Article Information

Papamentzelopoulou Myrto-Sotiria^1*, Liokari Emanouela², Mavrogianni Despoina^1,2, Dimitroulia Evagelia³, Stavros Sofoklis⁴, Potiris Anastasios⁴, Vrachnis Nikos⁴, Loutradis Dimitris^#5

¹1^st Department of Obstetrics and Gynaecology, “Alexandra” General Hospital, National and Kapodistrian University of Athens, Greece

²Diagnostic and Therapeutic Fertility Institute S.A., Athens, Greece

³Department of Microbiology and Biopathology, National and Kapodistrian University of Athens, Greece

⁴3^rd Department of Obstetrics and Gynecology, “Attikon” Hospital, National and Kapodistrian University of Athens, Greece

⁵Athens Medical School, National and Kapodistrian University of Athens, Greece

Corresponding Author: Papamentzelopoulou Myrto-Sotiria, Department of Obstetrics and Gynaecology, “Alexandra” General Hospital, National and Kapodistrian University of Athens, Greece.

Received: 29 July 2024; Accepted: 07 August 2024; Published: 16 August 2024

Citation: Papamentzelopoulou Myrto-Sotiria, Liokari Emanouela, Mavrogianni Despoina, Dimitroulia Evagelia, Stavros Sofoklis, Potiris Anastasios, Loutradis Dimitris. Intrafollicular Endocrine Milieu Hormonal Profile Using Four Different Ovarian Stimulation Protocols: HMG, HMG/hCG, rFSH, rFSH/hCG: A Single-Center Pilot Study. Journal of Women’s Health and Development. 7 (2024): 55-67.

Abstract

Keywords

Intrafollicular endocrine milieu; Hormonal profiling; HMG; hCG; rFSH

Article Details

Introduction

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are responsible for the initial recruitment, growth, selection, and dominance of ovarian follicles as well as for maturation and ovulation, respectively. Both gonadotropins act in synergy and have been used in various forms for hormonal controlled ovarian stimulation (COS) in assisted reproductive technology (ART) to increase follicle count [1-4]. Furthermore, human chorionic gonadotropin (hCG) is a hormone that presents biochemical similarities with LH, binds to the same receptor as LH, and therefore, it has also been used in ART. Despite their similarities, LH and hCG are two distinct hormones and activate different affairs through mechanisms that can vary significantly between the two molecules [5, 6]. Thuesen et al., have published that hCG supplementation was associated with a significant stimulatory effect on the intrafollicular androgen and estradiol (E2) levels, with a shift toward a more androgenic milieu. Good quality embryos were developed upon higher intrafollicular concentrations of E2 and progesterone. As demonstrated, the estrogen to androgen ratio was higher in the follicles with oocytes giving rise to good quality embryos, supporting the importance of aromatization in order for the follicles to produce high quality oocytes [7]. Notably, advanced age reflects a change in the structure of the FSH and LH molecules [8]. This may be related to the biological expression at the target cell level, where an imbalance in the FSH and LH ovarian receptors was found [9-11]. It is well recognized that an increase in the daily dose of gonadotropins compensates for the age-related decline in gonadotropin sensitivity. Granulosa cells in antral follicles develop LH receptors; hence, they become sensitive to LH/hCG stimulation. Thus, it could be hypothesized that granulosa cells, which are resistant to gonadotropin stimulation, might benefit from LH or hCG stimulation. Further, LH or hCG may have beneficial effects through stimulation of theca cells. Interestingly, in situations where the ovaries are older or less sensitive to gonadotropins (biologically older), there is increasing evidence that the supplementation with LH or hCG activity is beneficial in the successful pregnancy outcome [12-15].

