Intrafollicular Activin-A, Inhibin-B, and Follistatin may predict invitro fertilization outcomesin subfertile women.

Intrafollicular Activin-A, Inhibin-B, and Follistatin may predict invitro fertilization outcomesin subfertile women. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Intrafollicular Activin-A, Inhibin-B, and Follistatin may predict invitro fertilization outcomesin subfertile women. dimitrios karayiannis, Roxane Tenta, Meropi D. Kontogianni, Minas Mastrominas, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8907446/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Purpose Follicular microenvironment influence soocyte and embryo quality and therefore plays a critical role in assisted reproduction therapy outcomes. This study prospectively evaluated the association between follicular fluid (FF) concentrations of activin-A, follistatin,and inhibin-B and in vitro fertilization (IVF) outcome parameters. Methods Couples with primary infertility attending an Assisted Conception Unit in Athens, Greece, were recruited as part of an ongoing prospective cohort study evaluating the impact of dietary and lifestyle patterns on fertility. FF levels of activin-A, follistatin, and inhibin-B were measured in 86 women undergoing IVF/ICSI cycles, along with assessments of oocyte yield and quality, embryo number and quality, implantation success, clinical pregnancy, and live birth rates. FF samples were collected at oocyte retrieval from follicles ≥ 18 mm in diameter and analyzed using enzyme-linked immunosorbent assay. Associations between FF biomarkers and IVF outcomes were assessed using correlation analyses and multivariable-adjusted regression models. Results FF activin-A concentrations were positively correlated with both follistatin and inhibin-B levels (P < 0.001), while FF follistatin and inhibin-B levels were significantly associated with oocyte and embryo number and quality (P < 0.05). Multivariable-adjusted relative risk analyses demonstrated that higher FF activin-A levels were independently associated with improved reproductive outcomes. Specifically, each 10 pg/mL increase in FF activin-A was associated with a 58% higher likelihood of successful implantation [relative risk (95% confidence intervals) = 1.58 (1.03–2.45)] and a 69% higher likelihood of achieving a clinical pregnancy [1.69 (1.08–2.62)]. Conclusion In conclusion, FF activin-A, follistatin and inhibin-B levels may serve as predictive biomarkers of IVF outcomes in women undergoing assisted reproductive treatment. Activin A Inhibin B Follistatin Follicular fluid In vitro Fertilization Introduction The success of in vitro fertilization (IVF) and other assisted reproductive technologies (ARTs) is critically dependent on obtaining good-quality oocytes [ 1 ]. Oocyte quality and health depend on the microenvironment of the corresponding follicle where they develop[ 2 ]. This critical intrafollicular microenvironment for appropriate oocyte development is influenced by multiple endocrine and intraovarian paracrine interactions. Regarding the intraovarian effects, recent findings demonstrate a role for intrafollicular activin/follistatin/inhibin levels, which has received considerable attention due to their effects on follicle development [ 3 ]. Initial studies have explored the role of activins, inhibins and follistatin in reproductive and developmental biology, with most focusing on their serum levels [ 4 ]. Inhibins, activins, and follistatin preferentially influence Follicle-stimulating hormone (FSH) secretion and contribute to divergent release of Luteinizing hormone ( LH)and FSH throughout the menstrual cycle [ 5 , 6 ]. Inhibins primarily inhibit FSH release from pituitary gonadotrope cells, whereas activins stimulate FSH secretion. Follistatin, in turn, inhibits pituitary FSH secretion by binding to activin, thus rendering it inactive. In contrast to inhibins, which act primarily via endocrine signaling, activins and follistatin produced in the pituitary, influence FSH secretion via autocrine-paracrine signaling [ 7 – 9 ]. Follicular fluid (FF) composition differs compared to serum and undergoes physiological alterations during follicular development [ 10 ]. Hormone concentrations in FF can both directly (via genomic and non-genomic actions) and indirectly (via somatic cells within the follicle) influence oocyte differentiation [ 11 ]. Evaluation of the intrafollicular levels of activins, inhibins and follistatinis particularly useful to study their role in follicular development. Since most of these factors have a paracrine and/or autocrine mode of action, their intrafollicular levels reflect their role in follicular development better than circulating levels, which may be also influenced by extra ovarian production sites [ 3 ]. All these observations derive mainly from IVF treatment procedures, given the fact that IVF cycles offer the opportunity to measure a plethora of components contained in the FF. In addition, it is now feasible to investigate the association of factors such as activin/follistatin/inhibin and parameters like oocytes fertilization potential and the quality of embryos developed after conventional IVF or intracytoplasmic sperm injection. To date, few studies have investigated the interrelationship between intrafollicular levels of activins, inhibins and follistatin and their involvement in IVF outcomes [ 12 – 16 ]. The purpose of the present study was to evaluate FF levels of activin A, inhibin B, and follistatin in a sample of subfertile women undergoing IVF, to assess potential interplay among these factors, and to elucidate their role in IVF intermediate and clinical outcomes. Subjects and Methods Couples with primary infertility who sought evaluation and treatment in an Assisted Conception Unit in Athens, Greece (Embryogenesis Assisted Conception Unit, www.embryogenesis.gr ) were originally invited to participate in a prospective cohort study investigating how background diet and lifestyle patterns impact fertility [ 17 ]. Eligible female partners were ≤ 41 years of age, had a body mass index (BMI) of < 30 kg/m 2 , had no history of prior IVF attempts or pregnancies, and were not enrolled innatural-cycle protocols. Participants were also required to be free of endometriosis (based on transvaginal ultrasound assessment), prior ovarian surgery, diabetes mellitus, cardiovascular disease, hypertension, neoplasm, hypothyroidism or psychiatric disorders [ 18 ]. Of 244 eligible Greek couples evaluated from November 2013 until September 2018, 86 women consented to provide biological specimens (FF samples). At study enrollment, anthropometric assessments were conducted for all participants, alongside administration of a comprehensive questionnaire capturing demographic, reproductive, medical history, and lifestyle data. Habitual dietary and alcohol intake was evaluated using a validated 76-item semi-quantitative FFQ tailored for the Greek population, referencing the preceding 6 months prior to IVF. Physical activity levels were measured with the validated Greek version of the International Physical Activity Questionnaire (iPAQ), while anxiety was assessed using the Spielberger State-Trait Anxiety Inventory (STAI-Y) [ 17 , 18 ]. Biochemical analyses were performed in the Laboratory of Biology, Biochemistry and Physiology, in the Department of Nutrition & Dietetics at Harokopio University of Athens. All procedures adhered to the Helsinki Declaration, with written informed consent obtained from participants. The study protocol was approved by the Institutional Ethics Committee of Harokopio University (12/13-01-2013). FF and corresponding oocyte collection At the time of oocyte retrieval, the single dominant follicle (> 18 mm in diameter) was punctured with a 23G needle attached to a syringe. Follicular fluid (FF) was aspirated directly into an empty bottle without flushing medium and selected only if it contained one intact oocyte and was free of contamination by fresh blood. To minimize cross contamination between follicles, FF was obtained exclusively from one dominant follicle per ovary, while a separate needle was used for each ovary. Following needle withdrawal, the needle was flushed to retrieve any oocytes trapped in the dead space of the collection needle. To preclude contamination by flushing medium, only fluid from the initial aspirate from the dominant follicle was retained. Each FF sample was centrifuged immediately at 1,300 g for 10 minutes and the clear supernatants were collected and stored at -80°C until assayed. In vitro fertilization (IVF) procedure and outcome assessment Before initiating the IVF procedure, women underwent ovarian reserve testing and were assigned into one of the following ovarian stimulation protocols as clinically indicated: 1) Gonadotropin-Releasing Hormone (GnRH) antagonist protocol (Cetrotide, Orgalutran) and 2) Follicular-phase GnRH agonist/Flare protocol (Daronda, Arvekap). Subcutaneous recombinant FSH (Gonal-F, Puregon, Altrmon) and/or human menopausal gonadotropin (hMG; Menopur, Merional, Pergonal) were administered in all regimens with a maximum combined daily dose of 450 IU. Women were monitored during ovarian stimulation for serum estradiol (E2) and for follicle counts and size alterations. Human chorionic gonadotropin (hCG) was administered ~ 36 h before the scheduled egg retrieval procedure in order to induce ovulation. Oocyte retrieval was performed when follicle sizes reached 16–18 mm and when serum E2 concentrations reached at least 1800 pmol/L. Intracytoplasmic sperm injection (ICSI) was carried out for all IVF cycles in this study. Embryologists classified oocytes as germinal vesicle, metaphase-I or metaphase-II (oocytes-MII, presence of a polar body) and determined fertilization 17–20 h after insemination as the number of oocytes with two pronuclei. Fertilization rate was defined as the total number of fertilized oocytes divided by the number of oocytes-ΜII. The resulting embryos were monitored on day 3for cell number and morphological quality (1 = best to 5 = worst). Embryos that had reached 6–8 cells on day 3 were considered to be cleaving at a normal rate, whereas embryos with ≤ 5 cells or ≥ 9 were considered to be slow cleaving or to have accelerated cleavage, respectively. In the present analysis embryos were classified as high quality if they had at least 8 cells on day 3 and if they had reached a morphological quality score of 1 or 2. The maximum number of embryos transferred should abide by the Greek National Legislation for embryo transfer guidelines (Greek National Authority of Assisted Reproduction; Law 3305/01/2005; http://eaiya.gov.gr/en/law-fek/ ). Successful implantation was defined as serum β -hCG concentration > 20 IU/L measured at 14–21 days after egg retrieval. Clinical pregnancy was defined as the presence of an intrauterine pregnancy confirmed by ultrasound (presence of at least one gestational sac and cardiac activity at 6 weeks estimated gestational age), and live birth as the birth of a neonate on/or after 24 weeks of gestation. All clinical information, including infertility diagnosis, hormone levels and protocol type, was abstracted from the patient’s electronic medical records. The study’s primary outcomes were implantation, clinical pregnancy and live birth, whereas oocyte yield, fertilization rate and embryo quality parameters were used as intermediate