- Email: [email protected]
Blastocyst development in an extended embryo culture system after intracytoplasmic sperm injection
International Congress Series 1271 (2004) 155 – 158
www.ics-elsevier.com
Blastocyst development in an extended embryo culture system after intracytoplasmic sperm injection G.D. Adamson, M. Gvakharia * Fertility Physicians of Northern California, Palo Alto, CA, USA
Abstract. Introduction: The aim of this study was to determine the factors affecting blastocyst formation in an extended (5 – 6 days) embryo culture system after intracytoplasmic sperm injection (ICSI). Methods: The study included 77 consecutive couples (mean maternal age 34.5 years), undergoing IVF with the ICSI procedure and blastocyst culture for infertility treatment in a private clinic setting. These patients were divided into the following groups: group A, patients with total motile sperm count of 0 – 5 106 (n = 16); group B, patients with total motile sperm count of >5 106 (n = 61); group C, patients with normal sperm morphology (Krugers’s strict criteria) of 0 – 5% (n = 55); and group D, patients with normal sperm morphology of >5% (n = 22). A control group of 160 standard IVF patients not undergoing ICSI was also compared. Embryos were cultured for up to 6 days in a sequential embryo culture system (Irvine Scientific, CA). Development of zygotes to blastocysts in vitro was used as the main outcome measure for this study. Results: Overall, 853 oocytes were injected with normal fertilization and embryo cleavage rates of 74.5% and 99.2%, respectively. Overall, 631 supernumerary ICSI embryos were cultured for possible blastocyst development, resulting in 189 blastocysts (30% overall blastocyst development rate). Blastocyst development results in different groups were group A, 26%; group B, 32%; group C, 31%; and group D, 30%. There was no statistically significant difference in blastocyst development rates among these groups of patients. A blastocyst development rate of 38% was observed in the standard IVF group vs. 30% in IVF-ICSI group ( p < 0.05). Conclusions: Although the blastocyst development rate of ICSI embryos is statistically lower than the blastocyst development rate of non-ICSI embryos, this decrease is not statistically correlated with any of measured semen parameters. These results suggest that the ICSI procedure may have an adverse effect on the ability of the embryo to form a blastocyst in vitro. D 2004 Elsevier B.V. All rights reserved. Keywords: Blastocyst development; ICSI; Micromanipulation of gametes
1. Introduction Prolonged culture of embryos to blastocyst stage provides potential opportunities for optimized selection of viable embryos for transfer. Additionally, it has been hypothesized that blastocyst stage transfer may optimize implantation by better synchronization * Corresponding author. Tel.: +1-408-358-5624; fax: +1-408-358-5629. E-mail address: [email protected] (M. Gvakharia). 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.05.152
156
G.D. Adamson, M. Gvakharia / International Congress Series 1271 (2004) 155–158
between the endometrial lining and embryos [1,2]. The recent introduction of sequential embryo culture systems greatly improved embryo development to blastocyst stage in culture. However, some patients may still experience suboptimal rates of blastocyst development during their IVF cycles. Therefore, it is important to recognize the limitations of this process in order to minimize the occurrence of failed blastocyst development. Factors such as maternal age [3,4], number of oocytes retrieved, number of available zygotes and number of eight cell embryos [5,6] have been shown to affect blastocyst development in vitro. It is controversial, however, if method of fertilization (micromanipulation-assisted vs. conventional) has a measurable effect on blastocyst development [5,7,8]. The aim of this study was to compare blastocyst development rates between standard IVF and IVF-ICSI patients and to determine the factors affecting blastocyst formation in an extended (5 – 6 days) embryo culture system after intracytoplasmic sperm injection (ICSI). 