The main objective of this study was to characterize the profiles of FF biomarkers (leptin, VEGF, MMPs, and NO2−/NO3−) according to three different protocols for COS protocols in women with POR classified as POSEIDON Group 2.
First, this prospective clinical and research cohort study showed unequivocal and significant differences in FF biomarker concentrations among three commonly used COS protocols in POR, i.e., corifollitropin alfa + rFSH, rFSH + urinary menotropins (uhMG), and a fixed dose rFSH + rLH protocol. Importantly, Group C patients, who received a fixed and a lower dose of FSH and LH (in the forms of rFSH + rLH), showed significantly higher concentrations of all intrafollicular biomarkers compared to the other two groups. Patients in Group A were stimulated with pure FSH (no LH) in the forms of corifollitropin and rFSH, and patients in Group B received FSH and LH, and possibly hCG, all of them present in uhMG. The fundamental differences between the three stimulation protocols were higher doses of FSH in Groups A and B and the absence/presence, nature, and dose of the LH used.
To obtain a urinary menotropin preparation with equal proportions of FSH and LH, highly reproducible preparation procedures are needed. If the LH activity is very low, hCG is added to compensate. The pharmaceutical behind urinary menotropins (Merapur®; Ferring) has stated that it does not add external hCG to the drug, and that its purity is comparable to that obtained with recombinant gonadotropin products; however, in a study conducted by Van de Weijer et al., the analysis of the composition of this drug revealed the presence of not two but three hormones: FSH, LH, and hCG, in addition to a significant percentage of impurities (30%). These differences may have had an impact on the follicular fluid profiles of the patients studied herein .
FSH, LH, and hCG belong to a family of glycoproteins that are heterodimers of two subunits (α and β). These hormones have the same α subunit but differ in the β subunit, which determines the biological specificity of each molecule. LH and hCG have a rather similar β subunit, which only differs by the extension of the carboxyl-terminal peptide (CTP) from the hCG subunit. Corifollitropin alfa is composed of the union between the hCG CTP and the β subunit of the FSH .
Although the physiologic role of LH during the follicular phase of a natural cycle is unquestionable, the impact of LH addition during COS remains controversial . It has been documented that addition of LH leads to different patterns in the synthesis of follicular steroids, which may affect oocyte maturation and competence . For example, use of recombinant LH resulted in an LH-dose-dependent increase of follicular fluid E2, androstenedione, and testosterone, which in turn may affect follicular through different regulatory molecules. Furthermore, there appears to be an optimal level of LH action with low levels resulting in egg incompetence for meiosis and high levels leading to oocyte atresia, and more high-quality embryos were observed using highly purified sing pure FSH .
Additionally, there is growing evidence that the addition of LH has a positive effect in patients with age over 35 years, and patients diagnosed with POR, since it increases FSH receptor expression and growth, improves follicular recruitment, and it also reduces the granulosa cell apoptosis . The mechanism by which the addition of LH improves ovarian response in patients with POR is not clear. An excessive suppression of endogenous LH after downregulation with a GnRH analogue is a plausible explanation, while another is related to the presence of polymorphisms in the LH molecule (LH β-chain variant), reducing the bioactivity of the molecule or polymorphisms of the LH receptor .
Recombinant FSH is the preferred choice of gonadotropins in POR patients, and there is ample high-quality evidence showing that rFSH is superior to urinary FSH and human menopausal gonadotropin (hMG) as a means to increase the oocyte yield . This is crucial, since the POSEIDON criteria rely on oocyte numbers to increase the chances of having at least one euploid blastocyst for transfer. The most recent systematic review and a further meta-analysis have shown that adding rLH to COS protocols is beneficial for two subgroups of patients: (i) women with an adequate ovarian reserve having an unexpected hyporesponse to rFSH monotherapy, i.e., POSEIDON groups 1 and 2, and (ii) patients 36–39 years of age [38, 39]. Therefore, adding rLH to COS in POSEIDON groups 1 and 2 patients should be considered , and that was the rationale for introducing the combination of rFSH + rLH into our protocols.
The recommended maximum daily dose of rFSH is 300 IU, as higher doses do not increase neither the clinical pregnancy rate nor the live birth rate . Corifollitropin alfa, a long-acting rFSH, has the advantage of a rapid increase in FSH serum level, which leads to early recruitment and an increase in the number of preovulatory follicles [41, 42]. In a randomized control trial that included POR patients following Bologna criteria, corifollitropin alfa did not increase the pregnancy rate compared to rFSH; however, significantly more embryos were available for freezing, which could potentially increase the cumulative birth rate by increasing the chance of having at least one euploid embryo for transfer .