outcomes. Hormone assays Activin-A levels were measured using a commercially available Elisa Kit (CEA001Ra, Cloud-Clone Corp., Houston, USA), with a sensitivity of 5.7 pg/mL, intra assay coefficient of variation (CV) of < 10% and inter assay CV of < 12%.Inhibin-B levels were measured using a commercially available Elisa Kit (CEA760Hu,Cloud-Clone Corp., Houston, USA), with a sensitivity of 3.2 pg/mL, intra assay CV of < 10%, and inter assay CV of < 12%. Follistatin levels were measured using a commercially available Elisa Kit (DFN00, R&D Systems, Minneapolis, MN, USA), with a sensitivity of 29 pg/mL, intra assay CV of 2–2.7% and inter assay CV of 7.1–9.2%. All samples were diluted as necessary for values to be within the range of the standard curve (1:40 dilution of FF samples for follistatin, 1:30 dilution for activin-A and 1:5 dilution for inhibin-B) and in duplicate within the same assay to decrease inter assay variability. Total protein content was determined by the Bradford method[ 19 ].Absorbance was determined in a Power wave microplate spectrophotometer (Biotek Instruments, Inc.)at 450 nm. Statistical Analysis The normality of the distribution of the variables was assessed using the Shapiro-Wilks test. Continuous variables are presented as median (interquartile range, IQR), whereas categorical variables as absolute and relative frequencies. Associations between categorical variables were tested by χ 2 tests, while differences between categorical and several clinical and reproductive variables were tested using the non-parametric Mann-Whitney-U test. The association between FF hormones was assessed using Spearman's rank correlation (rho) coefficient because the data showed a non-normal distribution as determined by the Shapiro-Wilk test. Generalized linear models were used to test the associations between FF hormone levels and IVF outcomes. A Poisson distribution with log link function was used to test the association of number of total and mature oocytes, fertilized oocytes, and high-quality embryos (all count data), while a binomial distribution with logit link function was used for fertilization rate and clinical endpoints. The results are presented as relative risk (RR) and 95% confidence intervals (CIs). Statistical Package for Social Sciences software (SPSS, version 24.0, Chicago, Illinois, USA) was used for all statistical calculations. All reported P -values are based on two-sided tests and compared with a significant level of 5%. Results Study participants were aged25-41 years (median: 36 years) and had a mean BMI of 22.8 ± 2.9 kg/m 2 . Of the 86 women enrolled, 41 (47.6%) had successful implantation, 34 (39.5%) achieved a clinical pregnancy, and 32 (37.2%) gave live birth. Baseline demographic and clinical characteristics stratified by clinical pregnancy status are presented in Table 1 . No differences were observed between women with and without a clinical pregnancy with respect toΒΜΙ, physical activity levels, or anxiety scores. In contrast, women who achieved a clinical pregnancy had significantly higher number of retrieved oocytes, metaphase II (MII) oocytes, embryos, and high-quality embryos compared with those who did not (all P < 0.05). Table 1 Descriptive characteristics of women according to IVF clinical outcome (n = 86) IVF outcome Characteristic Clinical pregnancy, positive Clinical pregnancy, negative P-value Ν 34 52 Age, y 37 (33–39) 35 (33–37) 0.001 Body mass index, kg/m 2 22.3 (21.2–24.1) 22.1 (20.7–24.0) 0.899 Smoking status, n (%) Never Former Current 26 (81.3) 3 (9.4) 3 (9.4) 37(71.2) 2 (3.8) 13 (25.0) 0.160 Physical activity, metabolic equivalent of task(MET)-min/week 828.7 (506.2-1121.2) 896.0 (412.5-1054.5) 0.932 S-Anxiety (score range 20–80) a 46(32.2–53.7) 44.5(35.7–52.2) 0.806 T-Anxiety (score range 20–80) a 38(33.2–46.7) 41.5 (33.0–50.0) 0.747 Total energy intake, kcal/day 1820(1594–2041) 1798 (1546–2010) 0.728 Supplements use, n (%) 20 (58.8) 24 (46.0) 0.552 Reproductive characteristics and IVF outcome Family subfertility history , n (%) 4 (11.8) 20 (38.5) 0.007 Normal menstrual cycle, n (%) 23 (67.6) 36 (69.2) 0.877 Cause of infertility, n (%) Male factor Female factor Unexplained Polycystic ovary syndrome, n (%) 16 (47.1) 0 18 (52.9) 4 (11.8) 22 (42.3) 4 (7.7) 26 (50.0) 10 (19.2) 0.002 0.359 Oocytes produced, n Metaphase-II stage oocyte, n Fertilization rate, % Embryos produced, n High quality embryos, n 12 (9–16) 8 (6–12) 86.5(64.6–94.7) 7 (6–9) 3 (2–5) 9 (6–14) 6 (4–10) 81.6 (60.5–98.2) 5 (3–9) 1 (0–4) 0.010 0.015 0.284 0.019 0.046 Number of embryos transferred, n (%) No embryos transferred 1 embryo 2 embryos 3 + embryos 0 2 (5.9) 15 (44.1) 17 (50.0) 5 (9.6) 12 (23.1) 17 (32.7) 18 (34.6) 0.029 Follicular fluid hormone concentrations Activin-Α (pg/mL) 307.5 (264 -346.5) 313.5 (274–384) 0.336 Activin-Α(pg/mg total protein)† 49.4 (44.8–61.9) 53.2 (46.5–65.6) 0.059 Inhibin-Β(pg/mL) 45.5 (27.7–78.5) 39.5 (9.2–72.7) 0.281 Inhibin-Β(pg/mg total protein)† 7.7 (4.4–12.9) 6.8 (1.9–11.5) 0.293 Follistatin (ng/mL) 405.5 (204.7-849.5) 412.5 (183-788.7) 0.784 Follistatin(ng/mg total protein)† 65.8 (34.9-121.9) 65.6 (30.1-126.4) 0.747 Values represent median (IQR) or number of subjects (%). Differences in variables were tested using Mann-Whitney U test for continuous variables and or chi square test for categorical variables. a State (S)-Anxiety evaluates the current emotional state; Trait (T)-Anxiety evaluates relatively stable aspects of anxiety (how the respondent usually feels), with higher values suggesting higher levels of anxiety. b Type of supplements: multivitamins, iron, folic acid, vitamin C/other. †Values adjusted to follicular fluid total protein content. A total of 86 follicular fluid (FF) samples were collected to assess intrafollicular hormonal levels. The analysis revealed a significant positive correlation between activin-A and inhibin-Β (Spearman’s rho = 0.512, P < 0.001), between activin-A and follistatin (rho = 0.427, P < 0.001),as well as between inhibin-B and follistatin (rho = 0.604, P < 0.001).Furthermore, correlations between FF hormone levels and intermediate IVF outcomes demonstrated that both inhibin-B and follistatin concentrations were positively and significantly associated with the number of retrieved oocytes (rho = 0.316 and rho = 0.362, respectively), oocytes-MII (rho = 0.326 and r = 0.389), total embryos (rho = 0.329 and rho = 0.372), and high quality embryos (rho = 0.244 and rho = 0.210) (all P < 0.05). In contrast, activin-A levels were negatively correlated with fertilization rate (rho=-0.239, P = 0.03). In Table 2 , the results of the fully adjusted multivariable models for the study’s primary outcomes are presented. Regarding FF follistatin concentrations, an increase of 10 ng/mL was associated with a 1.02-fold increase (95% CI: 1.01–1.03) in the number of oocytes retrieved, a 1.03-fold increase (95%CI: 1.01–1.04) in oocytes-MII, and with higher numbers of total embryos and high quality embryos[RR (95%CI): 1.03 (1.01–1.04) and 1.03 (1.01–1.05), respectively]. Similarly, each 1 pg/mL increase in FF inhibin-B concentrations was associated with a 1.01-fold increase (95%CI: 1.00-1.02) in the number of oocytes retrieved and oocytes-MII, as well as a 1.03-fold increase (95%CI: 1.01–1.04) in high quality embryos. Finally, a 10 pg/mL increase in FF activin-A concentrations was associated with a 58%higher likelihood of successful implantation (RR = 1.58, 95%CI: 1.03–2.45) and a 69% higher likelihood of clinical pregnancy (RR = 1.69; 95%CI: 1.08–2.62). Table 2 Association between follicular fluid hormone levels and IVF clinical outcomes (n = 86 follicular fluid samples from the primary follicles of 86 women undergoing IVF) Activin-Α (per 10 pg/mL)† Inhibin-Β (per pg/mL)† Follistatin (per 10 ng/mL)† Intermediate outcomes Oocytes produced, n Model 1 0.99 (0.96–1.02) 1.01 (1.00-1.02)* 1.02 (1.01–1.03)* Model 2 0.99 (0.96–1.02) 1.01 (1.00-1.02)* 1.02 (1.01–1.04)* Model 3 0.98 (0.95–1.01) 1.01 (1.00-1.02)* 1.02 (1.01–1.03)* Oocytes-MII, n Model 1 0.98(0.94–1.02) 1.01 (1.00-1.02)* 1.02 (1.01–1.04)* Model 2 0.98(0.94–1.01) 1.01 (1.00-1.02)* 1.03 (1.01–1.04)* Model 3 0.97 (0.93–1.01) 1.01 (1.00-1.02)* 1.03 (1.01–1.04)* Embryos produced, n Model 1 0.98 (0.93–1.02) 1.02 (1.00-1.03)* 1.03 (1.01–1.04)* Model 2 0.97 (0.93–1.02) 1.02 (1.01–1.03)* 1.03 (1.01–1.04)* Model 3 0.96 (0.91–1.01) 1.02 (1.01–1.03)* 1.03 (1.01–1.04)* High quality embryos, n Model 1 0.91 (0.83-1.00) 1.02 (1.00-1.04)* 1.01 (0.99–1.04) Model 2 0.91 (0.84-1.00) 1.02 (1.00-1.04)* 1.02 (1.00-1.04)* Model 3 0.92 (0.84-1.00) 1.03 (1.01–1.04)* 1.03 (1.01–1.05)* Clinical endpoints Successful Implantation (Yes vs. No) Model 1 1.57 (1.06–2.32)* 0.98 (0.92–1.04) 1.01 (0.94–1.08) Model 2 1.51 (1.01–2.26)* 0.97 (0.91–1.04) 1.00 (0.92–1.08) Model 3 1.58 (1.03–2.45)* 0.96 (0.90–1.03) 0.98 (0.99–1.07) Clinical Pregnancy (Yes vs. No) Model 1 1.61 (1.07–2.43)* 0.94 (0.97–1.06) 1.02 (0.94–1.10) Model 2 1.58 (1.04 − 2.39)* 0.99 (0.93–1.05) 1.01 (0.93–1.09) Model 3 1.69 (1.08 − 2.62)* 0.98 (0.92–1.05) 1.01 (0.93–1.10) Live Birth (Yes vs. No) Model 1 1.45 (0.98–2.14) 0.99 (0.93–1.05) 1.02 (0.94–1.10) Model 2 1.41 (0.95 − 2.10) 0.98 (0.93–1.05) 1.01 (0.93–1.10) Model 3 1.47(0.96 − 2.26) 0.98 (0.92–1.05) 1.01 (0.93–1.10) Data represents relative risk (95% confidence interval). All analyses were conducted using generalized linear models. A Poisson distribution with log link function were used to test association of number of total and mature oocytes, embryos produced and high-quality embryos (all count data), while a binomial distribution with logit link function were used for clinical endpoints. Model 1 was adjusted for ovarian stimulation protocol and age. Model 2 was adjusted as for model 1 and for body mass index (continuous), smoking (never smoker vs ever smoking) and cause of infertility. Model 3 was adjusted as for model 2 and for stress and physical activity levels (all continuous). *P ≤ 0.05. † Values adjusted to follicular fluid total protein content. Abbreviations used : IVF = in vitro fertilization; Oocytes-MII=metaphase-II stage oocytes. Discussion The primary aim of the present study was to investigate the role of intrafollicular components of the activin-follistatin-inhibin axis in relation to oocyte competence, embryo development, and final IVF outcomes in subfertile women undergoing IVF/ICSI. Our findings indicated that higher FF concentrations of inhibin-B and follistatin were positively associated with key intermediate IVF outcomes, including the number of oocytes retrieved, the proportion of mature (MII) oocytes, as well as embryo number and quality. In contrast, FF activin-A levels were not linked to intermediate IVF outcomes but were significantly associated with increased implantation and clinical pregnancy rates, indicating a potentially distinct role in later stages of reproductive success. Collectively, these findings support the concept that the intrafollicular hormonal milieu plays a crucial role in follicular development, oocyte maturation, and embryo competence. Furthermore, the observed positive correlations among FF activin-A, inhibin-B, and follistatin reflect the tightly regulated local interplay of these factors within the ovarian follicle are consistent with previous experimental and human data demonstrating their coordinated expression during folliculogenesis [ 13 , 20 ]. In order to investigate whether