- Email: [email protected]
Abnormal sperm guidance in C. Elegans TGF-β SMAD mutants
development. Eggs are activated by repetitive increases in the intracellular concentration of free Ca2+, [Ca2+]i oscillations, which are triggered by the introduction during fertilization of the sperm-specific phospholipase C zeta 1 (PLCZ1). Recent studies have shown that sperm from patients lacking expression of PLCZ1 or expressing mutant forms of PLCZ1 fail to induce [Ca2+]i oscillations or egg activation. We first have purified recombinant human PLCZ1 protein and evaluated its [Ca2+] oscillation activity in mouse and human eggs with the view of investigating its application in the clinic for assisted oocyte activation in lieu of chemical agents. DESIGN: Experimental study. MATERIALS AND METHODS: Recombinant hPLCZ1 was synthesized using the E. coli system, and subjected to immunoblot analysis with antiPLCZ1 and anti-His tag antibodies. To evaluate the [Ca2+]i oscillatory activity, we performed microinjection of recombinant hPLCZ1into mouse or human mature eggs and [Ca2+]i monitoring. RESULTS: Injection of recombinant hPLCZ1 induced [Ca2+]i oscillations in a dose-dependent manner in both mouse and human eggs. These oscillations closely resemble those initiated by the sperm upon fertilization and triggered activation and cleavage in eggs of both species. U73122, a PLC inhibitor, blocked the ability of hPLCZ1 to initiate oscillations. CONCLUSION: Injection of recombinant protein could provide a biological solution in cases of ICSI failure or male factor infertility to induce artificial activation of eggs. 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).
M. Roldan, M. Martinez, S. Fortuno, N. Galindo, M. Munoz. IVI Alicante, Alicante, Spain. OBJECTIVE: To evaluate if seminal characteristics based on World Health Organization (WHO) 2010 criteria are related with parameters of embryo division kinetics measured with a time-lapse video system. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: We analyzed 721 embryos from oocyte donation. We performed both IVF (n ¼ 59) and ICSI (n ¼ 62) and classified sperm samples according WHO criteria into four categories related to concentration (<15 mill./ml; R15 mill/ml) and motility (<32% progressive; R32% progressive). We used the Embryoscope (Unisense Fertilitech, Aarhus, Denmark) to monitor the timing values of pronuclear fading (PNF), first and second cleavage (1st C, 2nd C), interval between them (IB), time of morula formation (M), blastocoel cavity formation (B) and expanded blastocyst (EB). T-test was applied with significance at P<0.05. RESULTS: When we analyzed embryo cleavage speed according sperm concentration and/or motility, we did not find significant differences (average values with 95% confidence intervals between brackets)
TABLE I PNF (h) <15 mill/ml (n ¼ 22)
P-463 Wednesday, October 19, 2011 ABNORMAL SPERM GUIDANCE IN C. ELEGANS TGF-b SMAD MUTANTS. K. K. McKnight, M. A. Miller. Obstetrics and Gynecology, Division of Reproductive Endocrinology, University of Alabama at Birmingham, Birmingham, AL; Cell Biology, University of Alabama at Birmingham, Birmingham, AL. OBJECTIVE: To determine if mutations in SMAD proteins in the TGFb signaling pathway result in sperm targeting defects in C. elegans. DESIGN: MitoTracker Red CMXRos sperm migration assays in C. elegans TGF- b SMAD mutants compared to wild-type. MATERIALS AND METHODS: MitoTracker Red CMXRos, a fluorescent dye that stains mitochondria, was used to label wild-type male sperm. Adult wild-type and SMAD mutant hermaphrodites (daf-3, daf8, and daf-14) were anesthetized with 0.1% tricaine and 0.01% tetramisole-HCl in M9 buffer for 30 minutes. The anesthetized worms were then transferred to a nematode growth media (NGM) plate containing 50-100 MitoTracker-stained wild-type males and were allowed to mate for 30 minutes. Mated hermaphrodites were removed from males for 1 hour and then mounted on a 2% agarose gel for microscopy. Imaging was performed using a Zeiss Axioshop 2 Plus with fluorescence and a 63X objective. Sperm accumulation was measured by calculating the percent of sperm found within a zone containing the fertilization site called the spermatheca (ST). 90% of sperm will accumulate at the ST within 1 hour of mating in wild-type worms. Student’s t-test was used to compare mean percent sperm accumulation in the ST of mutant and wild-type worms. RESULTS: daf-3 mutant hermaphrodites exhibited abnormal accumulation of wild-type sperm, with 77.8% reaching the ST compared to 91.1% in the wild-type (P¼0.002) after one hour. daf-8 mutants had a similar phenotype with 66.8% reaching the ST (P<0.001). daf-14 mutants also exhibited abnormal sperm accumulation with 49.4%% accumulating in the ST (P<0.001). CONCLUSION: Mutations in the C. elegans TGF-b SMAD proteins caused abnormal sperm accumulation in the reproductive tract, possibly due to abnormal oocyte prostaglandin synthesis. Further studies are needed to elucidate the mechanism by which TGF-b proteins influence sperm/oocyte interactions. Supported by: NIH R01 GM085105.