To address fertility issues, a variety of ovulation induction protocols have been introduced to reproductive medicine and assess the use of several forms of gonadotropins either of human purified urine or artificial recombinant origin. The urine analogues include Human Menopausal Gonadotropin (HMG) [16], urinary FSH (uFSH) [17] and urinary hCG (uhCG) [18]. On the other hand, the recombinant regimens that have been applied are recombinant FSH (rFSH) [19, 20], recombinant LH (rLH) [21], and recombinant hCG (rhCG) [22]. Selection of the most appropriate regimen among uFSH and rFSH still remains challenging with the currently available literature reporting no significant differences in the number of oocytes retrieved or pregnancy rates among the two types of FSH. The above gonadotropin preparations have been administered to induce COS in infertile women who undergo ART protocols. Hence, no concordance is available on the optimal regimens and preparations. Several studies support the addition of the LH-related regimens to FSH in COS, while LH, hCG, HMG and various combinations of them have been administered with or without FSH for COS [23-25]. Regarding combinations with hCG administration, Theofanakis et al., showed that the addition of hCG to rFSH was associated with better quality embryos and higher pregnancy rates, even in women over 40 years old, having higher basal FSH levels, and poor ovarian reserve [12]. The purpose of every ovulation induction protocol is to yield as many mature oocytes as possible; that is, to achieve the best possible synchronization of follicular maturation. For nuclear maturation, it has been suggested that the gap junction-mediated transmission of follicular-cell cyclic adenosine 3’-5’-monophosphate (cAMP) to the oocyte inhibits oocyte maturation [26-28], whereas gonadotropin stimulation ultimately terminates cumulus-oocyte communication and initiates resumption of meiosis by interruption of direct transfer of cAMP to the oocyte [27, 28]. Oocytes that mature in vitro appear to undergo nuclear and cytoplasmic maturation. For the assessment of their full in vitro maturation, it is necessary to examine not only their fertilizability, but also their potential for early embryonic development [26]. Moor et al., [29] and Ainsworth et al., [30] demonstrated that oocytes require a specific intrafollicular steroid environment for the inductive signals of meiotic resumption and the completion of the full maturational process. The aim of the present pilot study is the investigation of how hormonal follicular fluid (FF) profile derived from four different protocols of ovulation induction relates to the ovulation induction parameters, including number and quality of oocytes, and pregnancy rate.

Materials and Methods

Study population

The present pilot study was conducted from January 2022 until October 2022 at the Diagnostic and Therapeutic Fertility Institute S.A., Athens, Greece. The study group comprised 94 women that underwent controlled ovarian stimulation in a GnRH antagonist protocol with an age range of 24-45 years. Patient recruitment was accomplished using a computer-generated randomization table. Four different regimens were used: HMG (n=21) (Menopour Ferring), HMG/hCG (n=23), rFSH (n=29) (Gonal-F Merck) and rFSH/hCG (n=21). The addition of hCG (Pregnyl MSD) was a low dose of 100 IU/day. Protocol selection was based on age, Anti-Müllerian Hormone (AMH), FSH, LH, and antral follicle count (AFC). When the age of the patient was lower than 35 years rFSH was administered, while when the age was greater than 35 years HMG, rFSH+hCG, or HMG+hCG were administered. The rational for this decision was that women of advanced age are likely to achieve pregnancy using LH or hCG activity. Inclusion criteria were women with no uterine or ovarian anomalies, having normal hormonal profile (according to WHO guidelines), a regular menstrual cycle of 25-30 days and both ovaries intact. The indications for fertility treatment included male factor, tubal factor, and unexplained infertility. None of these women had been subjected to ovarian stimulation or any other hormonal treatment for at least three months before entering COS. For all participants, anthropometric data, such as age and BMI, and early follicular phase FSH, LH, PRL, AMH, TSH, T3, T4, TPO, TG, A, and DHEA-S levels within the preceding 6 months were recorded. The study protocol was approved by the review board of the Diagnostic and Therapeutic Fertility Institute S.A., (11/2020, date:20/12/2020). All participants provided informed consent for their medical records to be used in the study.