follicular activin/follistatin/inhibin concentration sinfluence oocyte maturation, FF hormone concentrations were compared with various parameters of oocyte quality. Both follistatin and inhibin-B concentrations showed a significant positive correlation withthe number of metaphase II (MII) oocytes, suggesting that these hormones might be involved in the preparation process for, or initiation of oocyte nuclear maturation. With respect to inhibin-B levels, multivariate analyses revealed significant associations with both the number and quality of oocytes and embryos. These findings are consistent with those reported by Chang et al. [ 21 ], who analyzed233 FF samples and demonstrated that inhibin-B levels were related to embryo quality scores on days 2 and 3 post-fertilization. Although the underlying mechanisms remain unclear, inhibin-B levels have been shown to increase as follicles grow and mature, supporting its potential role as an indicator of follicle quality. In agreement with this hypothesis, data from another study [ 22 ] indicated that higher FF inhibin-B concentrations were associated with increased fertilization and pregnancy rates. Collectively, these findings suggest that FF inhibin-B may serve as a reliable marker of follicular development and oocyte/embryo quality during controlled ovulation stimulation. Furthermore, the results of the present study are in accordance with evidence from experimental animal models demonstrating a role of follistatin in oocyte maturation and embryo quality[ 23 , 24 ]. Given the established association between follistatin and oocyte quality, its contribution to embryo quality is anticipated, as supported by data from bovine models indicating that follistatin mRNA and protein in early embryos are of oocyte origin [ 25 ]. Notably, follistatin levels are significantly higher in early-cleaving 2-cell stage bovine embryos that subsequently develop into blastocysts at higher rates (> 40%) compared with late-cleaving embryos (30–36 h post-insemination), which exhibit substantially lower blastocyst formation rates (< 10%)[ 24 ]. Previous studies have demonstrated that serum activin-A levels are markedly elevated in pregnant women of reproductive age compared with their non-pregnant counterparts[ 26 ].A key finding of the present study is the strong and independent association between intrafollicular activin-A concentrations and favorable clinical outcomes, particularly successful implantation and clinical pregnancy rates. Multivariable regression analysis identified activin-A as a robust predictor of oocyte developmental competence. Specifically, activin-A levels wihin the follicular microenvironment appear to function as a critical surrogate marker, in line with its established role in regulating granulosa cell differentiation and oocyte maturation. Our findings offer a more nuanced perspective compared to earlier literature. While Wen et al. [ 27 ]suggested that FF activin-A predominantly reflects follicular size rather than acting as an independent determinant of fertilization, our data indicate that its influence extends significantly toward post-fertilization milestones. Moreover, the observed stability of activin-A concentrations across different maternal age groups, a phenomenon corroborated by recent findings [ 28 ] in small antral follicles, suggests that the follicular regulatory machinery remains functionally resilient despite the quantitative decline in the ovarian reserve. Nevertheless, the integrity of this endocrine signature may be susceptible to external interference. A recent cohort study analysis[ 15 ] highlights that endocrine-disrupting chemicals can perturb the follicular hormonal milieu, potentially altering the predictive accuracy of Transforming Growth Factor-beta (TGF-β) superfamily members. Within this framework, our findings position activin-A not merely as a growth factor associated with follicular expansion, but as a superior biomarker of the oocyte’s 'compatibility' for successful gestation, potentially surpassing traditional steroidogenic markers in clinical prognostic value. Importantly, our findings differ from those reported by Bouzoni et al. [ 16 ], who observed no association between FF levels of activin-follistatin-inhibin axis components and embryo quality, and only a weak association between serum inhibin-B and embryo quality. Several methodological and population-related differences may explain this discrepancy. Bouzoni et al. investigated a cohort of healthy oocyte donors with relatively homogeneous baseline characteristics and primarily assessed embryo quality at the blastocyst stage, whereas our study focused on subfertile women and evaluated IVF outcomes longitudinally, encompassing implantation, clinical pregnancy, and live birth. Moreover, our analysis linked intrafollicular hormone concentrations from dominant follicles to detailed IVF outcomes across the entire treatment course, which may capture biologically relevant associations not evident when embryo quality alone is assessed. Additionally, differences in study populations, ovarian stimulation protocols, timing of outcome assessment, and analytical approaches are likely to contribute to the heterogeneity of findings across studies. As highlighted by Bouzoni et al., variability in reproductive endpoints, assay methodologies, and follicular sampling strategies complicates direct comparisons and may partly account for inconsistent results reported in the literature. The strengths of the present study include its prospective design, the assessment of intrafollicular hormone concentrations, and the evaluation of IVF outcomes beyond embryo morphology. Furthermore, the inclusion of exclusively non-obese women is a notable advantage, as it allows for a clearer interpretation of the role of these hormones in fertility outcomes. Recent evidence suggests that body weight significantly influences activin levels, which could otherwise confound observed associations [ 29 , 30 ]. A limitation of the study is that hormone levels were assessed only in the dominant follicle; however, previous research has demonstrated that activin-A and inhibin-B concentrations do not vary according to oocyte size or stage of maturation [ 12 ].In addition, the stability of intrafollicular hormone concentrations across different follicles and age groups, as reported by Wang et al., further alleviates this concern[ 28 ].Additional limitations include the exclusive use of ICSI, the restriction to a homogeneous Greek population, and the application of stringent inclusion criteria (age ≤ 41 years, BMI < 30 kg/m², primary infertility), which may limit the generalizability of the findings to conventional IVF settings and to more diverse or higher-risk populations. In conclusion, our findings indicate that FF follistatin and inhibin-B are closely associated with oocyte maturity and embryo quality, while FF activin-A is linked to implantation and clinical pregnancy rates. When viewed alongside recent evidence of preserved intrafollicular hormone secretion despite advancing maternal age, our results suggest that functional variability within the follicular microenvironment—rather than age-related hormonal decline—underlies differences in IVF success. Further studies are warranted to explore whether intrafollicular hormone profiling could form individualized strategies for optimizing assisted reproduction outcomes. Abbreviations ART Assisted reproductive technologies , BMI Body mass index , β -hCG β - chorionic gonadotropin , Ε2 Estradiol , ELISA Enzyme-linked immunosorbent assay , FF Follicular fluid , FSH Follicle-stimulating hormone , hMG Human menopausal gonadotropin , ICSI Intracytoplasmic sperm injection , IVF In vitro fertilization , LH Luteinizing hormone , MET M etabolic equivalent of task , Oocytes-MII Metaphase-II stage oocytes , PCOS Polycystic ovary syndrome , RR Relative Risk. Declarations Acknowledgements: The authors would like to thank the Embryogenesis Assisted Conception Unit clinic staff for their assistance in data collection. Funding Declaration : This work was partially supported by research grant from Harokopio University (KE321). Authors’ contribution: DK, MK and NY were involved in study concept and design. DK, and MM contributed to the acquisition of data. RT supervised samples analysis. DK and RT analyzed the data and drafted the manuscript. RT, MK, IL and NY supervised analysis and critically revised the manuscript. NY had the primary responsibility for final content. All authors haveread and approved the final manuscript. Conflict of interest: None declared. Compliance with Ethical Standards Disclosure of potential conflicts of interest : No Research involving Human Participants and/or Animals : Research involned Human Participants (n=86 women). Biochemical analyses were performed in the Laboratory of Biology, Biochemistry and Physiology, in the Department of Nutrition & Dietetics at Harokopio University of Athens. All procedures adhered to the Helsinki Declaration, with written informed consent obtained from participants. The study protocol was approved by the Institutional Ethics Committee of Harokopio University (12/13-01-2013). Informed consent : All procedures adhered to the Helsinki Declaration, with written informed consent obtained from participants. Trial registration number: NCT03050944 References Lazzaroni-Tealdi E et al (2015) Oocyte Scoring Enhances Embryo-Scoring in Predicting Pregnancy Chances with IVF Where It Counts Most. PLoS ONE 10(12):e0143632 Dumesic DA et al (2015) Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health. Fertil Steril 103(2):303–316 Wijayarathna R, de Kretser DM (2016) Activins in reproductive biology and beyond. Hum Reprod Update, 22(3) Funghi L et al (2018) Placental and maternal serum activin A in spontaneous and induced labor in late-term pregnancy. J Endocrinol Invest 41(2):171–177 Vale W et al (1986) Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature 321(6072):776–779 Ling N et al (1986) Pituitary FSH is released by a heterodimer of the beta-subunits from the two forms of inhibin. Nature 321(6072):779–782 Zhang L et al (2018) The emerging role of follistatin under stresses and its implications in diseases. Gene 639:111–116 Ueno N et al (1987) Isolation and partial characterization of follistatin: a single-chain Mr 35,000 monomeric protein that inhibits the release of follicle-stimulating hormone. Proc Natl Acad Sci U S A 84(23):8282–8286 Gosden R, Lee B (2010) Portrait of an oocyte: our obscure origin. J Clin Invest 120(4):973–983 Edwards RG (1974) Follicular fluid. J Reprod Fertil 37(1):189–219 Da Broi MG et al (2018) Influence of follicular fluid and cumulus cells on oocyte quality: clinical implications. J Assist Reprod Genet 35(5):735–751 Schneyer AL et al (2000) Dynamic changes in the intrafollicular inhibin/activin/follistatin axis during human follicular development: relationship to circulating hormone concentrations. J Clin Endocrinol Metab 85(9):3319–3330 Fujiwara T et al (2000) Analysis of follicular fluid hormone concentrations and granulosa cell mRNA levels for the inhibin-activin-follistatin system: relation to oocyte and embryo characteristics. Fertil Steril 74(2):348–355 Lawrenz B et al (2020) Inhibin A-A Promising Predictive Parameter for Determination of Final Oocyte Maturation in Ovarian Stimulation for IVF/ICSI. Front Endocrinol (Lausanne) 