EMBRYO BIOLOGY P-464 Wednesday, October 19, 2011 SPERM QUALITY AS PER WHO (2010) CRITERIA DOS NOT AFFECT EMBRYO DIVISION KINETICS. TIME-LAPSE ANALYSIS OF EMBRYO DEVELOPMENTAL KEY EVENTS. B. Gadea,
FERTILITY & STERILITYÒ
J15 mill/ml (n ¼ 96)
23.4 (22.8-24)
1stC (h)
2ndC (h)
IB (h)
M (h)
B(h)
EB (h)
26.2
38.7
12.7
90.8
103.6
119.0
(25.5-26.9)
(37.6-39.8)
(12.2-13.4)
(89.4-92.2)
(101.7-105.5)
(113.3-124.7)
24.2
27.2
39.1
13.5
92.5
103.8
117.3
(23.8-24.6)
(26.7-27.6)
(38.4-39.8)
(13.4-13.9)
(90.9-94.1)
(102.2-105.4)
(113.3-121.3)
<32 % progressive
24.3
27.1
39.9
12.0
93.1
104.6
123.1
(n ¼ 42)
(23.7-24.9)
(26.4-27.8)
(39.0-40.8)
(11.5-12.5)
(90.6-95.6)
(102.3-106.9)
(117.5-128.7)
J32% progressive
23.8
26.9
38.5
12.5
91.6
103.4
115.6
(n ¼ 78)
(23.4-24.2)
(26.4-27.4)
(37.8-39.3)
(12.1-12.8)
(90.1-93.1)
(101.8-105.0)
(111.9-119.3)
CONCLUSION: We analyzed the possible correlation between two basic sperm parameters, concentration and motility,with embryo kinetics of development and we found that in humans we could not detect paternal effects on embryo timing. We can conclude that semen quality per se is not directly affecting the rate of development.
