After cyclophosphamide exposure, granulosa cells recover their anti-müllerian hormone-producing ability but not their numbers.

Anti-müllerian hormone (AMH) produced by granulosa cells (GCs), reserves the ovarian follicle pool for future recruitment and ovulation. However, women who have undergone cyclophosphamide (Cy) treatment have decreased AMH levels due to damaged GCs. This study establishes flow cytometry protocols for identification of GCs and investigates the cause of the Cy-induced AMH decrease by analyzing the number of GCs and their AMH production at the single cell level. Over 2 weeks, C57BL/6 mice were intraperitoneally injected 6 times with 100 mg/kg Cy and sacrificed either immediately or 4 weeks after Cy treatment. Twenty-four hours post-Cy exposure, a decrease in serum AMH levels was seen due to a reduction in the number of follicle-stimulating hormone receptor (FSHR)+ AMH+ GCs and their ability to produce AMH. However, 4 weeks after Cy treatment, serum AMH levels were still decreased due to the decreased number of FSHR+ AMH+ GCs, however, their AMH-producing ability was unaltered. Consistently, in vitro, Cy-induced low AMH production in FSHR+ AMH+ hGL5 cells (immortalized human GCs) was restored 24 h after Cy treatment, although their numbers remained decreased. Thus, the surviving GCs after Cy exposure had intact AMH-producing ability. In future, an effort to minimize GC death by Cy treatment is required, while maintaining its therapeutic effects.