Research Overview*

Ovarian Tissue Cryopreservation and Grafting

Animal Studies

We are developing animal model to cryopreserve ovaries containing primordial follicles and study their fertilizing potential. Optimizing the cryopreservation protocol as well as the ischemia is critical to the success of the technique. Minimizing blood vessel damage and conserving the histological architecture of the vessel is being examined. We are also examining the ischemia-related follicular loss utilizing microvascular anastomosis for long-term survival and fertilizing potential using sheep as the study model. Whole ovaries from sheep with intact blood vessels will be cryopreserved. After thawing the ovary will be transplanted back microsurgically and observed for resumption of ovarian function. The hormonal profiles will be examined and the quality of the follicles evaluated. We will investigate the in-vitro fertilizing ability of these autotransplanted ovaries.

Another focus is to optimize the freezing protocols for isolated primordial follicles. The advantage of freezing primordial follicles over mature oocytes are its small size, few cytoplasmic organelles, absence of spindle structure, absence of zona pellucida and cortical granules. Using the murine and bovine models, we are examining the post thaw survival, histological changes, transplantation of isolated primordial follicles and their ability to progress during in-vitro maturation.

Furthermore we are developing new minimally invasive approaches to transplant the ovarian tissue. We are developing new methods to process and cryopreserve the ovarian tissue by vitrification which resulted in the creation of the "Ohio-Cryo method" for vitrification of ovarian tissue. Also new innovative methods for tissue re-engineering and enrichment of the ovarian grafts are currently being investigated to help improve ovarian graft post transplantation viability and functionality.

*No current research is being pursued in female infertility area.

We are currently investigating different methods for isolating primordial follicles. The objective is to implement these methods for restoring fertility. The goal is to optimize methods for isolating primordial follicles as well as identify protocols for cryopreservation of the isolated primordial follicles. Transplantation of the isolated primordial follicles is novel, and may be an alternative to restoring fertility in women following treatments that may compromise their fertility potential.

Immunological perturbations are characteristic of endometriosis. The role of cytokines in the pathogenesis of endometriosis is well known. We are investigating the ability of a group of serum and peritoneal fluid cytokines, such as IL-1, IL-6, IL-8, IL-12, IL-13, and TNF-a as markers in predicting endometriosis nonsurgically. Serum and peritoneal fluid for analysis are obtained in a prospective controlled trial from women while they are undergoing laparoscopy for pain, infertility, tubal ligation, or tubal reanastomosis.

We are examining the effect of exposure of spermatozoa to the peritoneal fluid from women with endometriosis. The aim of the study is to examine the extent of DNA damage as a result of coincubation with peritoneal fluid for its possible relationship with infertility in these women.

The effect of the peritoneal fluids' oxidative stress and TNF-a concentration is being tested on mouse embryo development. We are currently investigating the role of TNF-a in endometriosis by establishing its level in the peritoneal fluid of women with infertility. We are studying the effect of TNF-a on sperm-egg interaction, embryo development and in the pathophysiology of endometriosis. We are examining the use of potential agents such as Infliximab and pentoxifylline in reversing the toxic effects of TNF-a utilizing an in-vitro model. In addition, we are testing the reversibility of these effects by antioxidant supplementation with vitamin C and the addition of a TNF-a antibody such as Infliximab (Remicade) that binds to soluble and membrane forms of TNF-a and neutralize its biological effects. We feel that these novel therapeutic approaches may provide an evidence-based treatment modality for endometriosis that targets the exact etiological factors rather than the current symptomatic treatment.

The fertilization rates of oocytes matured in vitro are much lower than those in in-vivo stimulation cycles. Although a proportion of immature oocytes are able to mature in special culture media, the use of cocultures to improve maturation is novel. We are examining the effects of in vitro maturation of oocytes using various culture conditions and examining the oxidative stress generated in the conditioned media. Optimizing the culture conditions may help improve the maturation, morphological quality, and reduce apoptosis of immature oocytes. The goal is to develop optimal culture conditions that may subsequently help preserve the fertility potential in these patients.

In addition, ongoing studies are also aimed at examining the hypothesis that the pro-antioxidant profile of the various compartments of the bovine follicle is dynamically regulated during progressive stages of antral development. Markers examining the alterations in oxidative stress and antioxidant levels in bovine follicular fluid and conditioned media from antral follicles during different stages of development are being studied. Also identifying changes associated with the follicle size, presence of dominant follicle and the stage of the ovarian cycle are being examined.

The growth and development of the mammalian ovarian follicle is a unique and complex process. We are examining the prooxidant (reactive oxygen species, lipid peroxides) and antioxidant levels (total antioxidant capacity, superoxide dismutase, catalase) in the follicular fluid obtained from small (2-5mm), medium (6-8mm) and large (>10mm) bovine antral follicles.

The aim is to estimate and correlate the dynamic changes in the follicular fluid oxidative stress markers and antioxidants with the progressive stages of bovine follicular development, the stage of estrous cycle and with the follicle dominance. We are analyzing the potential correlation between the various markers of oxidative stress and the antioxidant protection with oocyte quality, developmental competence, fertilization capability and blastocyst development. Characterization of the antioxidant profiles in all the compartments of bovine antral follicles, before culture/after culture will help us understand the mechanisms of action/regulation of oxidative stress markers and antioxidants during in vitro maturation in the bovine. The clinical application of these studies will be to develop protocols to optimize the culture systems used for various ART procedures such as in-vitro maturation, IVF/ICSI by supplementation with non enzymatic and enzymatic forms of antioxidants.