Ovarian stimulation protocol

The study participants underwent controlled ovarian stimulation in a GnRH antagonist protocol according to the strict routine practice of our institute. On day 5 of the menstrual cycle, daily administration of GnRH-antagonist (Orgalutran, MDS Hellas) was initiated and maintained until triggering of final oocyte maturation with rhCG (Ovitrell, Merck Hellas). Gonadotropins were administered on day 2 at a dose of 200 IU which was adjusted according to ovarian response on a daily basis, 6 days after the onset of gonadotropins administration. hCG (Pregnyl, MSD Hellas) was administered intramuscularly at a dose of 100 IU per day along with gonadotropins, starting on day 2 of the menstrual cycle throughout the follicular phase, until the day of triggering of final oocyte maturation. Serum E2 levels were measured daily starting on day 5 of ovarian stimulation with gonadotropins (day 7 of the menstrual cycle) until the day of triggering the final oocyte maturation with subcutaneous administration of 250 μg rhCG (Ovitrell, Merck Hellas). Follicular tracking started on day 6 of stimulation (day 8 of the menstrual cycle) and subsequent ultrasound scans were performed every day until oocyte retrieval.

Follicular fluid sample

Follicular aspiration and oocyte retrieval were scheduled 36 hours after rhCG triggering via transvaginal ultrasound-guided puncture. The procedure was performed using a needle single lumen (Cook Medical, USA) with manual aspiration of each single follicle using a 20-mL syringe. Only FFs from follicles of approximately 12 mm and greater were aspirated, centrifuged and supernatants were aliquoted and stored at -20°C for subsequent analysis. IVF/ICSI procedure followed. Embryo quality evaluation was blinded according to treatment and included assessment of blastomere number, degree of fragmentation, blastomere uniformity, and multinucleation [26]. Embryo transfer was performed on day 5 after oocyte retrieval. Luteal phase support was provided with 200 mg of micronized progesterone administered intravaginally three times daily starting from the day after egg collection onwards and serum beta-hCG was measured 14 days later. Clinical pregnancy was defined as the presence of a gestational sac on ultrasound at 6 gestational weeks. Ultrasound scans, oocyte retrievals and embryo-transfers were conducted by either of the two fertility specialists of the institute. Similarly, oocyte grading, fertilization, early embryo development and embryo grading were performed by either of the two senior embryologists of the institute.

Follicular fluid hormonal determination

All hormone measurements in FFs, including progesterone (Prg), testosterone (T), androstenedione (A), hCG, FSH and estradiol (Ε2), were performed at the Laboratory Genes Lab (Athens, Greece). Estradiol and progesterone required a 1:1000 dilution. RIA method was applied for analysis of androstenedione. For all the other hormones COBAS 6000 analyzer (COBAS 6000, Roche Diagnostics) was used. The sensitivity for each measurement was as follows: hCG 0.1 IU/L; FSH, 0.1 IU/L; LH 0.1 IU/L; E2 0.02 nmol/L; progesterone 0.1 nmol/L; T 0.087 nmol/L; and androstenedione, 0.1 nmol/L. The ratios of E2 to Prg, E2 to A, E2 to T, and A to T were calculated to evaluate the activity of the major steroidogenic hormones in the follicle. The main outcome measures were intrafollicular hormone concentrations in relation to treatment groups, number and quality of oocytes, and pregnancy rate.

Statistical Analysis

The qualitative data were presented as frequency and percentage. Associations were explored by the Fisher’s exact test between protocol and categorical variables. The quantitative data were presented as mean, standard deviation (or 95% Confidence Interval for mean). Comparisons between protocols with regards to follicular fluid hormonal profiling were performed using Analysis of Variance (F-test or Kruskal-Wallis H test). In addition, the comparison between the protocols regarding the qualitative and quantitative variables was repeated for the age group >34 years. To investigate the relationship between oocyte quality and follicular fluid measurements, the Pearson's correlation coefficient was applied. Graphical display using scatter plots and applying a linear trend equation to the data was conducted. The coefficient of determination was used for the degree of equation fit to the data. The correlations of embryo quality with follicular fluid measurements through all four protocols were investigated using Mann-Whitney U test. Statistical analysis was performed on SPSS version 20 software. The criterion of statistical significance was set at 5%.

Results

Study group demographic and clinical characteristics

The study group consisted of 94 women with a mean age of 36.3 ± 4.5 years (min=24, max=45). Of them, 21 were treated with HMG, 23 with HMG/hCG, 29 with rFSH and 21 with rFSH/hCG. Demographic and clinical characteristics for the four distinct subgroups are presented in Table 1. Women treated with HMG/hCG were statistically significantly older (38.09 ± 4.25 years, p=0.000), while the group of women with rFSH treatment were statistically significantly younger (33.03 ± 3.57 years, p=0.000). Moreover, serum FSH level at day 3 [9.65 ± 4.60 IU/L, p=0.029] and the number of previous attempts (1.96 ± 1.46, p=0.000) were statistically significantly higher in the HMG/hCG treated group. On the contrary, all the other serum hormone levels did not present statistically significant differences among the four treatment groups.