11:307 Hoffmann-Dishon N et al (2024) Endocrine-disrupting chemical concentrations in follicular fluid and follicular reproductive hormone levels. J Assist Reprod Genet 41(6):1637–1642 Bouzoni E et al (2022) Embryo Quality May Be Associated With Serum Inhibin B Levels but Not With Serum or Follicular Fluid Levels of Other Components of the Activin-Follistatin-Inhibin Axis. Endocr Pract 28(10):1086–1090 Karayiannis D et al (2017) Association between adherence to the Mediterranean diet and semen quality parameters in male partners of couples attempting fertility. Hum Reprod 32(1):215–222 Karayiannis D et al (2018) Adherence to the Mediterranean diet and IVF success rate among non-obese women attempting fertility. Hum Reprod 33(3):494–502 Gotham SM, Fryer PJ, Paterson WR (1988) The measurement of insoluble proteins using a modified Bradford assay. Anal Biochem 173(2):353–358 Jeppesen JV et al (2012) Concentration of activin A and follistatin in follicular fluid from human small antral follicles associated to gene expression of the corresponding granulosa cells. Mol Cell Endocrinol 356(1–2):48–54 Chang CL et al (2002) The concentration of inhibin B in follicular fluid: relation to oocyte maturation and embryo development. Hum Reprod 17(7):1724–1728 Ocal P et al (2004) Follicular fluid concentrations of vascular endothelial growth factor, inhibin A and inhibin B in IVF cycles: are they markers for ovarian response and pregnancy outcome? Eur J Obstet Gynecol Reprod Biol 115(2):194–199 Fullerton PT Jr. et al (2017) Follistatin is critical for mouse uterine receptivity and decidualization. Proc Natl Acad Sci U S A 114(24):E4772–e4781 Lee KB et al (2009) Molecular determinants of oocyte competence: potential functional role for maternal (oocyte-derived) follistatin in promoting bovine early embryogenesis. Endocrinology 150(5):2463–2471 Patel OV et al (2007) Functional genomics studies of oocyte competence: evidence that reduced transcript abundance for follistatin is associated with poor developmental competence of bovine oocytes. Reproduction 133(1):95–106 Calvert ME et al (2022) Serum and urine profiles of TGF-β superfamily members in reproductive aged women. Clin Chim Acta 524:96–100 Wen X et al (2006) Follicular fluid levels of inhibin A, inhibin B, and activin A levels reflect changes in follicle size but are not independent markers of the oocyte's ability to fertilize. Fertil Steril 85(6):1723–1729 Wang NF et al (2025) Impact of female age on concentrations of reproductive hormones and oocyte-specific growth factors in follicular fluid from human small antral follicles. Hum Reprod 40(4):707–716 Dani C (2013) Activins in adipogenesis and obesity. Int J Obes (Lond) 37(2):163–166 Ongaro L, Bernard DJ (2025) Activin Actions in Adipocytes. J Clin Endocrinol Metab 110(7):1803–1810 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revisions 19 Mar, 2026 Reviewers agreed at journal 28 Feb, 2026 Reviewers invited by journal 25 Feb, 2026 Editor assigned by journal 19 Feb, 2026 First submitted to journal 18 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8907446","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596878507,"identity":"b16883fd-067f-4bd3-ba59-88bd5b9c7975","order_by":0,"name":"dimitrios karayiannis","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYBACxmYGZjDdz8DARqKWmQ3EagECiJYNB4jVwtzOnWzwcw+D7OYbyc8efKhgkOcXO0DIYbybE3ueMRhvu5FmbjjjDIPhzNkJhLUc4DnAkLjtRoKZNG8bQ4LBbSK0HPwD1LJ5Rvo34rUkg2zZIJFDgi3GMgcYjGeceVMmOeOMBGG/GPaf3Sz55gCDbH97+jaJDxU28vzShLQ0gKn/DAwCYJUS+JWDgDycxX+AsOpRMApGwSgYmQAAeJ5CdgRdKB0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4142-2392","institution":"Evaggelismos General Hospital: Geniko Nosokomeio Athenon O Euangelismos","correspondingAuthor":true,"prefix":"","firstName":"dimitrios","middleName":"","lastName":"karayiannis","suffix":""},{"id":596878508,"identity":"dd51a6f0-dce7-43a9-b5a6-8c519b39b990","order_by":1,"name":"Roxane Tenta","email":"","orcid":"","institution":"Harokopio University School of Health Science \u0026 Education: Charokopeio Panepistemio Schole Epistemon Ygeias kai Agoges","correspondingAuthor":false,"prefix":"","firstName":"Roxane","middleName":"","lastName":"Tenta","suffix":""},{"id":596878509,"identity":"654cc3aa-6d50-4cea-9a63-715ad1e310de","order_by":2,"name":"Meropi D. Kontogianni","email":"","orcid":"","institution":"Harokopio University School of Health Science \u0026 Education: Charokopeio Panepistemio Schole Epistemon Ygeias kai Agoges","correspondingAuthor":false,"prefix":"","firstName":"Meropi","middleName":"D.","lastName":"Kontogianni","suffix":""},{"id":596878510,"identity":"c31dd3b6-b321-4932-80fd-9490a105a16e","order_by":3,"name":"Minas Mastrominas","email":"","orcid":"","institution":"Embryogenesis","correspondingAuthor":false,"prefix":"","firstName":"Minas","middleName":"","lastName":"Mastrominas","suffix":""},{"id":596878511,"identity":"cabc09ce-9616-4376-be80-0e91af328c52","order_by":4,"name":"Nikos Yiannakouris","email":"","orcid":"","institution":"Harokopio University School of Health Science \u0026 Education: Charokopeio Panepistemio Schole Epistemon Ygeias kai Agoges","correspondingAuthor":false,"prefix":"","firstName":"Nikos","middleName":"","lastName":"Yiannakouris","suffix":""}],"badges":[],"createdAt":"2026-02-18 08:51:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8907446/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8907446/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104398961,"identity":"d48fb4e1-8576-402f-bc65-270da9a78c0f","added_by":"auto","created_at":"2026-03-11 12:04:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":727876,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8907446/v1/a5be7b66-8ca6-41da-9c65-e60b3f2a0893.pdf"}],"financialInterests":"","formattedTitle":"Intrafollicular Activin-A, Inhibin-B, and Follistatin may predict invitro fertilization outcomesin subfertile women.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe success of \u003cem\u003ein vitro\u003c/em\u003e fertilization (IVF) and other assisted reproductive technologies (ARTs) is critically dependent on obtaining good-quality oocytes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Oocyte quality and health depend on the microenvironment of the corresponding follicle where they develop[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This critical intrafollicular microenvironment for appropriate oocyte development is influenced by multiple endocrine and intraovarian paracrine interactions. Regarding the intraovarian effects, recent findings demonstrate a role for intrafollicular activin/follistatin/inhibin levels, which has received considerable attention due to their effects on follicle development [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInitial studies have explored the role of activins, inhibins and follistatin in reproductive and developmental biology, with most focusing on their serum levels [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Inhibins, activins, and follistatin preferentially influence Follicle-stimulating hormone (FSH) secretion and contribute to divergent release of Luteinizing hormone \u003cb\u003e(\u003c/b\u003eLH)and FSH throughout the menstrual cycle [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Inhibins primarily inhibit FSH release from pituitary gonadotrope cells, whereas activins stimulate FSH secretion. Follistatin, in turn, inhibits pituitary FSH secretion by binding to activin, thus rendering it inactive. In contrast to inhibins, which act primarily via endocrine signaling, activins and follistatin produced in the pituitary, influence FSH secretion via autocrine-paracrine signaling [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFollicular fluid (FF) composition differs compared to serum and undergoes physiological alterations during follicular development [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Hormone concentrations in FF can both directly (via genomic and non-genomic actions) and indirectly (via somatic cells within the follicle) influence oocyte differentiation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Evaluation of the intrafollicular levels of activins, inhibins and follistatinis particularly useful to study their role in follicular development. Since most of these factors have a paracrine and/or autocrine mode of action, their intrafollicular levels reflect their role in follicular development better than circulating levels, which may be also influenced by extra ovarian production sites [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. All these observations derive mainly from IVF treatment procedures, given the fact that IVF cycles offer the opportunity to measure a plethora of components contained in the FF. In addition, it is now feasible to investigate the association of factors such as activin/follistatin/inhibin and parameters like oocytes fertilization potential and the quality of embryos developed after conventional IVF or intracytoplasmic sperm injection.\u003c/p\u003e \u003cp\u003eTo date, few studies have investigated the interrelationship between intrafollicular levels of activins, inhibins and follistatin and their involvement in IVF outcomes [\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The purpose of the present study was to evaluate FF levels of activin A, inhibin B, and follistatin in a sample of subfertile women undergoing IVF, to assess potential interplay among these factors, and to elucidate their role in IVF intermediate and clinical outcomes.\u003c/p\u003e"},{"header":"Subjects and Methods","content":"\u003cp\u003eCouples with primary infertility who sought evaluation and treatment in an Assisted Conception Unit in Athens, Greece (Embryogenesis Assisted Conception Unit, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.embryogenesis.gr\u003c/span\u003e\u003cspan address=\"http://www.embryogenesis.gr\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were originally invited to participate in a prospective cohort study investigating how background diet and lifestyle patterns impact fertility [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Eligible female partners were \u0026le;\u0026thinsp;41 years of age, had a body mass index (BMI) of \u0026lt;\u0026thinsp;30 kg/m\u003csup\u003e2\u003c/sup\u003e, had no history of prior IVF attempts or pregnancies, and were not enrolled innatural-cycle protocols. Participants were also required to be free of endometriosis (based on transvaginal ultrasound assessment), prior ovarian surgery, diabetes mellitus, cardiovascular disease, hypertension, neoplasm, hypothyroidism or psychiatric disorders [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Of 244 eligible Greek couples evaluated from November 2013 until September 2018, 86 women consented to provide biological specimens (FF samples). At study enrollment, anthropometric assessments were conducted for all participants, alongside administration of a comprehensive questionnaire capturing demographic, reproductive, medical history, and lifestyle data. Habitual dietary and alcohol intake was evaluated using a validated 76-item semi-quantitative FFQ tailored for the Greek population, referencing the preceding 6 months prior to IVF. Physical activity levels were measured with the validated Greek version of the International Physical Activity Questionnaire (iPAQ), while anxiety was assessed using the Spielberger State-Trait Anxiety Inventory (STAI-Y) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiochemical analyses were performed in the Laboratory of Biology, Biochemistry and Physiology, in the Department of Nutrition \u0026amp; Dietetics at Harokopio University of Athens. All procedures adhered to the Helsinki Declaration, with written informed consent obtained from participants. The study protocol was approved by the Institutional Ethics Committee of Harokopio University (12/13-01-2013).