Animal Studies

We are developing animal model to cryopreserve ovaries containing primordial follicles and study their fertilizing potential. Optimizing the cryopreservation protocol as well as the ischemia is critical to the success of the technique. Minimizing blood vessel damage and conserving the histoarchitecutre of the vessel is being examined. We are also examining the ischemia-related follicular loss utilizing microvascular anastomosis for long-term survival and fertilizing potential using sheep as the study model. Whole ovaries from sheep with intact blood vessels will be cryopreserved. After thawing these will be transplanted back into the animal microsurgically. These will be observed for resumption of ovarian function. The hormonal profiles will be examined and the quality of the follicles evaluated. The in-vitro fertilizing ability of these autotransplanted ovaries will be studied.

Another focus is to optimize the freezing protocols for isolated primordial follicles. The advantage of freezing primordial follicles over mature oocytes are its small size, few cytoplasmic organelles, absence of spindle structure, absence of zona pellucida and cortical granules. Using the murine and bovine models, we are examining the post thaw survival, histological changes, transplantation of isolated primordial follicles and their ability to progress during in-vitro maturation.

Human Studies

Ovarian tissue banking is a developing technique aimed to preserve fertility in women of childbearing age undergoing chemotherapy. Successful ovarian transplantation and preservation of ovarian oocytes in the laboratory has been achieved using techniques that involve maintaining blood supply. We are developing techniques to optimize methods that are effective in the long-term cryopreservation of intact or larger ovarian segments. Minimizing ischemia time increases tissue survival. Initial studies using porcine model are encouraging. The focus is to examine intact large-sized ovaries from humans and overcome challenges posed by ischemia reperfusion injury, heat and mass transfer limitations, and more importantly intravascular ice-formation problems.

• The effect of peritoneal reactive oxygen species on fertility in patients with endometriosis, RPC # 5090, 1995, IRB # 2156
• Outcome of pregnancy and the role of ROS and total antioxidant status in the follicular fluid, RPC #6147, 1998
• Predicting fertilization in an IVF program by use of mannose receptor assay, RPC # 6238, 1999
• Role of cytokines and oxidative stress on fertility in laparoscopically retrieved peritoneal fluid from patients with endometriosis, RPC #6329, IRB #2156, 1999
• The role of oxidative stress in assisted reproduction, RPC # 6362,1999
• Effect of oxidative stress on in vitro mouse embryo development, RPC #6485, 2000
• Protective effect of heat shock proteins against reactive nitrogen species toxicity in in vitro fertilization, RPC #6706, 2001
• To develop a non-invasive diagnostic aid for endometriosis, RPC 6719, IRB # 4490, 2002
• Coculture with Vero cells and granulosa cells for in vitro maturation of human in immature ocytes . IRB # 4476, 2002
• Hemosiderin laden macrophages in the diagnosis of endometriosis and protective effect of antioxidant and anti-TNF-a antibody on embryo development, RPC #7235, IRB #5661, 2003
• Assessment of fertilizing potential of mouse germ cells matured in vitro. RPC 07531, 2004
• Autotransplantation of frozen-thawed whole sheep ovaries with microvascular anastomosis: Assessment of oocyte maturation, fertilization, and embryo development. RPC#07688, ARC#07686, 2004
• Does peritoneal fluid of patients with endometriosis and idiopathic infertility affect the spindle structure in metaphase II oocytes? The role of oxidative stress. RPC#07993, IRB #8407
• Alterations in the cytoskeleton of mouse oocyte induced by cryopreservation and oxidative stress. RPC#07832/ARC#07658
• Ovarian transplantation: assessment of vascular injury. RPC#07832/ARC
• Ovarian transplantation : Assessment of vascular injury. RPC#2006-1122/ARC
• Does peritoneal fluid of patients with endometriosis and idiopathic infertility affect the spindle structure ion metaphase II oocytes? The role of oxidative stress. RPC#2006-1159/IRB8407
• Effect of magnetically selected non-apoptotic sperm on fertilization and embryo development. RPC#2006-1159
• Antioxidant profiles of bovine antral follicle. RPC #2007-1042
• Role of L-carnitine as antioxidant, anti-apoptotic and anti-tumor necrosis a in mouse embryo cultures. RPC #1135 (Under review)
• Evaluation of orthotopic autologus transplantation of cryopreserved isolated primordial follicles. RPC #2007-1041
• Hormonal maintenance and stimulation of autotransplanted Vitrified Ovarian Tissue. RPC #2008-1032; ARC: 08139
• Evaluation of orthotopic autologous transplantation of cryopreserved isolated primordial follicles, hormonal maintenance and stimulation of autotransplanted vitrified ovarian tissue. ARC 08197
• Antioxidants combinations to minimize human sperm apoptosis and dna damage during cryopreservation. IRB #06-425
• Evaluation of primordial follicles count in human ovaries using stereoanalysis. IRB #07-100 (Exempt protocol)
• Comparative study of sperm vitrification and slow freezing. IRB #09-127.

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