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Sperm exposure to cyclophosphamide reduces preimplantation embryo development after ICSI
P-412 Wednesday, October 22, 2014 SPERM EXPOSURE TO CYCLOPHOSPHAMIDE REDUCES PREIMPLANTATION EMBRYO DEVELOPMENT AFTER ICSI. M. D. Johnson, C.-C. Lin, M. Sukhwani, S. Malik, K. E. Orwig. Department of Obstetrics, Gynecology and Reproductive Sciences, MageeWomens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA. OBJECTIVE: To determine the effect of cyclophosphamide administration on epididymal sperm and subsequent embryo development. DESIGN: Prospective Laboratory Study. MATERIALS AND METHODS: Sperm was collected from the cauda epididymis of adult male B6D2 mice. Seven days later intraperitoneal cyclophosphamide (CYC) (n¼4) or vehicle (VEH) (n¼4) were administered at 300 mg/kg). Mice were sacrificed seven days later to obtain sperm from the contralateral cauda epididymis. Sperm samples were cryopreserved at the time of collection and thawed prior to use for intracytoplasmic sperm injection (ICSI). Oocytes were collected from B6D2 female mice (n¼16) after controlled ovarian stimulation and divided equally into two groups. Half the oocytes from each female were fertilized with CYC or VEH exposed sperm using ICSI, the other half were fertilized with pre exposed sperm from the same male. Sperm motility was assessed at the time of ICSI. Embryos were monitored for in-vitro development for two days. The number of oocytes obtained, injected and survived as well as embryo development were compared among the four groups using a student’s t-test or ANOVA. RESULTS: Five hundred twenty-three embryos from eight in-vitro fertilization cycles with ICSI were monitored for preimplantation development. The mean number of oocytes surviving ICSI was 16.4 1.8 (94.4%) and was similar in all four groups (P>0.50). The mean motility was 26.5% 1.1% at the time of ICSI and was similar in all four groups (P>0.50). In the CYC arm, the number of 8-cell embryos was significantly lower for the Post exposure group than the Pre exposure group. 57% of 2-cell embryos in the CYC-Pre group progressed to 8-cell embryos, while only 19% of 2-cell embryos progressed to 8-cell embryos in the CYC-Post group (P¼0.002). There was no difference between the Pre and Post groups in the VEH arm (56% vs 47%, P¼0.39).
Percentage Embryo Development, by Treatment Group
VEH Pre
Post
CYC Pre vs. Post P-Value
2-cell / Oocytes 97.6 94.9 Survived 8-cell / 2-cell 56.0 46.6
Pre
Post
0.38
92.8 87.8
0.39
56.9 19.1
Pre vs. Post P-Value 0.38 .002
CONCLUSION: CYC treatment does not impact sperm motility, but significantly decreases preimplantation embryo development following ICSI. Supported by: NIH grant HD055475, Magee-Womens Research Institute and Foundation and gifts from Sylvia Bernassoli. P-413 Wednesday, October 22, 2014 ACUTE EFFECTS OF ALKYLATING CHEMOTHERAPY (CTX) ON SEMEN PARAMETERS AND PREIMPLANTATION EMBRYO DEVELOPMENT AFTER INTRACYTOPLASMIC SPERM INJECTION (ICSI) IN A MOUSE MODEL. S. Malik, C.-C. Lin, M. Sukhwani, M. D. Johnson, M. Yang, A. Althouse, K. E. Orwig. Magee Womens Research Institute, Pittsburgh, PA. OBJECTIVE: To determine the effect of alkylating CTx on semen parameters and preimplantation embryo development after ICSI. DESIGN: Prospective Laboratory Study.