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFF and corresponding oocyte collection\u003c/h2\u003e \u003cp\u003eAt the time of oocyte retrieval, the single dominant follicle (\u0026gt;\u0026thinsp;18 mm in diameter) was punctured with a 23G needle attached to a syringe. Follicular fluid (FF) was aspirated directly into an empty bottle without flushing medium and selected only if it contained one intact oocyte and was free of contamination by fresh blood. To minimize cross contamination between follicles, FF was obtained exclusively from one dominant follicle per ovary, while a separate needle was used for each ovary. Following needle withdrawal, the needle was flushed to retrieve any oocytes trapped in the dead space of the collection needle. To preclude contamination by flushing medium, only fluid from the initial aspirate from the dominant follicle was retained. Each FF sample was centrifuged immediately at 1,300 g for 10 minutes and the clear supernatants were collected and stored at -80\u0026deg;C until assayed.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIn vitro fertilization (IVF) procedure and outcome assessment\u003c/h3\u003e\n\u003cp\u003eBefore initiating the IVF procedure, women underwent ovarian reserve testing and were assigned into one of the following ovarian stimulation protocols as clinically indicated: 1) Gonadotropin-Releasing Hormone (GnRH) antagonist protocol (Cetrotide, Orgalutran) and 2) Follicular-phase GnRH agonist/Flare protocol (Daronda, Arvekap). Subcutaneous recombinant FSH (Gonal-F, Puregon, Altrmon) and/or human menopausal gonadotropin (hMG; Menopur, Merional, Pergonal) were administered in all regimens with a maximum combined daily dose of 450 IU. Women were monitored during ovarian stimulation for serum estradiol (E2) and for follicle counts and size alterations. Human chorionic gonadotropin (hCG) was administered\u0026thinsp;~\u0026thinsp;36 h before the scheduled egg retrieval procedure in order to induce ovulation. Oocyte retrieval was performed when follicle sizes reached 16\u0026ndash;18 mm and when serum E2 concentrations reached at least 1800 pmol/L.\u003c/p\u003e \u003cp\u003eIntracytoplasmic sperm injection (ICSI) was carried out for all IVF cycles in this study. Embryologists classified oocytes as germinal vesicle, metaphase-I or metaphase-II (oocytes-MII, presence of a polar body) and determined fertilization 17\u0026ndash;20 h after insemination as the number of oocytes with two pronuclei. Fertilization rate was defined as the total number of fertilized oocytes divided by the number of oocytes-ΜII. The resulting embryos were monitored on day 3for cell number and morphological quality (1\u0026thinsp;=\u0026thinsp;best to 5\u0026thinsp;=\u0026thinsp;worst). Embryos that had reached 6\u0026ndash;8 cells on day 3 were considered to be cleaving at a normal rate, whereas embryos with \u0026le;\u0026thinsp;5 cells or \u0026ge;\u0026thinsp;9 were considered to be slow cleaving or to have accelerated cleavage, respectively. In the present analysis embryos were classified as high quality if they had at least 8 cells on day 3 and if they had reached a morphological quality score of 1 or 2. The maximum number of embryos transferred should abide by the Greek National Legislation for embryo transfer guidelines (Greek National Authority of Assisted Reproduction; Law 3305/01/2005; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://eaiya.gov.gr/en/law-fek/\u003c/span\u003e\u003cspan address=\"http://eaiya.gov.gr/en/law-fek/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSuccessful implantation was defined as serum \u003cem\u003eβ\u003c/em\u003e-hCG concentration\u0026thinsp;\u0026gt;\u0026thinsp;20 IU/L measured at 14\u0026ndash;21 days after egg retrieval. Clinical pregnancy was defined as the presence of an intrauterine pregnancy confirmed by ultrasound (presence of at least one gestational sac and cardiac activity at 6 weeks estimated gestational age), and live birth as the birth of a neonate on/or after 24 weeks of gestation. All clinical information, including infertility diagnosis, hormone levels and protocol type, was abstracted from the patient\u0026rsquo;s electronic medical records. The study\u0026rsquo;s primary outcomes were implantation, clinical pregnancy and live birth, whereas oocyte yield, fertilization rate and embryo quality parameters were used as intermediate outcomes.\u003c/p\u003e\n\u003ch3\u003eHormone assays\u003c/h3\u003e\n\u003cp\u003eActivin-A levels were measured using a commercially available Elisa Kit (CEA001Ra, Cloud-Clone Corp., Houston, USA), with a sensitivity of 5.7 pg/mL, intra assay coefficient of variation (CV) of \u0026lt;\u0026thinsp;10% and inter assay CV of \u0026lt;\u0026thinsp;12%.Inhibin-B levels were measured using a commercially available Elisa Kit (CEA760Hu,Cloud-Clone Corp., Houston, USA), with a sensitivity of 3.2 pg/mL, intra assay CV of \u0026lt;\u0026thinsp;10%, and inter assay CV of \u0026lt;\u0026thinsp;12%. Follistatin levels were measured using a commercially available Elisa Kit (DFN00, R\u0026amp;D Systems, Minneapolis, MN, USA), with a sensitivity of 29 pg/mL, intra assay CV of 2\u0026ndash;2.7% and inter assay CV of 7.1\u0026ndash;9.2%. All samples were diluted as necessary for values to be within the range of the standard curve (1:40 dilution of FF samples for follistatin, 1:30 dilution for activin-A and 1:5 dilution for inhibin-B) and in duplicate within the same assay to decrease inter assay variability. Total protein content was determined by the Bradford method[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].Absorbance was determined in a Power wave microplate spectrophotometer (Biotek Instruments, Inc.)at 450 nm.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe normality of the distribution of the variables was assessed using the Shapiro-Wilks test. Continuous variables are presented as median (interquartile range, IQR), whereas categorical variables as absolute and relative frequencies. Associations between categorical variables were tested by χ\u003csup\u003e2\u003c/sup\u003e tests, while differences between categorical and several clinical and reproductive variables were tested using the non-parametric Mann-Whitney-U test. The association between FF hormones was assessed using Spearman's rank correlation (rho) coefficient because the data showed a non-normal distribution as determined by the Shapiro-Wilk test. Generalized linear models were used to test the associations between FF hormone levels and IVF outcomes. A Poisson distribution with log link function was used to test the association of number of total and mature oocytes, fertilized oocytes, and high-quality embryos (all count data), while a binomial distribution with logit link function was used for fertilization rate and clinical endpoints. The results are presented as relative risk (RR) and 95% confidence intervals (CIs). Statistical Package for Social Sciences software (SPSS, version 24.0, Chicago, Illinois, USA) was used for all statistical calculations. All reported \u003cem\u003eP\u003c/em\u003e-values are based on two-sided tests and compared with a significant level of 5%.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eStudy participants were aged25-41 years (median: 36 years) and had a mean BMI of 22.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 kg/m\u003csup\u003e2\u003c/sup\u003e. Of the 86 women enrolled, 41 (47.6%) had successful implantation, 34 (39.5%) achieved a clinical pregnancy, and 32 (37.2%) gave live birth. Baseline demographic and clinical characteristics stratified by clinical pregnancy status are presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. No differences were observed between women with and without a clinical pregnancy with respect to\u0026Beta;\u0026Mu;\u0026Iota;, physical activity levels, or anxiety scores. In contrast, women who achieved a clinical pregnancy had significantly higher number of retrieved oocytes, metaphase II (MII) oocytes, embryos, and high-quality embryos compared with those who did not (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u0026nbsp;\u003c/p\u003e\n\u003ctable id=\"Tab1\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDescriptive characteristics of women according to IVF clinical outcome (n\u0026thinsp;=\u0026thinsp;86)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eIVF outcome\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCharacteristic\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClinical pregnancy, positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClinical pregnancy, negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026Nu;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge, y\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37 (33\u0026ndash;39)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35 (33\u0026ndash;37)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBody mass index, kg/m\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.3 (21.2\u0026ndash;24.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.1 (20.7\u0026ndash;24.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.899\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmoking status, \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e\n \u003cp\u003eNever\u003c/p\u003e\n \u003cp\u003eFormer\u003c/p\u003e\n \u003cp\u003eCurrent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e26 (81.3)\u003c/p\u003e\n \u003cp\u003e3 (9.4)\u003c/p\u003e\n \u003cp\u003e3 (9.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e37(71.2)\u003c/p\u003e\n \u003cp\u003e2 (3.8)\u003c/p\u003e\n \u003cp\u003e13 (25.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e0.160\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhysical activity, metabolic equivalent of task(MET)-min/week\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e828.7 (506.2-1121.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e896.0 (412.5-1054.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.932\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS-Anxiety (score range 20\u0026ndash;80)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46(32.2\u0026ndash;53.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44.5(35.7\u0026ndash;52.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.806\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT-Anxiety (score range 20\u0026ndash;80)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38(33.2\u0026ndash;46.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.5 (33.0\u0026ndash;50.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.747\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal energy intake, kcal/day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1820(1594\u0026ndash;2041)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1798 (1546\u0026ndash;2010)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.728\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSupplements use,\u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20 (58.