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High magnification morphological selection of individual sperm cells for IVF-intracytoplasmic sperm injection
Abstracts - WARM: Beyond infertility: breakthroughs in reproductive medicine and technology
the acrosome, nucleus, postacrosomal fusogenic segment, centrosome, mitochondria and axoneme were documented by digital imaging. Conclusions: These images could be used to evaluate sperm samples prepared routinely for IVF-ICSI, particularly for single sperm visualized by high-powered light microscopy to assess nuclear morphology for intracytoplasmic morphologically selected sperm injection, which has been reported recently. High magnification morphological selection of individual sperm cells for IVF-intracytoplasmic sperm injection Bartoov B, Eltes F, Lederman H, Peer S, Ellenbogen A, Feldberg D, Yagoda A, Geva E, Kogosowski A, Bar I, Berkovitz A Male Fertility Laboratory, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Introduction: IVF-intracytoplasmic sperm injection (ICSI), the most effective treatment for male infertility, usually results in low live birth rate and unwanted embryos. Therefore, for medical, ethical, religious and legal reasons, it is essential to improve the IVF-ICSI outcome by selecting gametes with high developmental quality. However, most of the sperm quality characteristics cannot be tested, neither in real time nor for a single spermatozoon. To solve this problem, this study developed a method of sperm functional morphology, which is based on high-power light microscopy, real-time observation, motile sperm examination, and fine organellar morphology, called motile sperm organellar morphology examination (MSOME). Based on this method, it was found that out of the six sperm subcellular organelles observed, only the morphological normality of the sperm nucleus, defined by MSOME, had predictive value of 74% for pregnancy occurrence in IVF-ICSI. This study developed intracytoplasmic morphologically selected sperm injection (IMSI) based on MSOME and conducted a few random matched studies with the following aims: (i) to verify whether microinjection into retrieved oocytes of motile spermatozoa with morphologically normal nuclei, strictly defined by MSOME, improves the IVF-IMSI pregnancy outcome; (ii) to determine the effect of morphologically ‘second best’ spermatozoa on IVF-IMSI outcome in cases where sperm cells with strictly normal nucleus are not available; and (iii) to determine whether vacuolated spermatozoa with normal nuclear shape affect ICSI outcome. Materials and methods: MSOME was conducted in the Male Fertility Lab of Bar Ilan University, Israel, using an Olympus IX71 inverted microscope (microscope magnification ×150; video magnification ×44.45; actual magnification ×6300. For the IMSI procedure, five nuclear shape-specific malformations were analysed: small oval, large oval, narrow, wide and short; and two content malformations: vacuolated and regional disorders. For the matched control comparison study (n = 80) between IVF-ICSI and IMSI, 80 couples were recruited, who underwent these procedures. Another matched control comparison (n = 70) was conducted between embryos obtained from microinjection of morphologically best and second best spermatozoa. A third matched control comparison study was conducted (n = 22) comparing intact spermatozoa and vacuolated spermatozoa. Results: Comparison between the matched IMSI and ICSI groups in terms of number of retrieved and injected ova, fertilization rate, percentage of top embryos, number of
transferred embryos and implantation rate demonstrated that the percentage of top embryos and implantation rate were significantly higher in the IMSI group as compared with the ICSI one (F = 9.5 and F = 23.3 respectively, P ≤ 0.01). The pregnancy rate was significantly higher, and the abortion rate was significantly lower following IVF-IMSI, compared with the routine ICSI trial (chi-squared test = 20.1, P ≤ 0.01; and chi-squared test = 5.1, P ≤ 0.03, respectively). Comparison between the best and second-best matched study groups demonstrated that fertilization rate, percentage of top embryos, implantation rate, pregnancy rate and delivery rate per cycle were significantly higher, and the abortion rate was significantly lower in the ‘best’ group than in the ‘second best’ group (F = 10.5, P ≤ 0.01; F = 4.6, P ≤ 0.03; F = 23.4, P ≤ 0.01; chi-squared test = 15.5, P ≤ 0.05; chi-squared test = 19.6, P ≤ 0.01; and chi-squared test = 5.5, P ≤ 0.02 respectively). The underlying biochemistry of vacuolated spermatozoa is discussed. In the comparison between vacuolated and intact cells, both groups