While the decision to go to egg retrieval is based largely upon the size of the lead follicular group, an attempt is usually made to retrieve oocytes regardless of follicular size. Several studies have suggested that the chance of recovering an oocyte decreases with smaller follicular size, and our findings are consistent with this [1
]. A recent publication from our group has also shown that the odds of oocyte nuclear maturation and normal fertilization are decreased in oocytes derived from smaller follicles [10
]. Developmentally, each follicle contains an oocyte. Yet there are reports of no oocyte recovery from IVF cycles involving multiple mature sized follicles, and in virtually all ovarian stimulation cycles the rate of oocyte recovery is less than 100% relative to the number of observed follicles [11
]. Therefore, assuming a similar aspiration technique is used, one reason why no egg is retrieved from certain follicles is that the cumulus-oocyte complex remains attached to the follicular wall during aspiration. Breaking this connection is a vital process for normal ovulation, and aspirated follicles where no egg is retrieved may allow elucidation of this process. We performed a repeated measures logistic regression analysis examining the oocyte recovery rates at different follicle sizes. The results demonstrate a significant correlation with the odds of retrieving an oocyte decreasing as the follicle size decreases (Figure ).
Interestingly, we found that FSH levels were higher in follicles in which an oocyte was recovered, and this was significant after adjusting for the type of ovarian stimulation protocol. An adjustment for stimulation was made due to different management schemes used in the protocols, as commonly we step-down with down regulated protocols resulting in potential differences in follicular fluid FSH levels (Table ). We could not adequately compare the oocyte recovery between stimulation protocols due to sample size. However, the fact that follicle number is inversely predictive of oocyte recovery is consistent with follicular fluid FSH being associated with oocyte recovery, because down regulated cycles (which had on average lower follicular FSH) are typically prescribed in women with higher follicle numbers.
While FSH is an important contributor to oocyte recovery, it is only significant after adjustment for hCG (Table ). This is an interesting finding, since in spontaneous ovulation there is a surge in both FSH and LH. Whether the FSH and LH surges have overlapping, complementary or redundant functions is unknown. In high concentrations, FSH has been shown to induce ovulation by itself in certain animal models. In hypophysectomized rats, pure recombinant FSH as a large bolus has been shown to induce ovulation, although the dose of FSH required was larger than the dose of hCG needed to promote ovulation [5
]. In rhesus monkeys who were down regulated with GnRH agonist and undergoing IVF, recombinant FSH was shown to recapitulate some but not all of the characteristics of recombinant hCG. Specifically, r-hFSH was equivalent to r-hCG for the reinitiation of oocyte meiosis, fertilization and granulosa cell luteinization, but a midcycle FSH surge did not sustain normal luteal function [12
]. A role for FSH is supported by in vitro
experiments, where both FSH and LH can promote plasminogen activator activity in cultured granulosa cells [6
]. These results also have biochemical plausibility as both FSH and LH receptors primarily mediate downstream signaling events through activation of the stimulatory G protein Gs with resultant increases in intracellular cyclic AMP. Differences in signals from G protein coupled receptors (GPCRs) that activate the same G protein are presumably due to either different characteristics of cyclic AMP fluxes within the cell or the activation of alternate signaling pathways not mediated specifically by G protein interactions [14
]. Granulosa cell responsiveness to LH may be dependent upon whether the cells have been previously exposed to other hormones [15
]. Reich et al, showed in a mouse model that the potency of LH is enhanced in the presence of estradiol [6
]. Evidence in humans, at least in the natural cycle, also suggests that plasminogen activity is positively correlated with estradiol levels [16
]. However, this observation was not seen in the setting of ovarian stimulation and supraphysiologic estradiol levels, and it was suggested that a subtle balance exists between granulosa cell secretion of plasminogen activator and steroids that is disturbed during controlled ovarian hyper-stimulation [16
Although the precise functions of FSH and LH in ovulation are not completely known, in ovulation induction cycles, hCG, which activates LH receptors, is administered to promote the periovulatory events and induce ovulation. In the human menstrual cycle, there is a mid-cycle surge in both FSH and LH secretion in the periovulatory period. Presumably these hormones act synergistically to promote the optimum environment for final follicle maturation and ovulation. In our study, neither hCG nor FSH alone was significantly associated with oocyte recovery when analyzed independently. The excess amount of hCG administered and available at the follicular level may account for the absence of an association. Interestingly, when both FSH and hCG were added to the model, only FSH became an independent predictor of oocyte recovery during follicular aspiration. Although more studies are needed, it is possible that FSH participates in the ovulation process either directly, by stimulating plasminogen activity, or indirectly by enhancing the responsiveness to hCG (LH) via modulation of the follicular environment (i.e. the hormonal milieu).