Table 1: Demographic and clinical characteristics of the HMG, HMG/hCG, rFSH, rFSH/hCG-treated groups.

Follicular fluid hormone profile in HMG, HMG/hCG, rFSH, rFSH/hCG-treated groups

Hormonal profiling in the four treated groups presented statistically significant differences in terms of testosterone, androstenedione, estradiol and FSH levels, as demonstrated in Table 2. In details, testosterone, FSH, androstenedione and estradiol levels were significantly higher in HMG/hCG-treated group compared to the other three groups (697±448 ng/ml, p=0.000, 8.75±2.82 IU/L, p=0.002, 6.90±2.77 ng/ml, p=0.000, and 38696391 ± 170049486 ng/ml, p=0.000, respectively). On the other hand, progesterone and hCG levels did not differ significantly in the four groups. As presented in Table 3, the statistically significantly higher ratios for the combinations of Prg/T, Prg/A, Prg/E2, A/hCG, hCG/FSH, hCG/E2, FSH/E2 were found in the rFSH-treated group. Moreover, for Prg/FSH, T/FSH and A/FSH combinations, the higher ratios were detected in the rFSH/hCG-treated group, while for T/A, T/hCG and T/E2 combinations, the higher ratios were found in the HMG/hCG-treated group, revealing a stronger androgenic environment in the FF of women treated with HMG/hCG.

Table 2: Follicular fluid hormonal profile in HMG, HMG/hCG, rFSH, rFSH/hCG-treated groups.

Table 3: Follicular fluid hormonal profile ratios in HMG, HMG/hCG, rFSH, rFSH/hCG-treated groups.

Ovulation induction profiles and pregnancy rates in HMG, HMG/hCG, rFSH, rFSH/hCG-treated groups

As presented in Table 4, the numbers of follicles >12 mm (12.00 ± 2.59, p=0.000), mature oocytes (MII) (10.00 ± 2.87, p=0.000), and embryos (8.79 ± 3.06, p=0.000) obtained were statistically significantly higher in rFSH-treated group compared to the other three groups, as expected given the younger average age of rFSH-treated women. Moreover, those women that were treated with rFSH achieved a higher pregnancy rate (31.0%; 9/29) without, though, being statistically significant (p=0.669). Another observation is that in the HMG group with the older women of the study the pregnancy rate was higher (28.6%; 6/21) compared to the HMG/hCG (17.4%; 4/23) and rFSH/hCG (19.0%; 4/21) groups, and relatively similar with the pregnancy rate of the younger rFSH-treated women. As presented in Table 5, ovulation induction profiling and pregnancy rates in HMG, HMG/hCG, rFSH, rFSH/hCG-treated groups with age adjustment >34 years were recorded. In particular, the number of follicles >12 mm, the number of mature oocytes (MII), and the number of embryos were statistically significantly higher in rFSH-treated women, as also expected (11.82 ± 3.71, p=0.001; 11.27 ± 4.17, p=0.002; 11.45 ± 4.55, p=0.004, respectively). However, in the rFSH-group pregnancy rate was lower (1/11; 9.1%) compared to the other three groups, while the higher pregnancy rate was observed in the HMG-group (5/16; 31.2%); though, such finding did not reach statistical significance (p=0.531).

Table 4: Ovulation induction profiles and pregnancy rates in HMG, HMG/hCG, rFSH, rFSH/hCG-treated groups.