FERTILITY & STERILITYÒ
MATERIALS AND METHODS: Sperm was collected from the cauda epididymis of adult male C57BL/6 mice prior to CTx. After recovery, the mice were given intraperitoneal busulfan (55mg/kg) and sacrificed seven days later to obtain CTx exposed sperm from the contralateral epididymis. All sperm was cryopreserved at collection and thawed prior to use. Oocytes collected from individual C57BL/6 female mice were divided into two groups and fertilized by ICSI with motile sperm with normal morphology. Half were fertilized with pre-CTx sperm and the other half were fertilized with busulfan-exposed sperm from the same male. This design was repeated for 10 replicate males. Primary outcome measures were semen analyses and preimplantation embryo development after ICSI. Semen parameters were compared using pairwise comparisons and blastocyst development rate was analyzed using the Generalized Estimating Equations approach. RESULTS: Two hundred-forty embryos from ten cycles were monitored for preimplantation development to the blastocyst stage. CTx had a significant negative effect on sperm motility (38% 6.32SEM vs 19% 5.06; p¼0.03) and morphology (84% 1.58 vs 80 1.26; p¼0.02). However, overall sperm counts (100.36 8.8 vs 71.8 11.7 million; p¼NS) were not significantly affected. Chemotherapy did not have a significant effect on the rate of blastocyst development (p¼NS). In the pre-Ctx group, blastocyst development was 23.08% compared to 18.67% in the post-Ctx group. CONCLUSION: Alkylating CTx acutely impacts semen motility and morphology. Pre-implantation embryo development was not affected when CTx-exposed sperm was used for ICSI fertilization. Exome sequencing of the resultant blastocysts is in process to assess for genetic variability between the pre-CTx and post-CTx group. Supported by: NIH grant HD055475, Magee-Womens Research Institute and Foundation and gifts from Sylvia Bernassoli.
P-414 Wednesday, October 22, 2014 IMPACT OF INTERMENSTRUAL BLEEDING ON NATURAL FERTILITY. N. M. Crawford, K. Chantala, A. Z. Steiner. University of North Carolina, Chapel Hill, NC. OBJECTIVE: To determine the impact of intermenstrual and luteal phase bleeding on natural fertility. DESIGN: Prospective, time-to-conceive study. MATERIALS AND METHODS: Women, 30-44 years old, with no history of infertility, who were trying to conceive for less than 3 months, were enrolled and followed until pregnancy. Each day women recorded bleeding, ovulation test results, intercourse, and pregnancy test results for up to 4 months while attempting to conceive. For each cycle, presence of intermenstrual bleeding or luteal phase bleeding was determined. Intermenstrual bleeding was defined as any bleeding which occurred after completion of menses. Luteal phase bleeding was defined as any bleeding which occurred after ovulation and prior to the start of the next menstrual cycle. Cycles were restricted to 25 to 35 days in length to exclude anovulatory bleeding. We examined both the impact of bleeding on conception, defined as a positive pregnancy test, in the current cycle (current cycle fecundability) as well as the impact of bleeding on conception in the next cycle (future cycle fecundability). Discrete models were used to calculate fecundability ratios (FR) to compare subjects with bleeding to those without adjusting for maternal age. RESULTS: 1767 cycles from 537 women were included in the analysis. Intermenstrual bleeding occurred in 35% of cycles, and luteal phase bleeding occurred in 33% of cycles. Women, who had bleeding, tended to be younger (<35 years), Caucasian, and of a normal body mass index. Compared to subjects without any bleeding, women with intermenstrual bleeding had 0.29 times the odds of pregnancy (95% CI: 0.2-0.41) in that cycle. Furthermore, if bleeding occurred in the luteal phase of the cycle, women had 0.23 times the odds of pregnancy (95% CI: 0.16-0.35). However, if a woman had intermenstrual bleeding or luteal phase bleeding, her future cycle fecundability was not significantly different from women without bleeding (FR 1.06, 95% CI: 0.77-1.48, and FR: 1.24, 95% CI: 0.88-1.74, respectively). CONCLUSION: Intermenstrual bleeding significantly decreases the odds of pregnancy detection in that cycle; however, it does not appear to impact a woman’s future reproductive potential. Supported by: This work was funded by the NIH/NICHD grant R01067683.
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Percentage Embryo Development, by Treatment Group
VEH Pre
Post
CYC Pre vs. Post P-Value
2-cell / Oocytes 97.6 94.9 Survived 8-cell / 2-cell 56.0 46.6
Pre
Post
0.38
92.8 87.8
0.39
56.9 19.1
Pre vs. Post P-Value 0.38 .002
CONCLUSION: CYC treatment does not impact sperm motility, but significantly decreases preimplantation embryo development following ICSI. Supported by: NIH grant HD055475, Magee-Womens Research Institute and Foundation and gifts from Sylvia Bernassoli. P-413 Wednesday, October 22, 2014 ACUTE EFFECTS OF ALKYLATING CHEMOTHERAPY (CTX) ON SEMEN PARAMETERS AND PREIMPLANTATION EMBRYO DEVELOPMENT AFTER INTRACYTOPLASMIC SPERM INJECTION (ICSI) IN A MOUSE MODEL. S. Malik, C.-C. Lin, M. Sukhwani, M. D. Johnson, M. Yang, A. Althouse, K. E. Orwig. Magee Womens Research Institute, Pittsburgh, PA. OBJECTIVE: To determine the effect of alkylating CTx on semen parameters and preimplantation embryo development after ICSI. DESIGN: Prospective Laboratory Study.
