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INNOVATIONS IN FEMALE INFERTILITY RESEARCH

Cleveland Clinic's American Center for Reproductive Medicine
1993 - 2008

Oxidative stress and ART outcomes

Oxidative Stress in ART setting

Figure:
Oxidative Stress in ART setting

 

Assisted reproductive techniques (ART) have become the treatment of choice in many cases of male and female infertility. Despite technical advances in gamete handling and culture conditions, success rates after ART procedures are still unsatisfactory. Suboptimal oocyte and embryo quality is one of the many reasons contributing to poor pregnancy rates after in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). As our understanding of gamete and embryo metabolism has increased over recent years, we have come to the hypothesis that oxidative stress (OS) negatively impacts human embryos. OS has been implicated in arrested embryo development and poor quality embryos that develop in vitro. We reported that reactive oxygen species (ROS) are generated in culture media from developing embryos and are significantly high when there is low fertilization, low cleavage, low blastocyst development and increased fragmentation. This was specific in ICSI cycles and is explained by the potential antioxidant activity of cumulus cell mass around oocytes that are denuded during ICSI.

We measured levels of OS markers such as lipid peroxidation (LPO) and total antioxidant capacity (TAC) in the follicular fluid from women undergoing IVF treatment and found both markers correlated positively with pregnancy outcome. We demonstrated significantly increased levels of ROS and TAC in the follicular fluid of women undergoing IVF who became pregnant in contrast with those who did not. Follicular fluid ROS and lipid peroxidation levels may be viewed as markers for success with IVF.

  • PDF File (agradoc150.pdf 191 Kb)
    Bedaiwy MA, Falcone T, Al-Hussaini T, Abdel-Aleem A, Sharma RK, Worley SE, Thornton J, and Agarwal A. (2004):
    Differential growth of human embryos in vitro: role of reactive oxygen species.
    Fertil Steril 82:593-600.
     
  • PDF File (agradoc141.pdf)
    Pasqualotto EB, Agarwal A, Sharma RK, Izzo VM, Pinotti JA, Joshi NJ, and Rose BI. (2004):
    The effect of oxidative stress in follicular fluid on the outcome of assisted reproductive procedures.
    Fertil Steril 81:973-6.
     
  • PDF File (agradoc182.pdf 84 Kb)
    Agarwal A, Allamaneni, SSR, Nallella KP, George AT, and Mascha E. (2005):
    Correlation of reactive oxygen species (ROS) levels with fertilization rate following in vitro fertilization (IVF): A meta-analysis.
    Fertil Steril 84:228-31.
     
  • PDF File (agradoc253.pdf 101 Kb)
    Bedaiwy MA, Agarwal A, Said TM, Goldberg JM, Sharma RK, Worley S, and Falcone T. (2006):
    Role of total antioxidant capacity in the differential growth of human embryos in vitro.
    Fertil Steril 2006 Aug; 86(2):304-9.
     
  • PDF File (agradoc150.pdf 191 Kb)
    Bedaiwy, MA, Falcone, T, Al-Hussaini, T, Abdel-Aleem, A, Sharma, RK, Worley, SE, Thornton, J, and Agarwal, A. (2004):
    Differential growth of human embryos in vitro: role of reactive oxygen species.
    Fertil Steril 82:593-600.
     

Oxidative stress and oocytes maturation in vitro

Oxidative Stress and Oocyte Maturation

Figure:
Oxidative Stress and Oocyte Maturation

 

To further study the role of antioxidants in follicular development, we are investigating the correlation between follicular fluid proxidant-antioxidant levels in bovine antral follicles with stages of folliculogenesis and luteal phases of the ovulatory cycle. The clinical application of the translational studies will be the development of protocols to optimize the culture systems used for various assisted reproduction procedures such as in vitro maturation and in vitro fertilization/intracytoplasmic sperm injection by supplementation with non-enzymatic and enzymatic forms of antioxidants.

  • PDF File (07-A-952191-ASRM.pdf 41 Kb)
    Gupta S, Chowdary H, Koli R, Czerniak S, Combelles C, Agarwal A. :
    Association of catalase enzymatic activity in bovine follicular fluid with both the phases of folliculogenesis and the stages of the estrus cycle.
    Fertility and Sterility, Vol. 88, September, 2007. pp. S37.
     