Table 5: Ovulation induction profiles and pregnancy rates in HMG, HMG/hCG, rFSH, rFSH/hCG-treated groups with age adjustment >34 years

Follicular fluid hormonal profile in relation to pregnancy status in HMG, HMG/hCG, rFSH, rFSH/hCG-treated groups

The hormonal profile of follicular fluid in HMG, rFSH, HMG/hCG and rFSH/hCG-treated women with respect to pregnancy status is presented in Table 6. As shown, follicular fluid progesterone and hCG levels were statistically significantly lower in pregnant women treated with HMG compared to non-pregnant women who also received HMG (16184±3910 ng/ml vs 23338±8880 ng/ml, p=0.054 for progesterone, and 30.62±15.04 mIU/ml vs 63.00±25.61 mIU/ml, p=0.033 for hCG). For women treated with rFSH and proceeded to pregnancy, only follicular fluid progesterone levels were statistically significantly lower compared to the non-pregnant ones (11570±6049 ng/ml vs 25152±13829 ng/ml, p=0.051). On the contrary, women that received HMG in combination with hCG and achieved pregnancy had higher progesterone levels compared to non-pregnant women that were treated with the same regimen (24377±4944 ng/ml vs 14338±8634 ng/ml, p=0.034). As for rFSH/hCG-treated group, pregnant women had higher follicular fluid androstenedione levels compared to the non-pregnant women of the group (8.30±1.40 ng/ml vs 5.27±2.34 ng/ml, p=0.017).

Table 6: Follicular fluid hormonal profile in relation to pregnancy status in HMG, HMG/hCG, rFSH, rFSH/hCG-treated groups.

Follicular fluid profiling in relation to oocyte quality in HMG, HMG/hCG, rFSH, rFSH/hCG-treated groups

Next, the possible associations between testosterone and estradiol levels in follicular fluid of women treated with HMG/hCG, rFSH/hCG, HMG and rFSH, and mature oocyte (MII) quality were investigated via Pearson's correlation coefficient. As shown in Figures 1 and 2, high testosterone levels tended to be positively correlated with an increased number of mature oocytes in both HMG/hCG (r=0.309, p=0.162) and rFSH/hCG (r=0.253, p=0.295) protocols, respectively; however, such observations did not reach statistical significance. On the contrary, high levels of testosterone seemed to be slightly inversely related to maturation rate in HMG and rFSH protocols, respectively, whereas, no statistical significance was observed (data not shown). In addition, estradiol levels were weakly positively related to MII oocytes in both HMG/hCG (r=0.155, p=0.502, Fig.3) and rFSH/hCG (r=0.056, p=0.821, Fig.4) protocols, respectively; however, such findings were not statistically significant. Similarly, a weak negative not statistically significant correlation was detected between high estradiol levels and maturation rate in HMG and rFSH protocols, respectively (data not shown).

Figure 1: Testosterone vs mature oocytes (MII) in HMG/hCG protocol. Scatter plot showing the association between testosterone levels in follicular fluid of HMG/hCG-treated women and mature oocyte (MII) quality using Pearson's correlation coefficient.

Figure 2: Testosterone vs mature oocytes (MII) in rFSH/hCG protocol. Scatter plot showing the association between testosterone levels in follicular fluid of rFSH/hCG-treated women and mature oocyte (MII) quality using Pearson's correlation coefficient.

Figure 3: Estradiol vs mature oocytes (MII) in HMG/hCG protocol. Scatter plot showing the association between estradiol levels in follicular fluid of HMG/hCG-treated women and mature oocyte (MII) quality using Pearson's correlation coefficient.

Figure 4: Estradiol vs mature oocytes (MII) in rFSH/hCG protocol. Scatter plot showing the association between estradiol levels in follicular fluid of rFSH/hCG-treated women and mature oocyte (MII) quality using Pearson's correlation coefficient.

Follicular fluid hormonal profile in relation to quality of embryos in HMG, HMG/hCG, rFSH, rFSH/hCG-treated groups

The possible effect of the different gonadotropin preparations and follicular fluid profiling on the quality of embryos was consecutively studied. As presented in Table 7a, good quality embryos outperformed poor quality ones in all ovulation induction regimens, with HMG-treated group having the highest number of good quality embryos with only one poor quality embryo (HMG: 95.0% vs 5.0%, HMG/hCG: 65.2% vs 34.8%, rFSH: 74.1% vs 25.9%, and rFSH/hCG: 66.7% vs 33.3%). Moreover, none of the four different ovulation induction protocols affected the quality of embryos. Similarly, there were no statistically significant differences in the FF profile between good quality and poor quality embryos. The conclusion about the absence of correlation between FF profile and embryo quality was observed in all four protocols (Table 7b).