were statistically similar in number of retrieved and injected ova, fertilization rate, percentage of top embryos and number of transferred embryos. The group with vacuolated spermatozoa was found to have significantly higher abortion rate, and significantly lower pregnancy rate and delivery rate (100% versus 10%, 18% versus 45% and 0% versus 41%, respectively; P ≤ 0.01). Conclusions: ICSI-associated pregnancy rate may be affected by subtle morphological malformations of the sperm nucleus, which may remain undetected by the embryologist during the routine selection procedure. Microinjection of sperm cells with strictly normal nucleus, defined by MSOME, is an important factor in achieving durable pregnancy after ICSI. FSH therapy in male infertility Acosta AA Eastern Virginia Medical School, Emeritus Professor Human andrological sciences have gone through different stages of development: (i) clinical; (ii) andrology and male contraception, in which no reliable suppression of spermatogenesis without impairment of the endocrine function has been achieved as yet; (iii) endocrinological andrology, at the time of development of gonadotrophic and steroid hormones therapies; (iv) andrology and assisted reproduction; (v) andrology and assisted fertilization; and (vi) andrology and genetics, molecular biology and the ‘omics’. Each one of these periods made and left a profound impact in the andrological field. The clinical use of gonadotrophin therapy in the oligoasthenoteratozoospermic male population was started, mainly on an empirical basis, by the Italian school. The opening of the assisted reproduction and assisted fertilization fields forced the establishment of brand new criteria for the use and evaluation of spermatozoa in these two areas. Here again, although renewed and strenuous diagnostic efforts applicable to the new therapeutic methods were attempted, the use of gonadotrophin therapy in this field continued being done mostly on empirical grounds. Basic scientists of superb ability made strenuous efforts to clarify the testicular endocrine physiology that could be applied in the clinical field. Regrettably we still do not have a clear understanding of the extremely complex testicular function and of the endocrine, paracrine and autocrine testicular function control. A brief review of some of these efforts will be performed in this presentation. The results
S-7 Reproductive BioMedicine Online, Vol. 17, Suppl. 2, September 2008
the acrosome, nucleus, postacrosomal fusogenic segment, centrosome, mitochondria and axoneme were documented by digital imaging. Conclusions: These images could be used to evaluate sperm samples prepared routinely for IVF-ICSI, particularly for single sperm visualized by high-powered light microscopy to assess nuclear morphology for intracytoplasmic morphologically selected sperm injection, which has been reported recently. High magnification morphological selection of individual sperm cells for IVF-intracytoplasmic sperm injection Bartoov B, Eltes F, Lederman H, Peer S, Ellenbogen A, Feldberg D, Yagoda A, Geva E, Kogosowski A, Bar I, Berkovitz A Male Fertility Laboratory, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Introduction: IVF-intracytoplasmic sperm injection (ICSI), the most effective treatment for male infertility, usually results in low live birth rate and unwanted embryos. Therefore, for medical, ethical, religious and legal reasons, it is essential to improve the IVF-ICSI outcome by selecting gametes with high developmental quality. However, most of the sperm quality characteristics cannot be tested, neither in real time nor for a single spermatozoon. To solve this problem, this study developed a method of sperm functional morphology, which is based on high-power light microscopy, real-time observation, motile sperm examination, and fine organellar morphology, called motile sperm organellar morphology examination (MSOME). Based on this method, it was found that out of the six sperm subcellular organelles observed, only the morphological normality of the sperm nucleus, defined by MSOME, had predictive value of 74% for pregnancy occurrence in IVF-ICSI. This study developed intracytoplasmic morphologically selected sperm injection (IMSI) based on MSOME and conducted a few random matched studies with the following aims: (i) to verify whether microinjection into retrieved oocytes of motile spermatozoa with morphologically normal nuclei, strictly defined by MSOME, improves the IVF-IMSI pregnancy outcome; (ii) to determine the effect of morphologically ‘second best’ spermatozoa on IVF-IMSI outcome in cases where sperm cells with strictly normal nucleus are not