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24 (46.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.552\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eReproductive characteristics and IVF outcome\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFamily subfertility history ,\u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 (11.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20 (38.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal menstrual cycle, \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23 (67.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36 (69.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.877\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCause of infertility, \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e\n \u003cp\u003eMale factor\u003c/p\u003e\n \u003cp\u003eFemale factor\u003c/p\u003e\n \u003cp\u003eUnexplained\u003c/p\u003e\n \u003cp\u003ePolycystic ovary syndrome, \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e16 (47.1)\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003cp\u003e18 (52.9)\u003c/p\u003e\n \u003cp\u003e4 (11.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e22 (42.3)\u003c/p\u003e\n \u003cp\u003e4 (7.7)\u003c/p\u003e\n \u003cp\u003e26 (50.0)\u003c/p\u003e\n \u003cp\u003e10 (19.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003cp\u003e0.359\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOocytes produced, \u003cem\u003en\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eMetaphase-II stage oocyte, \u003cem\u003en\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eFertilization rate, %\u003c/p\u003e\n \u003cp\u003eEmbryos produced, \u003cem\u003en\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eHigh quality embryos, \u003cem\u003en\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12 (9\u0026ndash;16)\u003c/p\u003e\n \u003cp\u003e8 (6\u0026ndash;12)\u003c/p\u003e\n \u003cp\u003e86.5(64.6\u0026ndash;94.7)\u003c/p\u003e\n \u003cp\u003e7 (6\u0026ndash;9)\u003c/p\u003e\n \u003cp\u003e3 (2\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9 (6\u0026ndash;14)\u003c/p\u003e\n \u003cp\u003e6 (4\u0026ndash;10)\u003c/p\u003e\n \u003cp\u003e81.6 (60.5\u0026ndash;98.2)\u003c/p\u003e\n \u003cp\u003e5 (3\u0026ndash;9)\u003c/p\u003e\n \u003cp\u003e1 (0\u0026ndash;4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.010\u003c/p\u003e\n \u003cp\u003e0.015\u003c/p\u003e\n \u003cp\u003e0.284\u003c/p\u003e\n \u003cp\u003e0.019\u003c/p\u003e\n \u003cp\u003e0.046\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of embryos transferred, \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e\n \u003cp\u003eNo embryos transferred\u003c/p\u003e\n \u003cp\u003e1 embryo\u003c/p\u003e\n \u003cp\u003e2 embryos\u003c/p\u003e\n \u003cp\u003e3\u0026thinsp;+\u0026thinsp;embryos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003cp\u003e2 (5.9)\u003c/p\u003e\n \u003cp\u003e15 (44.1)\u003c/p\u003e\n \u003cp\u003e17 (50.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e5 (9.6)\u003c/p\u003e\n \u003cp\u003e12 (23.1)\u003c/p\u003e\n \u003cp\u003e17 (32.7)\u003c/p\u003e\n \u003cp\u003e18 (34.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e0.029\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eFollicular fluid hormone concentrations\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eActivin-\u0026Alpha; (pg/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e307.5 (264 -346.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e313.5 (274\u0026ndash;384)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.336\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eActivin-\u0026Alpha;(pg/mg total protein)\u0026dagger;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.4 (44.8\u0026ndash;61.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.2 (46.5\u0026ndash;65.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.059\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInhibin-\u0026Beta;(pg/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.5 (27.7\u0026ndash;78.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.5 (9.2\u0026ndash;72.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.281\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInhibin-\u0026Beta;(pg/mg total protein)\u0026dagger;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.7 (4.4\u0026ndash;12.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.8 (1.9\u0026ndash;11.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.293\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFollistatin (ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e405.5 (204.7-849.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e412.5 (183-788.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.784\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFollistatin(ng/mg total protein)\u0026dagger;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65.8 (34.9-121.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65.6 (30.1-126.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.747\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eValues represent median (IQR) or number of subjects (%). Differences in variables were tested using Mann-Whitney U test for continuous variables and or chi square test for categorical variables.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\u003csup\u003ea\u003c/sup\u003e State (S)-Anxiety evaluates the current emotional state; Trait (T)-Anxiety evaluates relatively stable aspects of anxiety (how the respondent usually feels), with higher values suggesting higher levels of anxiety.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\u003csup\u003eb\u003c/sup\u003e Type of supplements: multivitamins, iron, folic acid, vitamin C/other. \u0026dagger;Values adjusted to follicular fluid total protein content.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eA total of 86 follicular fluid (FF) samples were collected to assess intrafollicular hormonal levels. The analysis revealed a significant positive correlation between activin-A and inhibin-\u0026Beta; (Spearman\u0026rsquo;s rho\u0026thinsp;=\u0026thinsp;0.512, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), between activin-A and follistatin (rho\u0026thinsp;=\u0026thinsp;0.427, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001),as well as between inhibin-B and follistatin (rho\u0026thinsp;=\u0026thinsp;0.604, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).Furthermore, correlations between FF hormone levels and intermediate IVF outcomes demonstrated that both inhibin-B and follistatin concentrations were positively and significantly associated with the number of retrieved oocytes (rho\u0026thinsp;=\u0026thinsp;0.316 and rho\u0026thinsp;=\u0026thinsp;0.362, respectively), oocytes-MII (rho\u0026thinsp;=\u0026thinsp;0.326 and r\u0026thinsp;=\u0026thinsp;0.389), total embryos (rho\u0026thinsp;=\u0026thinsp;0.329 and rho\u0026thinsp;=\u0026thinsp;0.372), and high quality embryos (rho\u0026thinsp;=\u0026thinsp;0.244 and rho\u0026thinsp;=\u0026thinsp;0.210) (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, activin-A levels were negatively correlated with fertilization rate (rho=-0.239, P\u0026thinsp;=\u0026thinsp;0.03).\u003c/p\u003e\n\u003cp\u003eIn Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, the results of the fully adjusted multivariable models for the study\u0026rsquo;s primary outcomes are presented. Regarding FF follistatin concentrations, an increase of 10 ng/mL was associated with a 1.02-fold increase (95% CI: 1.01\u0026ndash;1.03) in the number of oocytes retrieved, a 1.03-fold increase (95%CI: 1.01\u0026ndash;1.04) in oocytes-MII, and with higher numbers of total embryos and high quality embryos[RR (95%CI): 1.03 (1.01\u0026ndash;1.04) and 1.03 (1.01\u0026ndash;1.05), respectively]. Similarly, each 1 pg/mL increase in FF inhibin-B concentrations was associated with a 1.01-fold increase (95%CI: 1.00-1.02) in the number of oocytes retrieved and oocytes-MII, as well as a 1.03-fold increase (95%CI: 1.01\u0026ndash;1.04) in high quality embryos. Finally, a 10 pg/mL increase in FF activin-A concentrations was associated with a 58%higher likelihood of successful implantation (RR\u0026thinsp;=\u0026thinsp;1.58, 95%CI: 1.03\u0026ndash;2.45) and a 69% higher likelihood of clinical pregnancy (RR\u0026thinsp;=\u0026thinsp;1.69; 95%CI: 1.08\u0026ndash;2.62).\u0026nbsp;\u003c/p\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAssociation between follicular fluid hormone levels and IVF clinical outcomes (n\u0026thinsp;=\u0026thinsp;86 follicular fluid samples from the primary follicles of 86 women undergoing IVF)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eActivin-\u0026Alpha;\u003c/p\u003e\n \u003cp\u003e(per 10 pg/mL)\u0026dagger;\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInhibin-\u0026Beta;\u003c/p\u003e\n \u003cp\u003e(per pg/mL)\u0026dagger;\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFollistatin\u003c/p\u003e\n \u003cp\u003e(per 10 ng/mL)\u0026dagger;\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eIntermediate outcomes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eOocytes produced, \u003cem\u003en\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99 (0.96\u0026ndash;1.02)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01 (1.00-1.02)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02 (1.01\u0026ndash;1.03)*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99 (0.96\u0026ndash;1.02)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01 (1.00-1.02)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02 (1.01\u0026ndash;1.04)*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98 (0.95\u0026ndash;1.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01 (1.00-1.02)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02 (1.01\u0026ndash;1.03)*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOocytes-MII, \u003cem\u003en\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98(0.94\u0026ndash;1.02)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01 (1.00-1.02)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02 (1.01\u0026ndash;1.04)*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98(0.94\u0026ndash;1.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01 (1.00-1.02)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03 (1.01\u0026ndash;1.04)*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.97 (0.93\u0026ndash;1.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01 (1.00-1.02)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03 (1.01\u0026ndash;1.04)*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eEmbryos produced, \u003cem\u003en\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98 (0.93\u0026ndash;1.02)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02 (1.00-1.03)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03 (1.01\u0026ndash;1.04)*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.97 (0.93\u0026ndash;1.02)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02 (1.01\u0026ndash;1.03)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03 (1.01\u0026ndash;1.04)*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.96 (0.91\u0026ndash;1.