Ovarian stimulation with excessive FSH doses causes cumulus cell and oocyte dysfunction in small ovarian reserve heifers.

Excessive FSH doses during ovarian stimulation in the small ovarian reserve heifer (SORH) cause premature cumulus expansion and follicular hyperstimulation dysgenesis (FHD) in nearly all ovulatory-size follicles with predicted disruptions in cell-signaling pathways in cumulus cells and oocytes (before ovulatory hCG stimulation). These observations support the hypothesis that excessive FSH dysregulates cumulus cell function and oocyte maturation. To test this hypothesis, we determined whether excessive FSH-induced differentially expressed genes (DEGs) in cumulus cells identified in our previously published transcriptome analysis were altered independent of extreme phenotypic differences observed amongst ovulatory-size follicles, and assessed predicted roles of these DEGs in cumulus and oocyte biology. We also determined if excessive FSH alters cumulus cell morphology, and oocyte nuclear maturation before (premature) or after an ovulatory hCG stimulus or during IVM. Excessive FSH doses increased expression of 17 cumulus DEGs with known roles in cumulus cell and oocyte functions (responsiveness to gonadotrophins, survival, expansion, and oocyte maturation). Excessive FSH also induced premature cumulus expansion and oocyte maturation but inhibited cumulus expansion and oocyte maturation post-hCG and diminished the ability of oocytes with prematurely expanded cumulus cells to undergo IVF or nuclear maturation during IVM. Ovarian stimulation with excessive FSH is concluded to disrupt cumulus cell and oocyte functions by inducing premature cumulus expansion and dysregulating oocyte maturation without an ovulatory hCG stimulus yielding poor-quality cumulus-oocyte complexes that may be incorrectly judged morphologically as suitable for IVF during ART.

Excessive follicle-stimulating hormone during ovarian stimulation of cattle may induce premature luteinization of most ovulatory-size follicles†.

High follicle-stimulating hormone (FSH) doses during ovarian stimulation are detrimental to ovulatory follicle function and decrease live birth rate in cattle and women. However, the mechanism whereby excessive FSH causes ovarian dysfunction is unknown. This study tested the hypothesis that excessive FSH during ovarian stimulation induces premature luteinization of ovulatory-size follicles. Small ovarian reserve heifers were injected twice daily for 4 days with 70 IU (N = 7 heifers) or 210 IU (N = 6 heifers) Folltropin-V [commercial FSH-enriched preparation of porcine pituitary glands with minor (<1%) luteinizing hormone (LH) contamination, cpFSH]. Ovulatory-size (≥10 mm) follicles were excised from ovaries after the last cpFSH injection and hormone concentrations in follicular fluid (FF) were determined using ELISA. Luteinization was monitored by assessing cumulus cell-oocyte complex (COC) morphology and measuring concentrations of estradiol (E), progesterone (P), and oxytocin (O) in FF. COCs were classified as having compact (cCOC) or expanded (eCOC) cumulus cell layers, and as estrogen-active (E:P in FF ≥1), estrogen-inactive (EI, E:P in FF ≤1 > 0.1), or extreme-estrogen-inactive (EEI, E:P in FF ≤0.1). A high proportion (72%) of ovulatory-size follicles in 210 IU, but not 70 IU, dose heifers displayed eCOCs. The high doses also produced higher proportions of EI or EEI follicles which had lower E:P ratio and/or E but higher P and/or O concentrations compared with the 70 IU dose heifers. In conclusion, excessive cpFSH doses during ovarian stimulation may induce premature luteinization of most ovulatory-size follicles in heifers with small ovarian reserves.

Limitations in use of ovarian reserve biomarkers to predict the superovulation response in small ovarian reserve heifers.

High FSH doses during superovulation of heifers with a small ovarian reserve increase the number of dysfunctional ovulatory-size follicles that do not ovulate in response to human chorionic gonadotropin (hCG). Thus, anti-Müllerian hormone (AMH) and antral follicle count (AFC), two well-established biomarkers of responsiveness of individuals to superovulation, are hypothesized to be positively linked to number of dysfunctional ovulatory-size follicles developing in response to superovulation with high FSH doses. To test this hypothesis, heifers with a small ovarian reserve were stimulated beginning on Day 1 of the estrous cycle with twice daily treatments for 4 days with each of four Folltropin-V (FSH) doses (35 IU, 70 IU (industry standard), 140 IU, or 210 IU) followed by prostaglandin F2α to regress corpora lutea (CL) from the previous estrous cycle and hCG to induce ovulation. Ovulatory-size follicles were classified as functional or dysfunctional based on whether they ovulated and formed CL in response to hCG. FSH dose did not impact the relationship between AMH, AFC and the number of functional or dysfunctional ovulatory-size follicles developing in response to superovulation. Thus, data from the four superovulations were averaged for each heifer. AMH and AFC were positively associated with the subsequent number of functional and dysfunctional ovulatory-size follicles and the proportion of ovulatory-size follicles that are dysfunctional after superovulation. Because measurements of AMH concentration and AFC predict the number but not functionality of ovulatory-size follicles, which may also impact oocyte quality, these ovarian reserve biomarkers are concluded to be unlikely useful to improve IVF or embryo transfer outcomes in heifers with a small ovarian reserve.