تأثیر یونومایسین به عنوان یک ماده مصنوعی فعال کننده تخمک بر نتایج لقاح پس از انجام تزریق سیتوپلاسمی اسپرم به درون تخمک

نوع مقاله: اصیل پژوهشی

نویسندگان

1 دانشیار گروه بافت شناسی و جنین شناسی، گروه علوم تشریح و بیولوژی مولکولی، دانشکده پزشکی، دانشگاه علوم پزشکی اصفهان، اصفهان، ایران.

2 پزشک عمومی، دانشکده پزشکی، دانشگاه علوم پزشکی اصفهان، اصفهان، ایران

3 دانشیار گروه جنین شناسی، گروه جنین شناسی و آندرولوژی پژوهشکده رویان، مرکز باروری و ناباروری اصفهان، دانشگاه علوم پزشکی اصفهان، اصفهان، ایران

4 مربی گروه بافت شناسی، گروه علوم تشریح، دانشکده پزشکی، دانشگاه علوم پزشکی شهرکرد، شهرکرد، ایران.

چکیده

مقدمه: جهت افزایش احتمال لقاح در مواردی که عدم موفقیت لقاح بعد از انجام تزریق سیتوپلاسمی اسپرم به درون تخمک (ICSI)، ناشی از فعال نشدن تخمک باشد، فعال کردن تخمک به صورت مصنوعی توصیه می شود. روش های فعال کردن مصنوعی تخمک از طریق استفاده از یک ماده شیمیایی یا به طریق الکتریکی می باشد. مطالعه حاضر با هدف بررسی تأثیر یونومایسین به عنوان یک ماده شیمیایی که به طور مصنوعی باعث فعال سازی تخمک می شود، بر میزان لقاح، تسهیم، رشد و نمو جنین و میزان بارداری بعد از انجام ICSI انجام شد.
روش‌کار: این مطالعه مداخله ای در سال 84-1383 بر روی 87 زوج نابارور مراجعه کننده به مرکز باروری و ناباروری اصفهان که علت ناباروری آنها فاکتورهای مردانه بود، انجام شد. مایع سمن از افراد مورد مطالعه جهت درمان ICSI جمع آوری شد. بخشی از آن جهت آنالیز معمول سمن که شامل بررسی غلظت، تحرک و مورفولوژی است، بر اساس معیارهای سازمان بهداشت جهانی استفاده شد و باقیمانده نمونه ها به روش با Pure Sperm شستشو داده شد و جهت انجام عمل ICSI مورد استفاده قرار گرفت. برای هر بیمار، اووسیت ها به طور تصادفی در دو گروه کنترل و تیمار قرار گرفتند. اووسیت های گروه تیمار به مدت 10 دقیقه در مجاورت با 10 میکرو مولار یونومایسین قرار گرفتند. پس از گذشت 18- 16 ساعت از تزریق به داخل اووسیت، میزان لقاح بر اساس مشاهده پیش هسته ها بررسی شد. میزان تسهیم و کیفیت جنین 48 و 72 ساعت پس از لقاح برای هر دو گروه بررسی و ثبت شد. داده ها پس از گردآوری با استفاده از نرم افزار آماریSPSS (نسخه 5/11) مورد تجزیه و تحلیل قرار گرفت. جهت مقایسه میانگین داده ها بین دو گروه از آزمون تی استفاده شد. میزان pکمتر از 05/0 معنی دار در نظر گرفته شد.
یافته‌ها: بین دو گروه از نظر میزان لقاح و درصد تسهیم 72 ساعت بعد از ICSI تفاوت معنی داری مشاهده شد (001/0>p). به علاوه در بیمارانی که گروه کنترل آنها فاقد لقاح بود، از گروه تیمار با یونومایسین، 2 عدد بارداری به دست آمد. در حالی که در گروهی که میزان لقاح پایین (33-1%) بود، میانگین میزان لقاح در گروه تیمار آنها (31/58%) نسبت به گروه کنترل (3/14%) به طور معنی داری افزایش یافت.
نتیجه‌گیری: فعال سازی مصنوعی تخمک با استفاده از یونومایسین نه تنها بر میزان موفقیت در لقاح، بلکه بر میزان تسهیم و همچنین رشد جنین اثر مثبت دارد.