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02 (1.01\u0026ndash;1.03)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03 (1.01\u0026ndash;1.04)*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eHigh quality embryos, \u003cem\u003en\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.91 (0.83-1.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02 (1.00-1.04)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01 (0.99\u0026ndash;1.04)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.91 (0.84-1.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02 (1.00-1.04)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02 (1.00-1.04)*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.92 (0.84-1.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03 (1.01\u0026ndash;1.04)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03 (1.01\u0026ndash;1.05)*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eClinical endpoints\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eSuccessful Implantation (Yes vs. No)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.57 (1.06\u0026ndash;2.32)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98 (0.92\u0026ndash;1.04)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01 (0.94\u0026ndash;1.08)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.51 (1.01\u0026ndash;2.26)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.97 (0.91\u0026ndash;1.04)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00 (0.92\u0026ndash;1.08)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.58 (1.03\u0026ndash;2.45)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.96 (0.90\u0026ndash;1.03)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98 (0.99\u0026ndash;1.07)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eClinical Pregnancy (Yes vs. No)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.61 (1.07\u0026ndash;2.43)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.94 (0.97\u0026ndash;1.06)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02 (0.94\u0026ndash;1.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.58 (1.04 \u0026minus;\u0026thinsp;2.39)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99 (0.93\u0026ndash;1.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01 (0.93\u0026ndash;1.09)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.69 (1.08 \u0026minus;\u0026thinsp;2.62)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98 (0.92\u0026ndash;1.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01 (0.93\u0026ndash;1.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eLive Birth (Yes vs. No)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.45 (0.98\u0026ndash;2.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99 (0.93\u0026ndash;1.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02 (0.94\u0026ndash;1.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.41 (0.95 \u0026minus;\u0026thinsp;2.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98 (0.93\u0026ndash;1.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01 (0.93\u0026ndash;1.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.47(0.96 \u0026minus;\u0026thinsp;2.26)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98 (0.92\u0026ndash;1.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01 (0.93\u0026ndash;1.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eData represents relative risk (95% confidence interval). All analyses were conducted using generalized linear models. A Poisson distribution with log link function were used to test association of number of total and mature oocytes, embryos produced and high-quality embryos (all count data), while a binomial distribution with logit link function were used for clinical endpoints. Model 1 was adjusted for ovarian stimulation protocol and age. Model 2 was adjusted as for model 1 and for body mass index (continuous), smoking (never smoker vs ever smoking) and cause of infertility. Model 3 was adjusted as for model 2 and for stress and physical activity levels (all continuous).\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e*P\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\u0026dagger; Values adjusted to follicular fluid total protein content.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAbbreviations used\u003c/span\u003e: IVF\u0026thinsp;=\u0026thinsp;in vitro fertilization; Oocytes-MII=metaphase-II stage oocytes.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe primary aim of the present study was to investigate the role of intrafollicular components of the activin-follistatin-inhibin axis in relation to oocyte competence, embryo development, and final IVF outcomes in subfertile women undergoing IVF/ICSI. Our findings indicated that higher FF concentrations of inhibin-B and follistatin were positively associated with key intermediate IVF outcomes, including the number of oocytes retrieved, the proportion of mature (MII) oocytes, as well as embryo number and quality. In contrast, FF activin-A levels were not linked to intermediate IVF outcomes but were significantly associated with increased implantation and clinical pregnancy rates, indicating a potentially distinct role in later stages of reproductive success. Collectively, these findings support the concept that the intrafollicular hormonal milieu plays a crucial role in follicular development, oocyte maturation, and embryo competence. Furthermore, the observed positive correlations among FF activin-A, inhibin-B, and follistatin reflect the tightly regulated local interplay of these factors within the ovarian follicle are consistent with previous experimental and human data demonstrating their coordinated expression during folliculogenesis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn order to investigate whether follicular activin/follistatin/inhibin concentration sinfluence oocyte maturation, FF hormone concentrations were compared with various parameters of oocyte quality. Both follistatin and inhibin-B concentrations showed a significant positive correlation withthe number of metaphase II (MII) oocytes, suggesting that these hormones might be involved in the preparation process for, or initiation of oocyte nuclear maturation. With respect to inhibin-B levels, multivariate analyses revealed significant associations with both the number and quality of oocytes and embryos. These findings are consistent with those reported by Chang et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], who analyzed233 FF samples and demonstrated that inhibin-B levels were related to embryo quality scores on days 2 and 3 post-fertilization. Although the underlying mechanisms remain unclear, inhibin-B levels have been shown to increase as follicles grow and mature, supporting its potential role as an indicator of follicle quality. In agreement with this hypothesis, data from another study [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] indicated that higher FF inhibin-B concentrations were associated with increased fertilization and pregnancy rates. Collectively, these findings suggest that FF inhibin-B may serve as a reliable marker of follicular development and oocyte/embryo quality during controlled ovulation stimulation.\u003c/p\u003e \u003cp\u003eFurthermore, the results of the present study are in accordance with evidence from experimental animal models demonstrating a role of follistatin in oocyte maturation and embryo quality[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Given the established association between follistatin and oocyte quality, its contribution to embryo quality is anticipated, as supported by data from bovine models indicating that follistatin mRNA and protein in early embryos are of oocyte origin [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Notably, follistatin levels are significantly higher in early-cleaving 2-cell stage bovine embryos that subsequently develop into blastocysts at higher rates (\u0026gt;\u0026thinsp;40%) compared with late-cleaving embryos (30\u0026ndash;36 h post-insemination), which exhibit substantially lower blastocyst formation rates (\u0026lt;\u0026thinsp;10%)[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that serum activin-A levels are markedly elevated in pregnant women of reproductive age compared with their non-pregnant counterparts[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].A key finding of the present study is the strong and independent association between intrafollicular activin-A concentrations and favorable clinical outcomes, particularly successful implantation and clinical pregnancy rates. Multivariable regression analysis identified activin-A as a robust predictor of oocyte developmental competence. Specifically, activin-A levels wihin the follicular microenvironment appear to function as a critical surrogate marker, in line with its established role in regulating granulosa cell differentiation and oocyte maturation. Our findings offer a more nuanced perspective compared to earlier literature. While Wen et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]suggested that FF activin-A predominantly reflects follicular size rather than acting as an independent determinant of fertilization, our data indicate that its influence extends significantly toward post-fertilization milestones. Moreover, the observed stability of activin-A concentrations across different maternal age groups, a phenomenon corroborated by recent findings [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] in small antral follicles, suggests that the follicular regulatory machinery remains functionally resilient despite the quantitative decline in the ovarian reserve. Nevertheless, the integrity of this endocrine signature may be susceptible to external interference. A recent cohort study analysis[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] highlights that endocrine-disrupting chemicals can perturb the follicular hormonal milieu, potentially altering the predictive accuracy of Transforming Growth Factor-beta (TGF-β) superfamily members. Within this framework, our findings position activin-A not merely as a growth factor associated with follicular expansion, but as a superior biomarker of the oocyte\u0026rsquo;s 'compatibility' for successful gestation, potentially surpassing traditional steroidogenic markers in clinical prognostic value.\u003c/p\u003e \u003cp\u003eImportantly, our findings differ from those reported by Bouzoni et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], who observed no association between FF levels of activin-follistatin-inhibin axis components and embryo quality, and only a weak association between serum inhibin-B and embryo quality. Several methodological and population-related differences may explain this discrepancy. Bouzoni et al. investigated a cohort of healthy oocyte donors with relatively homogeneous baseline characteristics and primarily assessed embryo quality at the blastocyst stage, whereas our study focused on subfertile women and evaluated IVF outcomes longitudinally, encompassing implantation, clinical pregnancy, and live birth. Moreover, our analysis linked intrafollicular hormone concentrations from dominant follicles to detailed IVF outcomes across the entire treatment course, which may capture biologically relevant associations not evident when embryo quality alone is assessed. Additionally, differences in study populations, ovarian stimulation protocols, timing of outcome assessment, and analytical approaches are likely to contribute to the heterogeneity of findings across studies. As highlighted by Bouzoni et al., variability in reproductive endpoints, assay methodologies, and follicular sampling strategies complicates direct comparisons and may partly account for inconsistent results reported in the literature.