Optimizing culture conditions and reduction of OS levels in ART

Interventions to overcome OS in ART setting

Figure:
Interventions to overcome OS in ART setting

 

Manipulation of gametes and embryos favors reactive oxygen species (ROS) production and may be responsible in part for lower grade embryos and consequent low pregnancy rates. Reduction of oxidative stress (OS) leads to improved assisted reproductive technology options. Avoiding OS during gamete and embryo culture is complex. The introduction of ROS scavengers is one option for controlling OS, and our program has been investigating the choice of the appropriate antioxidant and its concentration.

Human embryos generated from in vitro fertilization may exhibit varying degrees of cytoplasmic fragmentation, and increasing evidence demonstrates that cytoplasmic fragmentation in human embryos arises from apoptosis. Mitochondrial membrane potential and energy production in preimplantation embryos have recently been the focus of many studies. Review of the current literature reveals that carnitines have antiapoptotic, antioxidant, and anti TNF-α effects. Administration of acetyl-L-carnitine (ALC) to mouse fibroblasts in culture media has shown a reduction in apoptosis through the mitochondrial pathway. L-carnitine (LC) was observed to promote neuronal survival and mitochondrial activity in a concentration-dependent manner in primary cultured neurons from cerebral cortex of rat embryos. LC has been investigated for its role in antagonizing the effect of TNF-α in conditions such as liver cell damage, tumors, and inflammatory diseases. The main objective of our study is to optimize embryo quality and enhance ART procedure outcomes. This can be achieved by supplementation of in vitro culture media with LC. Supplementation with LC is a novel approach due to its antioxidant, anti-TNF-α, and antiapoptotic effects.

  • PDF File (agradoc168.pdf 391 Kb)
    Esfandiari N, Falcone T, Agarwal A, Attaran M, Nelson DR, Sharma RK. :
    Protein supplementation and the incidence of apoptosis and oxidative stress in mouse embryos.
    Obstet Gynecol. 2005 Mar;105(3):653-60.
     
  • PDF File (agradoc193.pdf 200 Kb)
    Esfandiari N, Falcone T, Goldberg JM, Agarwal A, and Sharma RK. (2005):
    Effects of peritoneal fluid from patients with endometriosis on pre-implantation mouse embryo development and apoptosis in vitro.
    Reprod Biomed Online 11:615-19.
     
  • Noriega J, Bedaiwy M, Sharma RK, Falcone T. (2004):
    Effect of tumor necrosis factor-a blocker (infliximab) on blastocyst development in vitro.
    Fertil Steril 81:1704-1706.
     
  • PDF File (agradoc197.pdf 62 Kb)
    Zhang X, Sharma RK, Agarwal A and Falcone T. (2005):
    Effect of pentoxifylline in reducing oxidative stress induced embryotoxicity.
    J Assist Reprod Genet 22:415-7.
     
  • PDF File (agradoc113.pdf 241 Kb)
    Wang X, Falcone T, Attaran M., Goldberg JM, Agarwal A., and Sharma, RK. (2002):
    Vitamin C and vitamin E supplementation reduce oxidative stress induced embryo toxicity and improve blastocyst development rate.
    Fertil Steril 78:1272-1277.
     
  • PDF File (07-A-950966-ASRM.pdf 35 Kb)
    Abdelrazik H, Agarwa, A, Mansour G, Gupta S, Sabanegh E, Sharma R. :
    Efficacy of L-carnitine in reversing the antiproliferative effects of TNF-a on mouse embryos in vitro.
    Fertility and Sterility, Vol. 88, September, 2007. pp. S124.
     
  • PDF File (07-A-950954-ASRM.pdf 28 Kb)
    Abdelrazik H, Agarwal A, Kader A, Eyada MMK, Sabanegh E, Sharma R. :
    L-carnitine as an antiapoptotic supplement in mouse embryo culture media.
    Fertility and Sterility, Vol. 88, September, 2007. pp. S321-S322.
     
  • Abdelrazik H, Sharma, R,Mahfouz R, Agarwal A. (2007):
    L carnitine (LC) decreases DNA damage and improves the in vitro blastocyst development rate in mouse embryos.
    Fertil Steril (In Press).
     

Women with cancer and systemic diseases: Fertility preservation options

 

With increasing cancer survival rates, there is an increased emphasis on quality of life, and several successful fertility preservation and post-treatment parenthood options are being investigated in our program. Young women with cancer who undergo chemotherapy or radiotherapy may lose their fertility. Ovarian tissue banking is emerging as an option for helping such women achieve motherhood.