2. Materials and methods 2.1. Patients The study included 77 couples undergoing IVF-ICSI for infertility treatment in our center and 160 couples undergoing standard IVF as a control group. There was no significant difference in mean maternal age (33.9 vs. 34.0), number of retrieved oocytes (18.5 vs. 16.6), fertilization rate (75% vs. 70%), embryo cleavage rate (99% vs. 98%), mean number of blastomeres (4.6 vs. 5.1) and grade of the embryos between standard IVF and IVF-ICSI groups. IVF-ICSI patients were further divided into following groups: group A, patients with total motile sperm count of 0 –5 106 (n = 16); group B, patients with total motile sperm count of >5 106 (n = 61); group C, patients with normal sperm morphology (Krugers’s strict criteria) of 0 –5% (n = 55); and group D, patients with normal sperm morphology of >5% (n = 22). Results were analyzed using v2 test with Yates correction. A p-value of < 0.05 was considered statistically significant. 2.2. Stimulation protocol and embryo culture Controlled ovarian hyperstimulation protocol consisted of initial treatment with gonadotropin-releasing hormone agonist followed by recombinant human FSH. Oocyte retrieval was performed 35 h after hCG injection. Sperm samples were analyzed in accordance with WHO protocols. Strict morphology of sperm was assessed according to Kruger’s strict criteria. Fertilization was assessed after 18 h of insemination/ICSI and only normally fertilized oocytes were cultured. Embryos were cultured for up to 6 days in a sequential embryo culture system (Irvine Scientific, CA). Development of zygotes to blastocysts in vitro was used as the main outcome measure for this study. 3. Results Overall, 853 oocytes were injected with normal fertilization and embryo cleavage rates of 75% and 99%, respectively. A total of 631 ICSI embryos were cultured for
G.D. Adamson, M. Gvakharia / International Congress Series 1271 (2004) 155–158
157
Table 1 Blastocyst development in standard IVF and IVF-ICSI patients
Embryos cultured Blastocysts developed Blastocyst development rate Statistical significance
IVF group n = 160
ICSI group n = 77
ICSI group A n = 16
ICSI group B n = 61
ICSI group C n = 55
ICSI group D n = 22
1472 560 38
631 189 30
147 38 26
484 151 31
470 146 31
161 43 28
p>0.05
p>0.05
p>0.05
p>0.05
p < 0.05
possible blastocyst development, resulting in 189 blastocysts (30% overall blastocyst development rate). Blastocyst development results in different groups were group A, 26%; group B, 32%; group C, 31%; and group D, 30%. There was no statistically significant difference in blastocyst development rates among these groups of patients (Table 1). A blastocyst development rate of 38% was observed in the standard IVF control group vs. 30% in IVF-ICSI group ( p < 0.05). 4. Discussion The objectives of this study were to study the possible effect of ICSI procedure on embryo development to blastocyst in vitro and to determine whether major sperm parameters influence blastulation rates after ICSI. Based on presented results, the blastocyst development rate of IVF embryos is statistically higher than blastocyst development rate of ICSI embryos. In order to analyze whether this impaired blastulation after ICSI was associated with sperm parameters, we identified four groups of ICSI patients as described in Materials and methods. There was no significant difference in blastocyst development in patient groups based on total motile sperm count or strict morphology (Table 1). In conclusion, although the blastocyst development rate of ICSI embryos is statistically lower than blastocyst development rate of non-ICSI embryos, this decrease is not statistically correlated with any of measured semen parameters. These results suggest that the ICSI procedure may have an adverse effect on the ability of the embryo to form a blastocyst in vitro. References [1] D.K. Gardner, et al., Culture and transfer of human blastocysts increases implantation rate and reduces the need for multiple embryo transfers, Fertil. Steril. 69 (1998) 84 – 88. [2] B. Behr, et al., Preliminary clinical experience with human blastocyst development in vitro without coculture, Hum. Reprod. 14 (1999) 454 – 457. [3] M.C.W. Scholtes, G.H. Zeilmaker, A prospective, randomized study of embryo transfer results after 3 or 5 days of embryo culture in in vitro fertilization, Fertil. Steril. 65 (1996) 1245 – 1248. [4] L. Janny, Y.J. Menezo, Maternal age effect on early human embryonic development and blastocyst formation, Mol. Reprod. Dev. 45 (1996) 31 – 37. [5] Z. Karaki, et al., Blastocyst culture and transfer: a step toward improved in vitro fertilization outcome, Fertil. Steril. 77 (2002 January) 114 – 118.
