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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.


Current Research Interests

Isolation of Primordial Follicles

Follicle

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.


Immunopathology of Endometriosis

Peritoneal Fluid Schematic
 
ROS

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.


Role of tumor necrosis factor TNF-a in endometriosis

Dr.ZhangX

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.


In Vitro Maturation of Immature Oocytes

Dr.EsfandiariN

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.


Correlation of follicular fluid proxidant-antioxidant levels in bovine antral follicles with stages of folliculogenesis

FollicleAntrum

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.


Oxidative Stress and its Impact on Oocyte Cytoskeleton

OocyteWhole

Oxidative stress as a result of free radicals formation is higher in in-vitro culture environment. This may damage both the spindle microtubules and microfilaments. Disruption of cytoskeletal architecture is believed to lead to disorganization of these cytoskeletal elements that may ultimately lead to chromosomal anomalies and failure of fertilization and development. Damaging effects of oxidative stress on spindle formation, microarchitecture and chromosome alignment are currently being studied utilizing hydrogen peroxide as an exogenous source in metaphase II oocytes obtained from mouse. In addition, studies are being conducted to test the reversibility of these effects by antioxidant supplementation. Establishing these basic concepts of microtubules may provide a novel protocol for improving oocyte and embryo quality for use in IVF-ET.

We are also examining the effect of peritoneal fluid from women with endometriosis on oocyte quality, i.e. spindle structure, both microtubule morphology and alterations in chromosomal alignment in effort to find the cause of infertility in women with endometriosis.


Ovarian Tissue Cryopreservation

Dr.FalconeT
 
Dr.Choi

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.


Improving Pregnancy Rates by Using Non-apoptotic MACS Separated Sperm

Sperm preparation by density gradient centrifugation is routinely used in artificial reproductive techniques (ART). However the limitation of this technique is that, it does not allow separation of non-apoptotic and apoptotic sperm. Increased number of apoptotic sperms may influence the fertilization and pregnancy rate. Our group has recently reported a novel technique to efficiently separate apoptotic and non-apoptotic sperm by utilizing the magnetic activated cell sorting (MACS).

The externalization of the phospholipid phosphatidylserine (EPS) from the inner to the outer leaflet of the plasma membrane is a feature of apoptosis and can be monitored by annexin V-binding. Colloidal superparamagnetic microbeads bind to annexin V labeled dead and apoptotic spermatozoa. These are retained within the column by an external magnetic field.. The effects of this technique on subsequent fertilization and development are not known. In the pilot study we will evaluate the effects of non-apoptotic sperm separated by MACS technique on number of implantations, pregnancy rate and fetal development using an in vivo mouse model and compare to those after sperm preparation by density gradient separation. The goal is to determine whether non-apoptotic sperm after MACS improve the pregnancy rate (number of implantations) and to establish that exposure of sperm to MACS is safe and is not associated with a higher rate of fetal malformations when compared to those separated by density gradient separation. We will examine the beneficial as well any potential adverse effects of MACS technique in addition to the alterations in the number of implantations and fetal characteristics after the exposure of sperms to MACS.


Vitrification

Vitrification is the ultra rapid method of freezing. We are investigating its use in different applications such as blastocysts cryopreservation and tissue vitrification.

Blastocysts cryopreservation:

We are investigating the vitrification of blastocysts and comparing the results to those by slow freezing. We have analyzed different factors that may affect the outcome of blastocysts' vitrification as well as investigated different modalities whether invasive or non-invasive that may improve the outcome.

Tissue vitrification:

This is a highly challenging area, we have developed a new method that can accommodate the vitrification of tissues or a high load of cells in a closed system. Our new method and device "The Ohio-Cryo" are being investigated for ovarian tissue vitrification, testicular tissue vitrification as well as sperm vitrification. The device has the potential of allowing vitrification of relatively large volume which was not possible earlier with the minimal volume vitrification principal.


Approved Research Projects

Dr.BedaiwyM
 
 
Lab
  • 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.

** Click on the RPC # to view information about a Research Project; click on the IRB # to view information on Institutional Review Board approval. The above information will open as a PDF in Adobe Acrobat on your PC.


Gynecology Research Alumni

Lab

 
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Last Update : June 03, 2009
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