Our group initiated this line of research in 2002, and we continue to investigate different fertility preservation options. We have extensively studied ovarian tissue cryopreservation and its effects on oocyte quality extensively. In our initial studies, we established freezing protocols and studied the cryobiology of the oocyte as well as any associated cellular changes and the effects of ischemia and post-transplant responses using animal models (porcine and ovine). We next established a technique of whole ovary cryopreservation with intact vascular pedicles in a sheep model. Cryopreserved whole ovary was thawed and transplanted back at a different site with microvascular anastomosis to minimize ischemic damage to the tissue. Patency and resumption of hormonal function were examined. We are presently conducting studies to optimize the fertilizing ability of oocytes retrieved from cryopreserved ovaries utilizing in vitro maturation of the primordial follicles. Various markers of angiogenic and apoptotic markers are also being investigated.

  • PDF File (otcdoc03.pdf 57 Kb)
    Jeremias E, Bedaiwy MA, Nelson D, Biscotti CV, and Falcone T. (2003):
    Assessment of tissue injury in cryopreserved ovarian tissue.
    Fertil Steril 79:651-653.
     
  • PDF File (agradoc125.pdf 176 Kb)
    Esfandiari N, Falcone T, Bedaiwy MA, Agarwal A, Jeremias E, and Sharma RK. (2003):
    Autologous transplantation of cryopreserved ovary induces the generation of antiovary antibodies in sheep.
    Fertil Steril 80:1062-1064.
     
  • PDF File (otcdoc02.pdf 415 Kb)
    Bedaiwy, MA, Jeremias, E, Gurunluoglu, R, Hussein, MR, Siemianow, M, Biscotti, C, and Falcone, T. (2003):
    Restoration of ovarian function after autotransplantation of intact frozen-thawed sheep ovaries with microvascular anastomosis.
    Fertil Steril 79:594-602.
     
  • PDF File (faltdoc065.pdf 44Kb)
    Bedaiwy MA and Falcone T. (2004):
    Ovarian tissue banking for cancer patients. Reduction of post-transplantation ischemic injury: intact ovary freezing and transplantation.
    Hum Reprod 19:1242-44.
     

Freezing of oocytes and blastocysts

DNA damage with Vitrification

Figure:
DNA damage with Vitrification

 

Blastocysts suffer from some degree of DNA damage to their blastomeres as a result of any cryopreservation protocol. The more expanded the blastocyst is, the more prone it is to damage during cryopreservation. Ultra-rapid or slow cryopreservation can induce some DNA damage to blastocysts. We are currently optimizing vitrification of oocytes/blastocysts. A combination of technologies for cryopreservation of ovarian tissue and oocytes represents an emerging option to preserve fertility in cancer patients.

  • PDF File (07-A-950968-ASRM.pdf 30 Kb)
    Kader, A, MD, Abdelrazik, H, MD, Sharma,R, Ph.D, Falcone, T, MD, Goldberg, J, MD, Agarwal, A, Ph.D, HCLD:
    Effect of zonal hatching on expanded and non-expanded blastocysts vitrification.
    Abstract # 950968, Oral Presentation
     
  • PDF File (07-A-951090-ASRM.pdf 36 Kb)
    Kader, A, MD, Abdelrazik,H, MD, Sharma, R, Ph.D, Falcone, T, MD, Goldberg, J, MD, Agarwal, A, Ph.D, HCLD:
    Vitrification versus slow cryopreservation of expanded and non expanded blastocysts - Effect on DNA damage.
    Abstract # 951090, Oral Presentation
     
  • PDF File (07-A-951105-ASRM.pdf 28 Kb)
    Kader, A, MD, Agarwal, A, Ph.D, HCLD, Abdelrazik, H, MD, Falcone, T, MD, Goldberg, J, MD, Sharma,R, Ph.D.:
    Improvement in expanded blastocyst vitrification outcome by the use of a pre-vitrification intervention and non-intervention technique.
    Abstract # 951105, Oral Presentation
     
  • PDF File (07-A-951116-ASRM.pdf 29 Kb)
    Kader,A, MD, Agarwal, A, Ph.D, HCLD, Abdelrazik, H, MD, Falcone, T, MD, Goldberg, J, MD, Sharma, R, Ph.D:
    Routine use of blastocele aspiration of expanded blastocysts and assisted hatching of non-expanded blastocysts before vitrification.
    Abstract # 951116, Oral Presentation
     

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Last Update : June 06, 2009
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