158
G.D. Adamson, M. Gvakharia / International Congress Series 1271 (2004) 155–158
[6] C. Racowsky, et al., The number of eight-cell embryos is a key determinant for selecting day 3 or day 5 transfer, Fertil. Steril. 73 (2000) 558 – 564. [7] Y. Shoukri, et al., Blastocyst development from supranumerary embryos after intracytoplasmic sperm injection: a paternal influence, Hum. Reprod. 13 (1998) 1632 – 1637. [8] J.C.M. Dumoulin, et al., Embryo development and chromosomal anomalies after ICSI: effect of the injection procedure, Hum. Reprod. 16 (2001) 306 – 312.
www.ics-elsevier.com
Blastocyst development in an extended embryo culture system after intracytoplasmic sperm injection G.D. Adamson, M. Gvakharia * Fertility Physicians of Northern California, Palo Alto, CA, USA
Abstract. Introduction: The aim of this study was to determine the factors affecting blastocyst formation in an extended (5 – 6 days) embryo culture system after intracytoplasmic sperm injection (ICSI). Methods: The study included 77 consecutive couples (mean maternal age 34.5 years), undergoing IVF with the ICSI procedure and blastocyst culture for infertility treatment in a private clinic setting. These patients were divided into the following groups: group A, patients with total motile sperm count of 0 – 5 106 (n = 16); group B, patients with total motile sperm count of >5 106 (n = 61); group C, patients with normal sperm morphology (Krugers’s strict criteria) of 0 – 5% (n = 55); and group D, patients with normal sperm morphology of >5% (n = 22). A control group of 160 standard IVF patients not undergoing ICSI was also compared. Embryos were cultured for up to 6 days in a sequential embryo culture system (Irvine Scientific, CA). Development of zygotes to blastocysts in vitro was used as the main outcome measure for this study. Results: Overall, 853 oocytes were injected with normal fertilization and embryo cleavage rates of 74.5% and 99.2%, respectively. Overall, 631 supernumerary ICSI embryos were cultured for possible blastocyst development, resulting in 189 blastocysts (30% overall blastocyst development rate). Blastocyst development results in different groups were group A, 26%; group B, 32%; group C, 31%; and group D, 30%. There was no statistically significant difference in blastocyst development rates among these groups of patients. A blastocyst development rate of 38% was observed in the standard IVF group vs. 30% in IVF-ICSI group ( p < 0.05). Conclusions: Although the blastocyst development rate of ICSI embryos is statistically lower than the blastocyst development rate of non-ICSI embryos, this decrease is not statistically correlated with any of measured semen parameters. These results suggest that the ICSI procedure may have an adverse effect on the ability of the embryo to form a blastocyst in vitro. D 2004 Elsevier B.V. All rights reserved. Keywords: Blastocyst development; ICSI; Micromanipulation of gametes
1. Introduction Prolonged culture of embryos to blastocyst stage provides potential opportunities for optimized selection of viable embryos for transfer. Additionally, it has been hypothesized that blastocyst stage transfer may optimize implantation by better synchronization * Corresponding author. Tel.: +1-408-358-5624; fax: +1-408-358-5629. E-mail address: [email protected] (M. Gvakharia). 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.05.152
156
G.D. Adamson, M. Gvakharia / International Congress Series 1271 (2004) 155–158
between the endometrial lining and embryos [1,2]. The recent introduction of sequential embryo culture systems greatly improved embryo development to blastocyst stage in culture. However, some patients may still experience suboptimal rates of blastocyst development during their IVF cycles. Therefore, it is important to recognize the limitations of this process in order to minimize the occurrence of failed blastocyst development. Factors such as maternal age [3,4], number of oocytes retrieved, number of available zygotes and number of eight cell embryos [5,6] have been shown to affect blastocyst development in vitro. It is controversial, however, if method of fertilization (micromanipulation-assisted vs. conventional) has a measurable effect on blastocyst development [5,7,8]. The aim of this study was to compare blastocyst development rates between standard IVF and IVF-ICSI patients and to determine the factors affecting blastocyst formation in an extended (5 – 6 days) embryo culture system after intracytoplasmic sperm injection (ICSI). 