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.

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.

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.

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.

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.