J. David Wininger

Development of antral follicles in cryopreserved cat ovarian tissue transplanted to immunodeficient mice

Ovarian cortex cryopreservation and xenotransplantation into immunodeficient mice represents a potential means for female germplasm conservation and an immediate model for investigation of folliculogenesis. The objectives of this study were to: (1) assess follicle survival after cryopreservation and transplantation of cat ovarian tissue into non-obese diabetic severely combined immunodeficient (NOD SCID) mice; and (2) evaluate the effects of gonadotropin treatments on follicular development in the transplanted tissue. Slices from the cat ovarian cortex were frozen and after thawing, transplanted under each kidney capsule of castrated male NOD SCID mice (eight xenografts in four mice). Sixty-two days after surgery, mice were randomly assigned (two per group) to gonadotropin-treated (eCG and hCG 88 h later) or control (saline-treated) groups. Twenty-four hours after the last injection, ovarian tissue was recovered and processed for histology. Fresh ovarian tissue from the same original source was similarly processed. Follicles were counted, measured, and classified as primordial, primary, secondary, or antral. Immunoreactive proliferating cell nuclear antigen (PCNA) stain was used to assess follicle viability. Microscopic examination revealed no evidence of necrosis or fibrosis. The grafts were well-vascularized, with follicles at all stages of development. Numbers of follicles in the transplanted tissue were markedly reduced compared to fresh tissue, with approximately 10% of follicles surviving freezing and transplantation procedures. Growing follicles positive for PCNA were found in all xenografts. Gonadotropin treatment did not alter the proportion of resting to growing follicles or mean follicle diameter by comparison with controls from untreated mice. By contrast, luteinization, but not ovulation, of antral follicles was observed only in grafts from treated mice. In summary, frozen-thawed cat ovarian cortex tissue not only survived xenotransplantation, it also contained follicles able to grow to antral stages. Exogenous gonadotropin treatment in this model resulted in luteinization of antral follicles but enhancement of follicular growth and ovulation did not occur.

The effects of different laser pulse lengths on the embryo biopsy procedure and embryo development to the blastocyst stage.

PurposeA laser is commonly used to remove a blastomere from an embryo for genetic testing. The laser uses intense heat which could possibly disrupt embryo development. It is the goal of this study to test the effects of different laser pulse lengths (and consequently heat) on the embryo biopsy procedure and embryo development.MethodsEach embryo biopsy was performed randomly utilizing laser pulse lengths of 0.604mS (group I), 0.708mS (group II), and 1.010mS (group III).ResultsFor groups I, II, and III, 83, 86, and 71 embryos were biopsied, respectively. There was no difference in day 5 embryo quality or lysed blastomeres between groups. Average number of blastomeres biopsied between group I (1.0 ± 0.0), II (1.0 ± 0.2), and III (1.1 ± 0.2) was significant (0.0001).ConclusionOur data demonstrates that laser pulse length does not influence the embryo biopsy procedure or embryo development.

The utility of embryo banking in order to increase the number of embryos available for preimplantation genetic screening in advanced maternal age patients

PurposeTo determine if embryo banking with PGS is more optimal than proceeding with PGS regardless of embryo number.MethodsPatients were divided into 2 groups, group 1 were those that banked embryos and proceeded through another round of IVF prior to PGS, and group 2 underwent PGS regardless of embryo number. Group 2 was divided into group 2A (patients with >10 embryos) and group 2B (patients who had <10 embryos).ResultsThere was no difference in embryos biopsied, normal embryos, number transferred, and pregnancy rate between group 1 and 2. A significant number of patients did not have a transfer in group 2B (6/11) compared to group 1 (3/19) (P = 0.0419). There was no significance between pregnancy rates per transfer between group 1 (6/16) and group 2B (2/5).ConclusionOur data suggests that banking will increase the odds of going to transfer but there was no increase in pregnancy rates.

Pregnancy after rebiopsy and vitrification of blastocysts following allele dropout after day 3 biopsy

OBJECTIVE To report a clinical pregnancy after rebiopsy and vitrification of blastocysts following allele dropout (ADO) of biopsied day 3 embryos. DESIGN Case report. SETTING Private center. PATIENT(S) Thirty-year-old woman and her 33-year-old husband who carries the single-gene condition paraganglioma. INTERVENTION(S) In vitro fertilization with day 3 embryo biopsy-ET-blastocyst biopsy and vitrification-subsequent frozen ET cycle. MAIN OUTCOME MEASURE(S) Results from preimplantation genetic diagnosis and pregnancy results after fresh and frozen ETs. RESULT(S) Nineteen oocytes were retrieved of which 13 were mature and 12 fertilized. Eleven embryos were biopsied on day 3: two were normal, five were affected, and four exhibited ADO. The two normal blastocysts were transferred, and three of the ADO blastocysts were biopsied and sent for reanalysis. The biopsied blastocysts were vitrified. No pregnancy resulted from the fresh ET. One of the biopsied blastocysts was normal, one received no result, and one exhibited ADO. A singleton clinical pregnancy resulted from a subsequent frozen ET of the thawed biopsied normal blastocyst. CONCLUSION(S) Rebiopsy and vitrification of blastocysts could be used in cases of ADO or lack of results after day 3 embryo biopsy.