Ovarian Preservation and Follicle Growth in the Grafts
We determined the state of grafted ovaries by the size and integrity of the follicles. Follicles were divided into seven groups according to their size, and both the viable and the atretic follicles were counted and graded. Grafts of half ovaries were assessed. In control ovaries, fixed immediately after the animal was killed (n = 3), all stages of follicular development were detected. In the controls, small atretic follicles were only rarely found at early stages of follicular development (Fig. 4A). In the subcutaneous ovarian grafts (n = 6; 2-24 days after transplantation), the largest viable follicles that were detected reached stage d (three layers of granulosa cells). A large number of degenerated primordial and primary follicles were detected (Fig. 4B).
The follicles in intramuscular grafts (n = 26; 1-31 days after transplantation), as the control intact ovary, ranged from the primordial to the antral stages. The number of degenerating primary follicles (stage b) was significantly lower and the number of viable follicles at more advanced stages was significantly higher in intramuscular relative to subcutaneous grafts (P < 0.05; two-tail, unpaired Mest; Fig. 4C). Distribution of follicles at various stages of development 1-3 days (Fig. 4D; n = 11), 6-7 days (Fig. 4E; n = 8), and 28-31 days (Fig. 4F; n = 7) after transplantation are presented. By grading the follicles according to their growth and viability, a marked difference was observed between the control ovaries (which scored 17.6), the subcutaneous grafts (which scored —35.2, i.e., very poor preservation), and the intramuscular grafts (which scored 6.7; Fig. 5). diabet glucotrol xl
FIG. 4. Distribution of follicles according to their size and condition from intact ovaries and from subcutaneous and intramuscular ovarian grafts. The follicles were counted and classified into seven categories according to their stage of development (a-g). Both viable (open bars) and atretic follicles (solid bars) were counted. The average number of follicles per square millimeter of ovary is presented (mean ± SEM). A) Follicle distribution of control 15-day-old intact Wistar rat ovary (n = 3). B) Follicle distribution in the subcutaneous grafts (2-24 days after transplantation; n = 6). C) Follicle distribution in the intramuscular grafts (1-31 days after transplantation; n = 26). D-F) Follicle distribution in the intramuscular transplantation according to the number of days after transplantation (1-3 d, n = 11 [D]; 6-7 d, n = 8 [E]; 28-31 d, n = 7 [F]). T-test analysis between the subcutaneous and intramuscular implants (B vs. C), P < 0.05 (asterisk, atretic stage b; diamond, viable stage d; x, viable stage e).
FIG. 5. Estimation of ovary state. Follicles were graded as indicated in Materials and Methods. The average grades ± SEM are presented for control 15-day-old intact Wistar rat ovary (n = 3); subcutaneous grafts 2-24 days after transplantation (n = 6); and intramuscular grafts 1-3 days after transplantation (n = 11), 6-7 days after transplantation (n = 8), and 2831 days after transplantation (n = 7). Asterisks indicate significant difference between the average grades of the different groups (P < 0.05).
FIG. 6. Distribution of intravenously administered contrast agent as detected by MRI and histology studies in intramuscular ovarian transplantation. All the pictures presented here were from one representative ovarian graft (1/3 ovary retrieved after 35 d). MRI of intramuscular ovarian transplantation site before administration of contrast agent. The femoral bone is indicated (arrow, A). Immediately after administration, the contrast agent was detected in large blood vessels (B, arrows) and after 32 min in the ovarian graft (C, arrowhead). D) Vascular permeability, namely the rate of change in contrast agent concentrations with time (APSMRI) was determined during the first 10 min after contrast administration. Pixels with significant permeability were overlaid on the anatomical image of 10 min after injection. The ovary is marked by an arrow. E) Histological analysis of the distribution and leakiness of blood vessels using avidin-FITC to detect the biotinylated contrast material (green). The avidin-FITC fluorescence is overlaid with SMC staining (aSMA, fluorescence of fast red) and DAPI stains of nuclei (blue).