FERTILITY & STERILITYÒ
MATERIALS AND METHODS: Sperm was collected from the cauda epididymis of adult male C57BL/6 mice prior to CTx. After recovery, the mice were given intraperitoneal busulfan (55mg/kg) and sacrificed seven days later to obtain CTx exposed sperm from the contralateral epididymis. All sperm was cryopreserved at collection and thawed prior to use. Oocytes collected from individual C57BL/6 female mice were divided into two groups and fertilized by ICSI with motile sperm with normal morphology. Half were fertilized with pre-CTx sperm and the other half were fertilized with busulfan-exposed sperm from the same male. This design was repeated for 10 replicate males. Primary outcome measures were semen analyses and preimplantation embryo development after ICSI. Semen parameters were compared using pairwise comparisons and blastocyst development rate was analyzed using the Generalized Estimating Equations approach. RESULTS: Two hundred-forty embryos from ten cycles were monitored for preimplantation development to the blastocyst stage. CTx had a significant negative effect on sperm motility (38% 6.32SEM vs 19% 5.06; p¼0.03) and morphology (84% 1.58 vs 80 1.26; p¼0.02). However, overall sperm counts (100.36 8.8 vs 71.8 11.7 million; p¼NS) were not significantly affected. Chemotherapy did not have a significant effect on the rate of blastocyst development (p¼NS). In the pre-Ctx group, blastocyst development was 23.08% compared to 18.67% in the post-Ctx group. CONCLUSION: Alkylating CTx acutely impacts semen motility and morphology. Pre-implantation embryo development was not affected when CTx-exposed sperm was used for ICSI fertilization. Exome sequencing of the resultant blastocysts is in process to assess for genetic variability between the pre-CTx and post-CTx group. Supported by: NIH grant HD055475, Magee-Womens Research Institute and Foundation and gifts from Sylvia Bernassoli.
P-414 Wednesday, October 22, 2014 IMPACT OF INTERMENSTRUAL BLEEDING ON NATURAL FERTILITY. N. M. Crawford, K. Chantala, A. Z. Steiner. University of North Carolina, Chapel Hill, NC. OBJECTIVE: To determine the impact of intermenstrual and luteal phase bleeding on natural fertility. DESIGN: Prospective, time-to-conceive study. MATERIALS AND METHODS: Women, 30-44 years old, with no history of infertility, who were trying to conceive for less than 3 months, were enrolled and followed until pregnancy. Each day women recorded bleeding, ovulation test results, intercourse, and pregnancy test results for up to 4 months while attempting to conceive. For each cycle, presence of intermenstrual bleeding or luteal phase bleeding was determined. Intermenstrual bleeding was defined as any bleeding which occurred after completion of menses. Luteal phase bleeding was defined as any bleeding which occurred after ovulation and prior to the start of the next menstrual cycle. Cycles were restricted to 25 to 35 days in length to exclude anovulatory bleeding. We examined both the impact of bleeding on conception, defined as a positive pregnancy test, in the current cycle (current cycle fecundability) as well as the impact of bleeding on conception in the next cycle (future cycle fecundability). Discrete models were used to calculate fecundability ratios (FR) to compare subjects with bleeding to those without adjusting for maternal age. RESULTS: 1767 cycles from 537 women were included in the analysis. Intermenstrual bleeding occurred in 35% of cycles, and luteal phase bleeding occurred in 33% of cycles. Women, who had bleeding, tended to be younger (<35 years), Caucasian, and of a normal body mass index. Compared to subjects without any bleeding, women with intermenstrual bleeding had 0.29 times the odds of pregnancy (95% CI: 0.2-0.41) in that cycle. Furthermore, if bleeding occurred in the luteal phase of the cycle, women had 0.23 times the odds of pregnancy (95% CI: 0.16-0.35). However, if a woman had intermenstrual bleeding or luteal phase bleeding, her future cycle fecundability was not significantly different from women without bleeding (FR 1.06, 95% CI: 0.77-1.48, and FR: 1.24, 95% CI: 0.88-1.74, respectively). CONCLUSION: Intermenstrual bleeding significantly decreases the odds of pregnancy detection in that cycle; however, it does not appear to impact a woman’s future reproductive potential. Supported by: This work was funded by the NIH/NICHD grant R01067683.
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