Table 7a: Good and poor quality embryo distribution in HMG, HMG/hCG, rFSH, rFSH/hCG-treated groups.

Table 7b: Follicular fluid hormonal profile in relation to quality of embryos in HMG, HMG/hCG, rFSH, rFSH/hCG-treated groups.

Discussion

To our knowledge, this is the first study that compares the endocrine profile of follicular fluid generated after gonadotrophin stimulation using four different regimens (HMG, HMG/hCG, rFSH and rFSH/hCG) in a GnRH antagonist protocol. As disclosed herein, testosterone, androstenedione, estradiol and FSH levels are higher in HMG/hCG-treated group. On the contrary, progesterone and hCG present similar levels in the four groups. Regarding the follicular fluid hormonal ratios, Prg/T, Prg/A, Prg/E2, A/hCG, hCG/FSH, hCG/E2, FSH/E2 ratios are higher in the rFSH-treated group, indicating a different balance of steroid/androgenic milieu of FF with rFSH regimen with lower FF levels of androgens and estradiol. Our results confirm the lower levels of follicular fluid androgens and estradiol in rFSH-treated women compared to women that received HMG, as disclosed in previous studies [13, 31]. Therefore, the strongest androgenic environment is observed in the HMG/hCG-treated group, which includes the oldest recruited women with the most previous failed attempts and the highest FSH level on day 3, suggesting that the LH/hCG activity enhances the androgenic environment of follicular fluid. This finding is in line with literature where the androgen production increased with the effect of LH or hCG supplementation and the FF environment became more androgenic. Moreover, a previous study demonstrated high androgen concentrations upon hCG supplementation, which resulted in a more androgenic milieu with good quality oocytes [7]. A recent study showed that the addition of hCG to rFSH in a short GnRH-agonist protocol, throughout the follicular phase, was associated with better quality embryos and higher pregnancy rates. The significance of these findings was enhanced by the fact that women who received hCG were significantly older and with higher basal FSH levels, thereby with expectant poorer ovarian reserve [14]. In our study such beneficial effect of hCG supplementation even in rFSH or HMG treatment was not observed, since the pregnancy rates were 19.0% and 17.4%, respectively, as compared with the pregnancy rate of 28.6% in the HMG-treated group. A potential explanation of this observation could be the type of ovulation induction; herein GnRH antagonist protocol was used, while Partsinevelos et al., used GnRH analogs short protocol [14]. On the other hand, it was recently demonstrated that the addition of low-dose hCG (100 IU of hCG/day) from the start of stimulation with rFSH in a GnRH agonist short protocol did not increase significantly the rate of top-quality embryos and clinical outcomes [15].

The present study also discloses that follicular fluid progesterone levels are lower in HMG and rFSH-treated women that achieved pregnancy compared to women receiving the same regimens that did not proceed to pregnancy. On the contrary, HMG/hCG-treated women that achieved pregnancy have higher progesterone levels in the follicular fluid compared to non-pregnant ones, which could be attributed to the fact that hCG readily stimulates progesterone production in normally functioning corpus luteum [32]. Therefore, our data suggest that the low progesterone concentrations in the follicular fluid endocrine milieu itself of both HMG and rFSH-treated women are responsible for the higher pregnancy rates, whereas, upon hCG co-administration progesterone levels in the follicular fluid of pregnant women are increased. However, the potential effect of follicular progesterone level on fertilization is still under debate. In a recent study, the progesterone levels were significantly higher in follicular fluid in women that received only hCG compared to women that received either GnRH-agonist or both GnRH-agonist and hCG trigger; however, when patients were divided into pregnant and non-pregnant, progesterone concentrations in the follicular fluid were comparable [33]. In a recent meta-analysis, fertilized oocytes demonstrated to be derived from follicles with higher levels of follicular fluid progesterone; however, Nagy et al., suggest that due to the various progesterone measurement methods and different treatment protocols the results must be interpreted with caution [34]. HMG-treated women that achieved pregnancy had lower follicular fluid hCG levels compared to the non-pregnant women of the group. Such observation could be attributed to the continuously administrations of LH as composition of HMG during the follicular phase that could change the sensitivity of follicular cells [35]. Our data indicate that desensitization of granulosa cells by LH may play a positive role and that the low hCG concentration in the follicular fluid has beneficial effect in pregnancy rate. It should be mentioned that the HMG group, which presents herein a better clinical demographic profile, has the highest pregnancy rate. Such observation is confirmed by Smitz et al., who showed that HMG stimulation significantly increased intrafollicular testosterone and estradiol levels due to LH activity, resulting in high pregnancy rates [13]. For patients >34 years old, the higher numbers of collected oocytes, mature oocytes, and embryos are found in rFSH-treated women, who have the higher Prg/E2 ratio, confirming that the progesterone/estradiol ratio may be the best indicator of maturity [36] and oocyte fertilization potential [37]. However, in the rFSH-group, pregnancy rate is lower (9.1%) compared to the other three groups, while the higher pregnancy rate is observed in the HMG-group (31.2%). Such finding is confirmed by previous studies that highlight the importance of LH in older women during ovulation induction [38-41].