کلیدواژه‌ها


عنوان مقاله [English]

Effect of Ionomycin as an Artificial Oocyte Activator on Fertilization Outcome by ICSI

نویسندگان [English]

  • Shahnaz Razavi 1
  • Zeinab Javdan 2
  • Mohammad Hossein Nasr Esfahani 3
  • Masoud Sadeghi 4
1 Associate Professor of Histology and Embryology, Department of Anatomy and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
2 General Practitioner, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
3 Associate Professor of Embryology, Department of Embryology and Andrology of Royan Institute, Fertility and Infertility Center, Isfahan University of Medical Sciences, Isfahan, Iran.
4 Lecturer of Histology, Department of Anatomy, Faculty of Medicine, University of Shahrekord, Shahrekord, Iran
چکیده [English]

Introduction: To increase the likelihood of fertilization in cases of failed oocytes activation after Intracytoplasmic sperm injection (ICSI) due to oocyte lack of activation, artificially activating oocyte is recommended. Artificial oocyte activation methods include: electrical and chemical activation. The aim of this study was to evaluate the efficiency of Ionomycin as a chemical substance that artificially activates oocyte on fertilization,cleavage rate and embryonic development, and pregnancy rates after ICSI was performed.
Methods: This interventional study was held on 87 couples with male factor etiology referring to Fertility and Infertility center for treatment in 2004-2005.Semen sample was collected from participants for ICSI treatment. Routine semen analysis includingexamination of the concentration, motility and morphology, according to World Health Organization criteria, was carried out on part of it. The remaining samples were washed with Pure Sperm method and used to perform ICSI. After oocyte collection, the oocytes were randomly divided into two groups: control and artificial oocyte activation (AOA). The AOA group was chemically activated by exposure to 10μM Ionomycin for 10 minutes. Around 16-18 hours after ICSI, fertilization was assessed by presence of pronuclei. The rate of cleavage and quality of embryo were evaluated and recorded 48 and 72 hours after ICSI .Collected data were analyzed by using statistical software SPSS version 11.5. In order to compare the mean of data between two groups T-test was held.P value less than 0.05 was considered statistically significant.
Results: Two group (AOA and control) hadsignificant differences between mean of fertilization and cleavage rates (P<0.001) 72 hours after ICSI. Also in patients who had no fertilization in their control group of AOA group two pregnancies were recorded. In the group whose participants had poor fertilization rate (1-3%), the mean fertilization rate in the treatment group (58.31%) was significantly increased than the control group (14.3%).
Conclusion: Artificial the oocyte activation using Ionomycin not only improves fertilization but also has positive effect on cleavage rates and embryo quality which in turn affect the implantation and pregnancy rate.

کلیدواژه‌ها [English]