P-465 Wednesday, October 19, 2011 THE EFFECT OF TIMING OF EMBRYONIC PROGRESSION ON CHROMOSOMAL ABNORMALITY: DOES DELAYED BLASTULATION MEAN MORE ANEUPLOIDY? L. L. Kroener, G. Ambartsumyan, C. Briton-Jones, D. L. Hill. Obstetrics and Gyencology, Division of REI, University of California Los Angeles, Los Angeles, CA; Southern California Reproductive Center, Beverly Hills, CA. OBJECTIVE: To evaluate the relationship between aneuploidy and morphology in embryos blastulated on day 5 versus day 6. DESIGN: Historical cohort study. MATERIALS AND METHODS: Ninty-eight in-vitro fertilization (IVF) patients who underwent preimplantation genetic screening (PGS) with Comparative Genomic Hybridization (CGH) were included in this analysis. Cycles with day 3 transfers were excluded. Day 6 blastocysts were defined as embryos that blastulated on day 6. Aneuploidy rates were calculated for day 5 and day 6 embryos and stratified based on stage and morphologic grade (A-D). Grade A and B embryos were categorized as ‘‘good’’ quality whereas grades C and D were categorized as ‘‘poor’’ quality. Student’s t-test assuming unequal variance was used for comparison. RESULTS: Average maternal age was 39. No significant difference in aneuploidy was found between day 5 and day 6 blastocysts (0.69 vs 0.73) and between day 5 morulas and day 5 blastocysts (0.72 vs 0.69). There was a significant difference in aneuploidy rates between good quality day 5 blastocysts and day 5 morulas (0.58 vs 0.71). Of interest was the finding that good quality day 5 morulas were as likely to be aneuploid as poor quality day 5 blastocysts. Of the 65 cycles observed to have good quality day 5 morulas, only in 20 cycles the morulas progressed to
S243
M. Roldan, M. Martinez, S. Fortuno, N. Galindo, M. Munoz. IVI Alicante, Alicante, Spain. OBJECTIVE: To evaluate if seminal characteristics based on World Health Organization (WHO) 2010 criteria are related with parameters of embryo division kinetics measured with a time-lapse video system. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: We analyzed 721 embryos from oocyte donation. We performed both IVF (n ¼ 59) and ICSI (n ¼ 62) and classified sperm samples according WHO criteria into four categories related to concentration (<15 mill./ml; R15 mill/ml) and motility (<32% progressive; R32% progressive). We used the Embryoscope (Unisense Fertilitech, Aarhus, Denmark) to monitor the timing values of pronuclear fading (PNF), first and second cleavage (1st C, 2nd C), interval between them (IB), time of morula formation (M), blastocoel cavity formation (B) and expanded blastocyst (EB). T-test was applied with significance at P<0.05. RESULTS: When we analyzed embryo cleavage speed according sperm concentration and/or motility, we did not find significant differences (average values with 95% confidence intervals between brackets)
TABLE I PNF (h) <15 mill/ml (n ¼ 22)
P-463 Wednesday, October 19, 2011 ABNORMAL SPERM GUIDANCE IN C. ELEGANS TGF-b SMAD MUTANTS. K. K. McKnight, M. A. Miller. Obstetrics and Gynecology, Division of Reproductive Endocrinology, University of Alabama at Birmingham, Birmingham, AL; Cell Biology, University of Alabama at Birmingham, Birmingham, AL. OBJECTIVE: To determine if mutations in SMAD proteins in the TGFb signaling pathway result in sperm targeting defects in C. elegans. DESIGN: MitoTracker Red CMXRos sperm migration assays in C. elegans TGF- b SMAD mutants compared to wild-type. MATERIALS AND METHODS: MitoTracker Red CMXRos, a fluorescent dye that stains mitochondria, was used to label wild-type male sperm. Adult wild-type and SMAD mutant hermaphrodites (daf-3, daf8, and daf-14) were anesthetized with 0.1% tricaine and 0.01% tetramisole-HCl in M9 buffer for 30 minutes. The anesthetized worms were then transferred to a nematode growth media (NGM) plate containing 50-100 MitoTracker-stained wild-type males and were allowed to mate for 30 minutes. Mated hermaphrodites were removed from males for 1 hour and then mounted on a 2% agarose gel for microscopy. Imaging was performed using a Zeiss Axioshop 2 Plus with fluorescence and a 63X objective. Sperm accumulation was measured by calculating the percent of sperm found within a zone containing the fertilization site called the spermatheca (ST). 90% of sperm will accumulate at the ST within 1 hour of mating in wild-type worms. Student’s t-test was used to compare mean percent sperm accumulation in the ST of mutant and wild-type worms. RESULTS: daf-3 mutant hermaphrodites exhibited abnormal accumulation of wild-type sperm, with 77.8% reaching the ST compared to 91.1% in the wild-type (P¼0.002) after one hour. daf-8 mutants had a similar phenotype with 66.8% reaching the ST (P<0.001). daf-14 mutants also exhibited abnormal sperm accumulation with 49.4%% accumulating in the ST (P<0.001). CONCLUSION: Mutations in the C. elegans TGF-b SMAD proteins caused abnormal sperm accumulation in the reproductive tract, possibly due to abnormal oocyte prostaglandin synthesis. Further studies are needed to elucidate the mechanism by which TGF-b proteins influence sperm/oocyte interactions. Supported by: NIH R01 GM085105.