Second, we investigated the individual variations among the studied biomarkers according to COS protocols. High levels of follicular leptin were previously shown to negatively correlate with follicular PO2, resembling a possible marker of follicular hypoxia and suboptimal embryonic development . Moreover, it has been described that the average leptin concentration is higher in FF of oocytes that present two pronuclei compared to those that had no evidence of fertilization . In the present study, we found significant differences in this hormone levels between the A vs C and B vs C groups. LH and hCG are characterized by specific molecular and biochemical features; they interact with distinct binding sites of the same receptor, resulting in lower dissociation rate by hCG than LH binding . Gonadotropin-specific ligand-receptor features imply different gene expression and intracellular signaling in vitro. According to Casarini et al. (2016), downstream signaling pathways of gonadotropins consist of LH-related proliferative and anti-apoptotic signals vs. high steroidogenic potential and pro-apoptotic activity of hCG in human primary granulosa cells . Here, we found a positive correlation between leptin and number of captured oocytes and leptin versus number of metaphase II eggs (Group B). Several reports have demonstrated that a narrow-leptin range is necessary to maintain a normal reproductive function, and that concentrations below or above these levels could interfere with the function of the hypothalamic-pituitary-gonadal axis .
Multiple studies have established that FF VEGF levels are significantly correlated with the degree of follicular peripheral vascularization [4, 11, 12, 46]. However, its correlation with reproductive outcomes is still controversial. Higher levels of intrafollicular VEGF have been observed in oocytes with fertilization failure. Furthermore, they have been negatively associated with the estrogen peak, number of metaphase II oocytes, and embryonic morphology [10, 24, 29]. Conversely, Monteleone et al. reported that VEGF levels are positively correlated with the degree of peripheral vascularization; additionally, oocytes obtained from follicles with a higher degree of vascularization had a higher fertilization rate and a better embryonic quality . Similar results were obtained in this study, where a positive correlation between VEGF and number of captured oocytes was found in Group C (rFSH + rLH). Our results provide further support to the hypothesis raised by Monteleone et al. about the crucial role of vascularization in follicular development. Group C had statistically significant higher levels of VEGF compared to the other groups. Min-zhi-Gao et al. support this concept as they found a positive correlation between VEGF and the dose of gonadotropins administered .
Barrionuevo et al. concluded that elevated levels of nitric oxide in FF may be useful for predicting a poor embryo outcome measured as the mature-oocytes potential to fertilize . Here, a negative correlation between nitric oxide with follicles with a diameter > 17 mm was found in Group B. In the same way, Revelli et al. pointed that excessive nitric oxide production in the microenvironment surrounding the oocyte could trigger apoptosis inside the follicle before fertilization, which affects the oocyte development . Likewise, serum nitric oxide was associated with implantation failure. This could be explained as high levels of nitric oxide in follicular fluid could generate an inflammatory reaction that negatively affects fertilization . Taken together, these data suggest that an excessive production of NO in the microenvironment surrounding the oocyte could trigger apoptosis within the follicle before fertilization, thus affecting oocyte development . FF concentrations of nitrites/nitrates could eventually be used a negative markers of oocyte quality; however, their assessment is rather tricky, and their intrafollicular levels were not found to correlate with IVF outcome ; thus, it is unlikely that the measurement of NO or its derivates will be used to identify optimal oocytes.
Matrix metalloproteinases (MMPs) catalyze the normal turnover of extracellular matrix (ECM) macromolecules . MMPs are membrane bound and depict gelatinase activity and hydrolyze gelatin into polypeptides, peptides, and amino acids that can then be secreted through the cellular membrane. MMP-2 and MMP-9 belong to gelatinases and facilitate both gelatin and collagen-binding through three fibronectin type-II-like repeat domains inserted in the catalytic domain of the structure . The rupture of the basal membrane of granulosa cells and the collagen matrix fragmentation at the follicular apex occur through the action of a cascade of proteolytic events that involve an increase in MMP activity before ovulation . Here, we showed that levels of Pro-MMP-2 in FF were negatively correlated with serum estradiol levels on the day of rhCG application and the number of captured oocytes in all groups. Nevertheless, Atabakhsh et al. did not observe a correlation between MMP-2 activity with oocyte count and fertilization rate. Moreover, Yang et al. concluded that higher levels of MMP-2 in IVF/ICSI cycles were positively correlated with the oocyte maturation and fertilization rates and proposed this metalloproteinase as a possible marker of oocyte maturation rate [19, 51].
We also found a negative correlation between MMP-9 levels with fertilization rate in Group C. Nevertheless, Atabakhsh et al. reported no significantly correlation between these two variables but demonstrated a positive correlation between MMP-9 and oocyte morphology . We observed a negative correlation between MMP-9 with number of follicles > 17 mm in Group A. To allow accurate remodeling and limit the site and extent of proteolytic degradation by MMPs, there must be a delicate balance between the activity of MMPs and their inhibitors .
Third, we evaluated the association between the FF biomarker profiles and clinical outcomes. Results of rate of metaphase II oocytes, fertilization rate, transfer rate, pregnancy rate, AND live birth rate were not different among groups, although the sample sizes regarding pregnancy were limited.
Although POR typically consist of a nonhomogeneous group of patients, the restriction of patients’ enrolment to POSEIDON Group 2 eliminates many of those concerns as all studied subgroups groups were similar in terms of age, AMH values, antral follicular count, and number of eggs recovered. However, we acknowledge that this study has some limitations. They include a relatively small sample size, its non-randomized design, and the fact that the total doses of gonadotropins used in the three arms were not equivalent. Future prospective, controlled, and randomized studies may be necessary to validate these results.