  • Failed fertilization
  • ICSI
  • Ionomycin
  • Oocyte activation
1. Flaherty SP, Payne D, Matthews CD. Fertilization failures and abnormal fertilization after intracytoplasmic
sperm injection. Hum Reprod 1998 Apr;l3 Suppl 1:155-64.
2. Lopes S, Jurisicova A, Casper RF. Gamete-specific DNA fragmentation in unfertilized human oocytes after
intracytoplasmic sperm injection. Hum Reprod 1998 Mar;13(3):703-8.
3. Sakkas D, Urner F, Bianchi PG, Bizzaro D, Wagner I, Jaquenoud N, et al. Sperm chromatin anomalies can
influence decondensation after intracytoplasmic sperm injection. Hum Reprod 1996 Apr;11(4):837-43.
4. Schmiady H, Tandler-Schneider A, Kentenich H. Premature chromosome condensation of the sperm nucleus
after intracytoplasmic sperm injection. Hum Reprod 1996 Oct;11(10):2239-45.
5. Nasr-Esfahani MH, Razavi S, Mardani M , Shirazi R , Javanmardi S. The effects of failed oocyte activation and
sperm protamine deficiency on fertilization post ICSI. Reprod Biomed Online 2007 Apr;14(4):422-9.
6. Sousa M, Tesarik J. Ultrastructural analysis of fertilization failure after intracytoplasmic sperm injection. Hum
Reprod 1994 Dec;9(12):2374-80.
7. Dozortsev D, De Sutter P, Dhont M. Behaviour of spermatozoa in human oocytes displaying no or one
pronucleus after intracytoplasmic sperm injection. Hum Reprod 1994 Nov;9(11):2139-44.
8. Tesarik J, Sousa M. More than 90% fertilization rates after intracytoplasmic sperm injection and artificial
induction of oocyte activation with calcium ionophore. Fertil Steril 1995 Feb;63(2):343-9.
9. Swann K, Lai FA.A novel signalling mechanism for generating Ca2+ oscillations at fertilization in mammals.
Bioessays 1997 May;19(5):371-8.
10. Schultz RM, Kopf GS. Molecular basis of mammalian egg activation. Curr Top Dev Biol 1995;30:21-62.
11. Tesarik J, Mendoza C, Greco E. The activity (calcium oscillator?) responsible for human oocyte activation after
injection with round spermatids is associated with spermatid nuclei. Fertil Steril 2000 Dec;74(6):1245-7.
12. Tesarik J, Rienzi L, Ubaldi F, Mendoza C, Greco E. Use of a modified intracytoplasmic sperm injection
technique to overcome sperm-borne and oocyte-borne oocyte activation failures. Fertil Steril 2002
Sep;78(3):619-24.
13. Nasr-Esfahani MH, Razavi S, Mozdarani H, Mardani M , Azvagi H. Relationship between protamine deficiency
with fertilization rate and incidence of sperm premature chromosomal condensation post-ICSI. Andrologia 2004
Jun;36(3):95-100.
14. Nasr-Esfahani MH, Naghshizadian N, Imani H, Razavi S, Mardani M, Kazemi S, et al. Can sperm protamine
deficiency induce sperm premature chromosomal condensation? Andrologia 2006;38(3):92-8.
15. Rawe VY, Olmedo SB, Nodar FN, Doncel GD, Acosta AA, Vitullo AD. Cytoskeletal organization defects and
abortive activation in human oocytes after IVF and ICSI failure. Mol Hum Reprod 2000 Jun;6(6):510-6.
16. Collas P, Fissore R, Robl JM, Sullivan EJ , Barnes FL. Electrically induced calcium elevation, activation, and
parthenogenetic development of bovine oocytes. Mol Reprod Dev 1993 Feb;34(2):212-23 .
17. Cummins JM, Pember SM, Jequier AM, Yovich JL, Hartmann PE. A test of the human sperm acrosome
reaction following ionophore challenge: relationship to fertility and other seminal parameters. J Androl 1991
Mar-Apr;12(2):98-103.
18. Fenichel P, Donzeau M, Farahifar D, Basteris B, Ayraud N, Hsi BL. Dynamics of human sperm acrosome
reaction: relation with in vitro fertilization. Fertil Steril 1991 May;55(5):994-9.
19. Mahutte NG , Arici A. Failed fertilization: is it predictable? Curr Opin Obstet Gynecol 2003 Jun;15(3):211-8.
 