EMBRYO BIOLOGY P-464 Wednesday, October 19, 2011 SPERM QUALITY AS PER WHO (2010) CRITERIA DOS NOT AFFECT EMBRYO DIVISION KINETICS. TIME-LAPSE ANALYSIS OF EMBRYO DEVELOPMENTAL KEY EVENTS. B. Gadea,
FERTILITY & STERILITYÒ
J15 mill/ml (n ¼ 96)
23.4 (22.8-24)
1stC (h)
2ndC (h)
IB (h)
M (h)
B(h)
EB (h)
26.2
38.7
12.7
90.8
103.6
119.0
(25.5-26.9)
(37.6-39.8)
(12.2-13.4)
(89.4-92.2)
(101.7-105.5)
(113.3-124.7)
24.2
27.2
39.1
13.5
92.5
103.8
117.3
(23.8-24.6)
(26.7-27.6)
(38.4-39.8)
(13.4-13.9)
(90.9-94.1)
(102.2-105.4)
(113.3-121.3)
<32 % progressive
24.3
27.1
39.9
12.0
93.1
104.6
123.1
(n ¼ 42)
(23.7-24.9)
(26.4-27.8)
(39.0-40.8)
(11.5-12.5)
(90.6-95.6)
(102.3-106.9)
(117.5-128.7)
J32% progressive
23.8
26.9
38.5
12.5
91.6
103.4
115.6
(n ¼ 78)
(23.4-24.2)
(26.4-27.4)
(37.8-39.3)
(12.1-12.8)
(90.1-93.1)
(101.8-105.0)
(111.9-119.3)
CONCLUSION: We analyzed the possible correlation between two basic sperm parameters, concentration and motility,with embryo kinetics of development and we found that in humans we could not detect paternal effects on embryo timing. We can conclude that semen quality per se is not directly affecting the rate of development.
P-465 Wednesday, October 19, 2011 THE EFFECT OF TIMING OF EMBRYONIC PROGRESSION ON CHROMOSOMAL ABNORMALITY: DOES DELAYED BLASTULATION MEAN MORE ANEUPLOIDY? L. L. Kroener, G. Ambartsumyan, C. Briton-Jones, D. L. Hill. Obstetrics and Gyencology, Division of REI, University of California Los Angeles, Los Angeles, CA; Southern California Reproductive Center, Beverly Hills, CA. OBJECTIVE: To evaluate the relationship between aneuploidy and morphology in embryos blastulated on day 5 versus day 6. DESIGN: Historical cohort study. MATERIALS AND METHODS: Ninty-eight in-vitro fertilization (IVF) patients who underwent preimplantation genetic screening (PGS) with Comparative Genomic Hybridization (CGH) were included in this analysis. Cycles with day 3 transfers were excluded. Day 6 blastocysts were defined as embryos that blastulated on day 6. Aneuploidy rates were calculated for day 5 and day 6 embryos and stratified based on stage and morphologic grade (A-D). Grade A and B embryos were categorized as ‘‘good’’ quality whereas grades C and D were categorized as ‘‘poor’’ quality. Student’s t-test assuming unequal variance was used for comparison. RESULTS: Average maternal age was 39. No significant difference in aneuploidy was found between day 5 and day 6 blastocysts (0.69 vs 0.73) and between day 5 morulas and day 5 blastocysts (0.72 vs 0.69). There was a significant difference in aneuploidy rates between good quality day 5 blastocysts and day 5 morulas (0.58 vs 0.71). Of interest was the finding that good quality day 5 morulas were as likely to be aneuploid as poor quality day 5 blastocysts. Of the 65 cycles observed to have good quality day 5 morulas, only in 20 cycles the morulas progressed to
S243