fragmentation index). PCA showed spectra from sperm with nDNA fragmentation were clearly distinguishable and significantly different from those with undamaged nDNA. Percentage of sperm identified as having damage correlated with FLAOT determination. Local spectral angle analysis, focussing on differences in main spectral peak due to DNA PO4 framework, found a clear shift from customary position at 1092 cm-1 towards 1042 cm-1, a translocation consistent with alterations
Vol. 96., No. 3, Supplement, September 2011
in the molecule’s vibrational capabilities due to perturbations in the DNA backbone. CONCLUSION: Oxidative nDNA fragmentation is manifest by a distinct Raman spectral profile which can be readily identified and used to select sperm with undamaged DNA. Supported by: CeRA internal funding.
P-435 Wednesday, October 19, 2011 IMMUNOREACTIVITY TEST OF SPERM AGAINST PLCZ1: EFFECTIVE DIAGNOSTIC TEST FOR MALE INFERTILITY IN ART. S.-Y. Yoon, T. H. Kim, M. K. Kim, H. H. Seuk, D. R. Lee, T. K. Yoon. Fertility Center of CHA Gangnam Medical Center, CHA University, Seoul, Korea. OBJECTIVE: Phospholipase C zeta 1 (PLCZ1), a strong candidate of egg activating sperm factor induces [Ca2+]i-oscillations in mammalian eggs that activate embryo development. Human sperm devoid PLCZ1 fails to induce [Ca2+]-oscillation in egg and lead to failure of egg activation and pregnancy. Here we produced human PLCZ1-specific antibody and evaluated the correlation of immunoreactivity of PLCZ1 in sperm and fertilization rate in ICSI patients. DESIGN: Study for new diagnostic test for male factor. MATERIALS AND METHODS: Two antibodies were raised in rabbits or rats against human PLCZ. After the completion of ICSI, the remaining patients’ sperm were washed in Dulbecco’s PBS (DPBS) and for immunofluorescence or western blotting. Fertilization rate was assessed 16–19 hours after ICSI by PN formation. We examined the immunoreactivity of sperm sample which showed normal fertilization rate (more than 90% after ICSI) for positive control. From western blot, intensity of PLCZ1 bands were quantified relatively compare to positive control by densitometer. From immunofluorescence, equatorial PLCZ1 staining on the sperm head were scored and compared to control sperm sample. The relevance of band intensity or immunofluorescence score and fertilization rate were investigated. RESULTS: We showed that repeated ICSI failure associated with egg activation failure is linked to the immunoreactivity of sperm against PLCZ1. We also presented evidence that PLCZ1-deficient or low expression of PLCZ1 reproduces the ICSI failure or low fertilization rate. CONCLUSION: According to these result, immunoreactivity of sperm against PLCZ1 could be an effective diagnostic test for male infertility in ART. Supported by: This work was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family affairs, Republic of Korea (A084923).
P-436 Wednesday, October 19, 2011 PHOSPHALIPASE C ZETA (PLCz) SPERM ANALYSIS IN PATIENTS WITH FAILED OR LOW FERTILIZATION AFTER ICSI. T. J. Jellerette-Nolan, H. C. Lee, M. Arny, D. Grow, R. Fissore. Reproductive Biology and Infertility Research, Department of Obstetrics and Gynecology, Baystate Medical Center, Sprignfield, MA; Animal Biotechnology and Biomedical Sciences, Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA. OBJECTIVE: To determine PLC zeta status in patients’ sperm that had either one or two failed or low fertilization ICSI cycles. DESIGN: Case series. MATERIALS AND METHODS: Following institutional IRB approval, infertile patients < 43 years of age undergoing Assisted Reproductive Technologies for intracytoplasmic sperm injection (ICSI) were consented for PLC zeta analysis of the husbands’ sperm after one or two failed or low fertilization ICSI cycles; the presence of protein was assessed by Western blot analysis. Monitoring of Ca2+ with fura-2 acetoxymethylester was performed after ICSI with human sperm into mouse eggs. RESULTS: Three couples presenting to Baystate Medical IVF clinic in 2010-2011 had either one or two failed or low fertilization ICSI cycles. Baystate Medical IVF treats 500 cycles per year; these three couples represent 1% of the patient population, which falls inline with national statistics for failed fertilization after ICSI. Couple 1: Female: 39yo, two previous ICSI cycles where 0/6 and 2/8 eggs fertilized, G1P0, husband’s SAN was within normal range; Mot: 47%; [23] Morph: 4.5. Couple 2: Female: 35yo, one previous cycle where 0/6 eggs fertilized, G0P0, Husband’s SAN was within normal range; Mot: 55%; [73] Morph: 5. Couple 3: Fe-
FERTILITY & STERILITYÒ
male: 42yo, one previous cycle 1/13 eggs fertilized. G0P0, husband’s SAN was not in normal range; Mot: 17%; [4.2] Morph: 2.0. The husband’s sperm was analyzed for calcium release and PLC zeta protein concentration; all sperm samples showed deficient calcium release patterns after ICSI into mouse eggs when compare to control. Western blot analysis revealed low PLC zeta content in all three patients’ sperm samples; patient 3< patient 2< patient 1. CONCLUSION: Our preliminary data demonstrate an association between poor ICSI outcome and sperm deficient in PLC zeta interestingly this is not always associated with poor SAN results. Supported by: This work was supported by Baystate Medical Center OB/ GYN dept.