20. Florke-Gerloff S, Topfer-Petersen E, Muller-Esterl W, Mansouri A, Schatz R, Schirren C, et al. Biochemical and
genetic investigation of round-headed spermatozoa in infertile men including two brothers and their father.
Andrologia 1984 May-Jun;16(3):187-202.
21. Escalier D. Failure of differentiation of the nuclear-perinuclear skeletal complex in the round-headed human
spermatozoa. Int J Dev Biol 1990 Jun;34(2):287-97.
22. Courtot AM . Presence and localization of the 60 KD calicin in human spermatozoa presenting postacrosomal
sheath defects: preliminary results. Mol Reprod Dev 1991 Mar;28(3):272-9.
23. Tesarik J, Testart J. Treatment of sperm-injected human oocytes with Ca2+ ionophore supports the development
of Ca2+ oscillations. Biol Reprod 1994 Sep;51(3):385-91.
24. Nakagawa K, Yamano S, Moride N, Yamashita M, Yoshizawa M, Aono T. Effect of activation with Ca
ionophore A23187 and puromycin on the development of human oocytes that failed to fertilize after
intracytoplasmic sperm injection. Fertil Steril 2001 Jul;76(1):148-52.
25. Eldar-Geva T, Brooks B, Margalioth EJ, Zylber-Haran E, Gal M , Silber SJ. Successful pregnancy and delivery
after calcium ionophore oocyte activation in a normozoospermic patient with previous repeated failed
fertilization after intracytoplasmic sperm injection. Fertil Steril 2003 Jun;79 Suppl 3:1656-8.
26. Ebner T, Moser M, Sommergruber M, Jesacher K , Tews G. Complete oocyte activation failure after ICSI can be
overcome by a modified injection technique. Hum Reprod 2004 Aug;19(8):1837-41.
27. Murase Y, Araki Y, Mizuno S, Kawaguchi C, Naito M, Yoshizawa M, et al. Pregnancy following chemical
activation of oocytes in a couple with repeated failure of fertilization using ICSI: case report. Hum Reprodm
2004 Jul;19(7):1604-7.
28. Chi HJ, Koo JJ, Song SJ, Lee JY, Chang SS. Successful fertilization and pregnancy after intracytoplasmic sperm
injection and oocyte activation with calcium ionophore in a normozoospermic patient with extremely low
fertilization rates in intracytoplasmic sperm injection cycles. Fertil Steril 2004 Aug;82(2):475-7.
29. World Health Organization. WHO Laboratory Manual for the examination of human semen and sprem–cervical
mucus interaction. 4th ed. Cambridge:om:Cambridge University Press;1999 .
30. Kruger TF, Menkveld R, Stander FS, Lombard CJ, Van der Merwe JP, van Zyl JA , et al. Sperm morphologic
features as a prognostic factor in in vitro fertilization. Fertil Steril 1986 Dec;46(6):1118-23.
31. Parrington J, Swann K, Shevchenko VI, Sesay AK, Lai FA. Calcium oscillations in mammalian eggs triggered
by a soluble sperm protein. Nature 1996 Jan 25;379(6563):364-8 .
32. Kimura Y, Yanagimachi R, Kuretake S, Bortkiewicz H, Perry AC, Yanagimachi H. Analysis of mouse oocyte
activation suggests the involvement of sperm perinuclear material. Biol Reprod 1998 Jun;58(6):1407-15.
33. Rybouchkin AV, Van der Straeten F, Quatacker J, De Sutter P, Dhont M. Fertilization and pregnancy after
assisted oocyte activation and intracytoplasmic sperm injection in a case of round-headed sperm associated with
deficient oocyte activation capacity. Fertil Steril 1997 Dec;68(6):1144-7.
34. Yanagida K, Katayose H, Yazawa H, Kimura Y, Sato A, Yanagimachi R. Successful fertilization and pregnancy
following ICSI and electrical oocyte activation. Hum Reprod 1999 May;14(5):1307-11.