2. Materials and methods 2.1. Patients The study included 77 couples undergoing IVF-ICSI for infertility treatment in our center and 160 couples undergoing standard IVF as a control group. There was no significant difference in mean maternal age (33.9 vs. 34.0), number of retrieved oocytes (18.5 vs. 16.6), fertilization rate (75% vs. 70%), embryo cleavage rate (99% vs. 98%), mean number of blastomeres (4.6 vs. 5.1) and grade of the embryos between standard IVF and IVF-ICSI groups. IVF-ICSI patients were further divided into following groups: group A, patients with total motile sperm count of 0 –5 106 (n = 16); group B, patients with total motile sperm count of >5 106 (n = 61); group C, patients with normal sperm morphology (Krugers’s strict criteria) of 0 –5% (n = 55); and group D, patients with normal sperm morphology of >5% (n = 22). Results were analyzed using v2 test with Yates correction. A p-value of < 0.05 was considered statistically significant. 2.2. Stimulation protocol and embryo culture Controlled ovarian hyperstimulation protocol consisted of initial treatment with gonadotropin-releasing hormone agonist followed by recombinant human FSH. Oocyte retrieval was performed 35 h after hCG injection. Sperm samples were analyzed in accordance with WHO protocols. Strict morphology of sperm was assessed according to Kruger’s strict criteria. Fertilization was assessed after 18 h of insemination/ICSI and only normally fertilized oocytes were cultured. Embryos were cultured for up to 6 days in a sequential embryo culture system (Irvine Scientific, CA). Development of zygotes to blastocysts in vitro was used as the main outcome measure for this study. 3. Results Overall, 853 oocytes were injected with normal fertilization and embryo cleavage rates of 75% and 99%, respectively. A total of 631 ICSI embryos were cultured for
G.D. Adamson, M. Gvakharia / International Congress Series 1271 (2004) 155–158
157
Table 1 Blastocyst development in standard IVF and IVF-ICSI patients
Embryos cultured Blastocysts developed Blastocyst development rate Statistical significance
IVF group n = 160
ICSI group n = 77
ICSI group A n = 16
ICSI group B n = 61
ICSI group C n = 55
ICSI group D n = 22
1472 560 38
631 189 30
147 38 26
484 151 31
470 146 31
161 43 28
p>0.05
p>0.05
p>0.05
p>0.05
p < 0.05
possible blastocyst development, resulting in 189 blastocysts (30% overall blastocyst development rate). Blastocyst development results in different groups were group A, 26%; group B, 32%; group C, 31%; and group D, 30%. There was no statistically significant difference in blastocyst development rates among these groups of patients (Table 1). A blastocyst development rate of 38% was observed in the standard IVF control group vs. 30% in IVF-ICSI group ( p < 0.05). 4. Discussion The objectives of this study were to study the possible effect of ICSI procedure on embryo development to blastocyst in vitro and to determine whether major sperm parameters influence blastulation rates after ICSI. Based on presented results, the blastocyst development rate of IVF embryos is statistically higher than blastocyst development rate of ICSI embryos. In order to analyze whether this impaired blastulation after ICSI was associated with sperm parameters, we identified four groups of ICSI patients as described in Materials and methods. There was no significant difference in blastocyst development in patient groups based on total motile sperm count or strict morphology (Table 1). In conclusion, although the blastocyst development rate of ICSI embryos is statistically lower than blastocyst development rate of non-ICSI embryos, this decrease is not statistically correlated with any of measured semen parameters. These results suggest that the ICSI procedure may have an adverse effect on the ability of the embryo to form a blastocyst in vitro. References [1] D.K. Gardner, et al., Culture and transfer of human blastocysts increases implantation rate and reduces the need for multiple embryo transfers, Fertil. Steril. 69 (1998) 84 – 88. [2] B. Behr, et al., Preliminary clinical experience with human blastocyst development in vitro without coculture, Hum. Reprod. 14 (1999) 454 – 457. [3] M.C.W. Scholtes, G.H. Zeilmaker, A prospective, randomized study of embryo transfer results after 3 or 5 days of embryo culture in in vitro fertilization, Fertil. Steril. 65 (1996) 1245 – 1248. [4] L. Janny, Y.J. Menezo, Maternal age effect on early human embryonic development and blastocyst formation, Mol. Reprod. Dev. 45 (1996) 31 – 37. [5] Z. Karaki, et al., Blastocyst culture and transfer: a step toward improved in vitro fertilization outcome, Fertil. Steril. 77 (2002 January) 114 – 118.
158
G.D. Adamson, M. Gvakharia / International Congress Series 1271 (2004) 155–158
[6] C. Racowsky, et al., The number of eight-cell embryos is a key determinant for selecting day 3 or day 5 transfer, Fertil. Steril. 73 (2000) 558 – 564. [7] Y. Shoukri, et al., Blastocyst development from supranumerary embryos after intracytoplasmic sperm injection: a paternal influence, Hum. Reprod. 13 (1998) 1632 – 1637. [8] J.C.M. Dumoulin, et al., Embryo development and chromosomal anomalies after ICSI: effect of the injection procedure, Hum. Reprod. 16 (2001) 306 – 312.