Concerning hormonal profiling and MII oocyte quality, high testosterone and estradiol levels in FF tend to be associated with increased number of mature oocytes in HMG/hCG and rFSH/hCG-treated women, while a slightly inverse relation was observed between high levels of FF testosterone and estradiol and maturation rate in HMG and rFSH-treated women. A possible explanation of the increased maturation rate in HMG/hCG and rFSH/hCG protocols is the beneficial effect of hCG administration on triggering final oocyte maturation [42]. However, there are contradictory disclosures upon follicular fluid hormonal profiling and oocyte maturity, with the majority supporting the beneficial effect of high testosterone and estradiol levels on the maturation rate of oocytes [7, 13, 43]. A recent study also displayed an increase in the estradiol levels in the FF with mature oocytes [44]. On the contrary, as disclosed in a previous study, E2 and testosterone levels were significantly higher in FF containing immature oocytes than in FF containing mature oocytes [45]. Upon further analysis on embryo quality, no association between follicular fluid hormonal profiling and embryo quality is demonstrated in all four ovulation induction protocols. Our observation is in line with an earlier study wherein embryo quality was not related to levels of follicular estradiol and progesterone [46]. Another study indicated that no robust association between follicular steroid hormones and embryo quality could be established [47]. Certain limitations are identified in our pilot study, which include the small number of the recruited patients and the significantly younger age of women that consisted the rFSH group. Another factor that along with the hormone levels in follicular fluid may influence main outcome measures, such as oocyte quality and the subsequent achievement of pregnancy, is the type of ovulation induction; herein, we used a GnRH antagonist protocol. Nevertheless, we tried by applying age adjustment and strict patient inclusion criteria to minimize potential biases.

To conclude, the strongest androgenic environment is observed in the HMG/hCG-treated group, which includes the older women of the study with the most previous failed attempts and the highest FSH level on day 3. Moreover, low progesterone concentrations in the follicular fluid of both HMG and rFSH-treated women are correlated with higher pregnancy rates, while hCG co-administration with either HMG or rFSH enhances oocyte maturation. For age >34 years, HMG-treated women have the higher pregnancy rate compared to rFSH-treated ones. However, more prospective studies with larger sample sizes are needed to provide firm evidence on the actual impact of the different gonadotropin preparations in ART protocols. Subsequently, the assisted reproduction experts will be provided with new, individualized therapeutic approaches based on the follicular fluid hormonal profiling of each patient; thus, increasing the probability for successful pregnancy.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the review board of the Diagnostic and Therapeutic Fertility Institute S.A., (11/2020, date:20/12/2020).

Acknowledgements

We would like to acknowledge the contribution of all patients in the present pilot study.

Author contributions

The conception and design of the study was performed by LD. The acquisition of data was carried out by MD, DE, VN, SS and PA, while MD, LE, PMS provided expertise on the analysis and interpretation of the data. PMS, MD, LD were responsible for drafting the article and revising it. The final approval of the submitted version was performed by LD. All authors approved the final version of the paper.

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