P-437 Wednesday, October 19, 2011 IS OBESITY DELETERIOUS TO MALE FERTILITY POTENTIAL? S. Belloc, J. de Mouzon, I. Lichtblau, M. Cohen-Bacrie, S. Alvarez, P. Cohen-Bacrie. ART Unit, Laboratoire d’Eylau-Unilabs, Paris, France; INSERM, Unite Statistique, Paris, France; ART Unit, Clinique de la Muette, Paris, France. OBJECTIVE: Obesity is a well established risk factor for many diseases (hypertension, diabetes.). In women, obesity is frequently related to ovarian function troubles. In men, obesity has been shown to be associated to a decrease in testosterone levels. However, its impact on semen quality has been scarcely studied, with few publications. We analysed, on an important sample, the relationship between semen parameters and Body Mass Index (BMI), recorded at the time of examination. DESIGN: Retrospective study of an observational cohort of 5874 spermograms in a private ART Unit. MATERIALS AND METHODS: Self-reported men height and weight values have been recorded at each spermogram since October 2010 and until April 15, 2011. In case of several measurements, only the first one was included in the study, allowing to compare 400 obese men (BMI > 30 Kg/ m2) and 3415 normal weight men (18-25 Kg/m2). The following parameters were analyzed: semen volume, sperm concentration, total sperm count, motility, vitality and normal morphology (WHO criteria). Men’s age was also entered. Statistical analysis was performed using variance, chi-square, and multivariate logistic models. P< 0.05 was considered statistically significant. RESULTS: The rate of azoospermia increased from 1.0% to 3.8% between normal and obese men (P<0.01). Semen volume decreased from 3.3 1.6 to 3.0 1.6 ml (P<0.001), concentration from 60.9 61.9 to 48.8 53.8 millions/ml (P<0.001), and total sperm count from 184 194 to 135 157 million sperm (P<0.001). A similar decrease was found for overall motility (39.8 16.6 vs. 36.8 14.7 %) and progressive motility (36.8 16.7 vs. 33.5 15.0 % P<0.001). Logistic models including men’s age (strongly related to BMI and sperm concentration) confirmed the results of monovariate analysis. CONCLUSION: Sperm production is clearly altered in obese men quantitatively (sperm count) and qualitatively (motility). Male obesity needs to be taken into account in infertility diagnosis.
P-438 Wednesday, October 19, 2011 ASSESSMENT OF THE SPERM CENTROSOME. Q. V. Neri, V. Scala, Z. Rosenwaks, G. D. Palermo. The Ronald O. Perelman & Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY. OBJECTIVE: Different assays are available to screen the male gamete but only sperm morphology, including high magnification techniques, has been used to ascertain indirect information on the centrosome without actually assessing for its presence and integrity. DESIGN: We carried out an assay to screen for presence and integrity of the sperm centrosome in infertile and fertile men. MATERIALS AND METHODS: The centrin immunofluorescence localization at the edges of the proximal centriole allowed the angle measurement of the centriolar axis against the axonemal axis passing through the basal body. The centrosome was considered intact when 2 signals were observed. The angle generated between the proximal centriole and the flagellum was measured in control and study groups. An attempt to obtain sperm centrioles devoid of nuclear DNA was performed by mechanical dissection of spermatozoa.
S235