available; and (iii) to determine whether vacuolated spermatozoa with normal nuclear shape affect ICSI outcome. Materials and methods: MSOME was conducted in the Male Fertility Lab of Bar Ilan University, Israel, using an Olympus IX71 inverted microscope (microscope magnification ×150; video magnification ×44.45; actual magnification ×6300. For the IMSI procedure, five nuclear shape-specific malformations were analysed: small oval, large oval, narrow, wide and short; and two content malformations: vacuolated and regional disorders. For the matched control comparison study (n = 80) between IVF-ICSI and IMSI, 80 couples were recruited, who underwent these procedures. Another matched control comparison (n = 70) was conducted between embryos obtained from microinjection of morphologically best and second best spermatozoa. A third matched control comparison study was conducted (n = 22) comparing intact spermatozoa and vacuolated spermatozoa. Results: Comparison between the matched IMSI and ICSI groups in terms of number of retrieved and injected ova, fertilization rate, percentage of top embryos, number of
transferred embryos and implantation rate demonstrated that the percentage of top embryos and implantation rate were significantly higher in the IMSI group as compared with the ICSI one (F = 9.5 and F = 23.3 respectively, P ≤ 0.01). The pregnancy rate was significantly higher, and the abortion rate was significantly lower following IVF-IMSI, compared with the routine ICSI trial (chi-squared test = 20.1, P ≤ 0.01; and chi-squared test = 5.1, P ≤ 0.03, respectively). Comparison between the best and second-best matched study groups demonstrated that fertilization rate, percentage of top embryos, implantation rate, pregnancy rate and delivery rate per cycle were significantly higher, and the abortion rate was significantly lower in the ‘best’ group than in the ‘second best’ group (F = 10.5, P ≤ 0.01; F = 4.6, P ≤ 0.03; F = 23.4, P ≤ 0.01; chi-squared test = 15.5, P ≤ 0.05; chi-squared test = 19.6, P ≤ 0.01; and chi-squared test = 5.5, P ≤ 0.02 respectively). The underlying biochemistry of vacuolated spermatozoa is discussed. In the comparison between vacuolated and intact cells, both groups were statistically similar in number of retrieved and injected ova, fertilization rate, percentage of top embryos and number of transferred embryos. The group with vacuolated spermatozoa was found to have significantly higher abortion rate, and significantly lower pregnancy rate and delivery rate (100% versus 10%, 18% versus 45% and 0% versus 41%, respectively; P ≤ 0.01). Conclusions: ICSI-associated pregnancy rate may be affected by subtle morphological malformations of the sperm nucleus, which may remain undetected by the embryologist during the routine selection procedure. Microinjection of sperm cells with strictly normal nucleus, defined by MSOME, is an important factor in achieving durable pregnancy after ICSI. FSH therapy in male infertility Acosta AA Eastern Virginia Medical School, Emeritus Professor Human andrological sciences have gone through different stages of development: (i) clinical; (ii) andrology and male contraception, in which no reliable suppression of spermatogenesis without impairment of the endocrine function has been achieved as yet; (iii) endocrinological andrology, at the time of development of gonadotrophic and steroid hormones therapies; (iv) andrology and assisted reproduction; (v) andrology and assisted fertilization; and (vi) andrology and genetics, molecular biology and the ‘omics’. Each one of these periods made and left a profound impact in the andrological field. The clinical use of gonadotrophin therapy in the oligoasthenoteratozoospermic male population was started, mainly on an empirical basis, by the Italian school. The opening of the assisted reproduction and assisted fertilization fields forced the establishment of brand new criteria for the use and evaluation of spermatozoa in these two areas. Here again, although renewed and strenuous diagnostic efforts applicable to the new therapeutic methods were attempted, the use of gonadotrophin therapy in this field continued being done mostly on empirical grounds. Basic scientists of superb ability made strenuous efforts to clarify the testicular endocrine physiology that could be applied in the clinical field. Regrettably we still do not have a clear understanding of the extremely complex testicular function and of the endocrine, paracrine and autocrine testicular function control. A brief review of some of these efforts will be performed in this presentation. The results
S-7 Reproductive BioMedicine Online, Vol. 17, Suppl. 2, September 2008