35. Bartoov B, Berkovitz A, Eltes F, Kogosowski A, Menezo Y , Barak Y. Real-time fine morphology of motile
human sperm cell is associated with IVF-ICSI outcome. J Androl 2002 Jan-Feb;23(1):1-8.
36. Battaglia DE, Koehler JK, Klein NA, Tucker MJ. Failure of oocyte activation after intracytoplasmic sperm
injection using round-headed sperm. Fertil Steril 1997 Jul;68(1):118-22.
37. Kim ST, Cha YB, Park JM, Gye MC. Successful pregnancy and delivery from frozen-thawed embryos after
intracytoplasmic sperm injection using round-headed spermatozoa and assisted oocyte activation in a
globozoospermic patient with mosaic Down syndrome. Fertil Steril 2001 Feb;75(2):445-7.
38. Lu Q, Zhao Y, Gao X, Li Y, Ma S, Mullen S, et al. Combination of calcium ionophore A23187 with puromycin
salvages human unfertilized oocytes after ICSI. Eur J Obstet Gynecol Reprod Biol 2006 May 1;126(1):72-6.
39. Winston N, Johnson M, Pickering S, Braude P. Parthenogenetic activation and development of fresh and aged
human oocytes. Fertil Steril 1991 Nov;56(5):904-12.
40. Taylor DM, Ray PF, Ao A, Winston RM, Handyside AH. Paternal transcripts for glucose-6-phosphate
dehydrogenase and adenosine deaminase are first detectable in the human preimplantation embryo at the threeto
four-cell stage. Mol Reprod Dev 1997 Dec;48(4):442-8 .
41. Araki Y, Yoshizawa M, Abe H, Murase Y, Araki Y.Use of mouse oocytes to evaluate the ability of human
sperm to activate oocytes after failure of activation by intracytoplasmic sperm injection. Zygote 2004
May;12(2):111-6.
42. Heindryckx B, Van der Elst J, De Sutter P, Dhont M. Treatment option for sperm- or oocyte-related fertilization
failure: assisted oocyte activation following diagnostic heterologous ICSI. Hum Reprod 2005 Aug;20(8):2237-41.
43. Henkel R, Muller C, Miska W, Schill WB, Kleinstein J, Gips H. Acrosin activity of human spermatozoa by
means of a simple gelatinolytic technique: a method useful for IVF. J Androl 1995 May-Jun;16(3):272-7.
44. Nasr-Esfahani MH, Razavi S, Tavalaee M. Failed fertilization post ICSI and spermiogenic defects. Fertil Steril
2007 Apr;89(4):892-8.
45. Oikawa T, Takada N, Kikuchi T, Numabe T, Takenaka M, Horiuchi T. Evaluation of activation treatments for
blastocyst production and birth of viable calves following bovine intracytoplasmic sperm injection. Anim
Reprod Sci 2005 Apr;86(3-4):87-94.
46. Chian RC, Tan SL, Sirard MA. Protein phosphorylation is essential for formation of male pronucleus in bovine
oocytes. Mol Reprod Dev 1999 Jan;52(1):43-9.
 
47. Moaz MN, Khattab S, Foutouh IA, Mohsen EA. Chemical activation of oocytes in different types of sperm
abnormalities in cases of low or failed fertilization after ICSI: a prospective pilot study. Reprod Biomed Online
2006 Dec;13(6):791-4.
48. Berkovitz A, Eltes F, Yaari S, Katz N, Barr I, Fishman A, et al. The morphological normalcy of the sperm
nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm. Hum Reprod
2005 Jan;20(1):185-90.
49. Heindryckx B, Lierman S, Combelles CM, Cuvelier CA, Gerris J, De Sutter P. Aberrant spindle structures
responsible for recurrent human metaphase I oocyte arrest with attempts to induce meiosis artificially. Hum
Reprod 2011 Apr;26(4):791-800 .
50. Yuzpe AA, Liu Z, Fluker MR. Rescue intracytoplasmic sperm injection (ICSI)-salvaging in vitro fertilization
(IVF) cycles after total or near-total fertilization failure. Fertil Steril 2000 Jun;73(6):1115–9.