Introduction:
How cyclophosphamide affects quiescent primordial follicles is unclear. The "burnout theory" suggests that growing follicle destruction reduces primordial follicle activation inhibitors, causing continuous recruitment.
Aim:
To study the in vitro effects, cyclophosphamide active metabolites [4-hydroxycyclophosphamide (4hc) and phosphoramide mustard (PM)], have on human follicles.
Material & Methods:
Ten frozen-thawed human ovarian samples were sliced. One slice was fixed immediately [uncultured controls] and other slices were cultured for 48hrs with culture medium [cultured controls] or with 4hc/PM (3mM, 10mM) [treated samples]. Samples were removed from culture every 24hrs, and spent media were collected at 48hrs. Specimen evaluation included follicular counts and classification, Ki67 immunohistochemistry, apoptosis assay, and measurement of 17b-estradiol (E2) and antimullerian hormone (AMH) in spent media.
Results:
Samples treated for 48hrs had a lower primordial follicle ratio (19-33%) in parallel with a higher primary/secondary follicle ratio (~53%) compared with cultured controls (48% and 32%, respectively). At 24hrs these contrasts were less pronounced and no differences were found between the two concentrations. Most atretic follicles in treated samples were primary/secondary. E2 and AMH levels were more than doubled in treated samples, compared with untreated samples. There were no traces of apoptosis in cultured follicles including those treated. All activated follicles including treated samples were stained positively for ki67 in granulosa cells.
Conclusions:
Cyclophosphamide metabolites seem to activate primordial follicles. In turn, these growing follicles may undergo non-apoptotic atresia causing inhibition removal and further growth of additional follicles. Our findings support the "burnout theory" as the cyclophosphamide follicular toxicity mechanism.