The association between higher FSH concentrations within follicles from which an oocyte was retrieved may suggest a role for increased vascularity around these follicles, since the follicular FSH is ultimately serum-derived [12
]. Following ovarian stimulation, follicles have different levels of oxygen tension [17
]. It is possible that while the amount of hCG is in excess, the FSH concentration may be at a threshold and would be vulnerable to changes in vascularity. However, if vascularity completely explained the correlation between FSH and oocyte recovery, other serum hormones impacted by changes in vascularity would be expected to correlate with oocyte recovery as well. Our data suggest there is an effect of FSH beyond that due to vascularity alone since prolactin, which is transported via the serum, has no association with oocyte recovery. This implies that other mechanisms may regulate FSH levels within the follicle. For example, the healthier follicles may be secreting factors such as VEGF, which is known to increase capillary permeability and thereby allow for enhanced diffusion of larger molecules such as FSH [18
]. Another possibility is there may be sequestration or preferential uptake of FSH in healthier metabolically active follicles.
Our analysis shows estradiol levels are inversely associated with oocyte recovery when measured alone or after adjusting for hCG and FSH. Physiologically, follicular estradiol is derived from androgen precursors and its production is influenced by FSH-induced aromatase activity [2
]. In our study, while the association of FSH and estradiol was significant, the ratio of estradiol/testosterone was not predictive of oocyte recovery, nor did it modify the effect of FSH. Estradiol production normally decreases after the midcycle surge of LH as a result of luteinization [2
]. It is possible that high concentrations of estradiol are a surrogate marker for inadequate response of the granulosa cells to hCG and thus indicate decreased ability of the oocyte to detach from the follicular wall.
A limitation of this study is that these findings are associative and cause and effect cannot be determined. Additionally, the relationship between plasma FSH and follicular fluid FSH at the time of hCG trigger or retrieval would be interesting to explore. Our data set is limited to plasma FSH levels obtained when the patient is not receiving exogenous gonadotropins and therefore we cannot explore this relationship. Another limitation is the numbers of patients analyzed within the subgroups of follicle size are limited (Table ). However, the results have biological plausibility, and provide a valid answer to the clinical question of whether there are any biochemical predictors of oocyte recovery.
The relationship between FSH and oocyte recovery was independent of the sex steroids, but the association was strengthened in the presence of progesterone which itself had a negative effect on oocyte recovery (Table ). This suggests that oocyte recovery could be improved if we can devise ways to increase follicular FSH without increasing progesterone. HCG is primarily responsible for luteinization of granulosa cells and subsequent progesterone production. High progesterone levels in this context may be an indicator of excess hCG or relative lack of FSH. Based on these data, and the hypothesis that a mid-cycle bolus of FSH will enhance oocyte recovery by promoting cumulus-oocyte complex release from the follicle wall, we have instituted a randomized, controlled study to evaluate the effects of FSH administration at the time of the hCG trigger during IVF. More studies are needed to determine the biological basis for the impact of FSH on oocyte recovery and to determine whether modifying gonadotropin administration can improve oocyte recovery rates.