\u003c/p\u003e \u003cp\u003eThe strengths of the present study include its prospective design, the assessment of intrafollicular hormone concentrations, and the evaluation of IVF outcomes beyond embryo morphology. Furthermore, the inclusion of exclusively non-obese women is a notable advantage, as it allows for a clearer interpretation of the role of these hormones in fertility outcomes. Recent evidence suggests that body weight significantly influences activin levels, which could otherwise confound observed associations [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. A limitation of the study is that hormone levels were assessed only in the dominant follicle; however, previous research has demonstrated that activin-A and inhibin-B concentrations do not vary according to oocyte size or stage of maturation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].In addition, the stability of intrafollicular hormone concentrations across different follicles and age groups, as reported by Wang et al., further alleviates this concern[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].Additional limitations include the exclusive use of ICSI, the restriction to a homogeneous Greek population, and the application of stringent inclusion criteria (age\u0026thinsp;\u0026le;\u0026thinsp;41 years, BMI\u0026thinsp;\u0026lt;\u0026thinsp;30 kg/m\u0026sup2;, primary infertility), which may limit the generalizability of the findings to conventional IVF settings and to more diverse or higher-risk populations.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings indicate that FF follistatin and inhibin-B are closely associated with oocyte maturity and embryo quality, while FF activin-A is linked to implantation and clinical pregnancy rates. When viewed alongside recent evidence of preserved intrafollicular hormone secretion despite advancing maternal age, our results suggest that functional variability within the follicular microenvironment\u0026mdash;rather than age-related hormonal decline\u0026mdash;underlies differences in IVF success. Further studies are warranted to explore whether intrafollicular hormone profiling could form individualized strategies for optimizing assisted reproduction outcomes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eART \u003cem\u003eAssisted reproductive technologies\u003c/em\u003e,\u003c/p\u003e\u003cp\u003eBMI \u003cem\u003eBody mass index\u003c/em\u003e,\u003c/p\u003e\u003cp\u003e\u003cem\u003eβ\u003c/em\u003e-hCG \u003cem\u003eβ\u003c/em\u003e-\u003cem\u003echorionic gonadotropin\u003c/em\u003e,\u003c/p\u003e\u003cp\u003eΕ2 \u003cem\u003eEstradiol\u003c/em\u003e,\u003c/p\u003e\u003cp\u003eELISA \u003cem\u003eEnzyme-linked immunosorbent assay\u003c/em\u003e,\u003c/p\u003e\u003cp\u003eFF \u003cem\u003eFollicular fluid\u003c/em\u003e,\u003c/p\u003e\u003cp\u003eFSH \u003cem\u003eFollicle-stimulating hormone\u003c/em\u003e,\u003c/p\u003e\u003cp\u003ehMG \u003cem\u003eHuman menopausal gonadotropin\u003c/em\u003e,\u003c/p\u003e\u003cp\u003eICSI \u003cem\u003eIntracytoplasmic sperm injection\u003c/em\u003e,\u003c/p\u003e\u003cp\u003eIVF \u003cem\u003eIn vitro fertilization\u003c/em\u003e,\u003c/p\u003e\u003cp\u003eLH \u003cem\u003eLuteinizing hormone\u003c/em\u003e,\u003c/p\u003e\u003cp\u003eMET M\u003cem\u003eetabolic equivalent of task\u003c/em\u003e,\u003c/p\u003e\u003cp\u003eOocytes-MII \u003cem\u003eMetaphase-II stage oocytes\u003c/em\u003e,\u003c/p\u003e\u003cp\u003ePCOS \u003cem\u003ePolycystic ovary syndrome\u003c/em\u003e,\u003c/p\u003e\u003cp\u003eRR \u003cem\u003eRelative Risk.\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e The authors would like to thank the Embryogenesis Assisted Conception Unit clinic staff for their assistance in data collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThis work was partially supported by research grant from Harokopio University (KE321).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution:\u0026nbsp;\u003c/strong\u003eDK, MK and NY were involved in study concept and design. DK, and MM contributed to the acquisition of data. RT supervised samples analysis. DK and RT analyzed the data and drafted the manuscript. RT, MK, IL and NY supervised analysis and critically revised the manuscript. NY had the primary responsibility for final content. All authors haveread and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eNone declared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure of potential conflicts of interest\u003c/strong\u003e: No\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch involving Human Participants and/or Animals\u003c/strong\u003e: Research involned Human Participants (n=86 women).\u0026nbsp;Biochemical analyses were performed in the Laboratory of Biology, Biochemistry and Physiology, in the Department of Nutrition \u0026amp; Dietetics at Harokopio University of Athens.\u0026nbsp;All procedures adhered to the Helsinki Declaration,\u0026nbsp;with written informed consent obtained from participants. The study protocol was approved by the Institutional Ethics Committee of Harokopio University (12/13-01-2013).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e : All procedures adhered to the Helsinki Declaration, with written informed consent obtained from participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial registration number:\u0026nbsp;\u003c/strong\u003eNCT03050944\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLazzaroni-Tealdi E et al (2015) Oocyte Scoring Enhances Embryo-Scoring in Predicting Pregnancy Chances with IVF Where It Counts Most. PLoS ONE 10(12):e0143632\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDumesic DA et al (2015) Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health. Fertil Steril 103(2):303\u0026ndash;316\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWijayarathna R, de Kretser DM (2016) Activins in reproductive biology and beyond. Hum Reprod Update, 22(3)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFunghi L et al (2018) Placental and maternal serum activin A in spontaneous and induced labor in late-term pregnancy. J Endocrinol Invest 41(2):171\u0026ndash;177\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVale W et al (1986) Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature 321(6072):776\u0026ndash;779\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLing N et al (1986) Pituitary FSH is released by a heterodimer of the beta-subunits from the two forms of inhibin. Nature 321(6072):779\u0026ndash;782\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L et al (2018) The emerging role of follistatin under stresses and its implications in diseases. Gene 639:111\u0026ndash;116\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUeno N et al (1987) Isolation and partial characterization of follistatin: a single-chain Mr 35,000 monomeric protein that inhibits the release of follicle-stimulating hormone. Proc Natl Acad Sci U S A 84(23):8282\u0026ndash;8286\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGosden R, Lee B (2010) Portrait of an oocyte: our obscure origin. J Clin Invest 120(4):973\u0026ndash;983\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdwards RG (1974) Follicular fluid. J Reprod Fertil 37(1):189\u0026ndash;219\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDa Broi MG et al (2018) Influence of follicular fluid and cumulus cells on oocyte quality: clinical implications. J Assist Reprod Genet 35(5):735\u0026ndash;751\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchneyer AL et al (2000) Dynamic changes in the intrafollicular inhibin/activin/follistatin axis during human follicular development: relationship to circulating hormone concentrations. J Clin Endocrinol Metab 85(9):3319\u0026ndash;3330\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujiwara T et al (2000) Analysis of follicular fluid hormone concentrations and granulosa cell mRNA levels for the inhibin-activin-follistatin system: relation to oocyte and embryo characteristics. Fertil Steril 74(2):348\u0026ndash;355\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawrenz B et al (2020) Inhibin A-A Promising Predictive Parameter for Determination of Final Oocyte Maturation in Ovarian Stimulation for IVF/ICSI. Front Endocrinol (Lausanne) 11:307\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoffmann-Dishon N et al (2024) Endocrine-disrupting chemical concentrations in follicular fluid and follicular reproductive hormone levels. J Assist Reprod Genet 41(6):1637\u0026ndash;1642\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBouzoni E et al (2022) Embryo Quality May Be Associated With Serum Inhibin B Levels but Not With Serum or Follicular Fluid Levels of Other Components of the Activin-Follistatin-Inhibin Axis. Endocr Pract 28(10):1086\u0026ndash;1090\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarayiannis D et al (2017) Association between adherence to the Mediterranean diet and semen quality parameters in male partners of couples attempting fertility. Hum Reprod 32(1):215\u0026ndash;222\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarayiannis D et al (2018) Adherence to the Mediterranean diet and IVF success rate among non-obese women attempting fertility. Hum Reprod 33(3):494\u0026ndash;502\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGotham SM, Fryer PJ, Paterson WR (1988) The measurement of insoluble proteins using a modified Bradford assay. Anal Biochem 173(2):353\u0026ndash;358\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJeppesen JV et al (2012) Concentration of activin A and follistatin in follicular fluid from human small antral follicles associated to gene expression of the corresponding granulosa cells. Mol Cell Endocrinol 356(1\u0026ndash;2):48\u0026ndash;54\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang CL et al (2002) The concentration of inhibin B in follicular fluid: relation to oocyte maturation and embryo development. Hum Reprod 17(7):1724\u0026ndash;1728\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOcal P et al (2004) Follicular fluid concentrations of vascular endothelial growth factor, inhibin A and inhibin B in IVF cycles: are they markers for ovarian response and pregnancy outcome? Eur J Obstet Gynecol Reprod Biol 115(2):194\u0026ndash;199\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFullerton PT Jr. et al (2017) Follistatin is critical for mouse uterine receptivity and decidualization. Proc Natl Acad Sci U S A 114(24):E4772\u0026ndash;e4781\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee KB et al (2009) Molecular determinants of oocyte competence: potential functional role for maternal (oocyte-derived) follistatin in promoting bovine early embryogenesis. Endocrinology 150(5):2463\u0026ndash;2471\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel OV et al (2007) Functional genomics studies of oocyte competence: evidence that reduced transcript abundance for follistatin is associated with poor developmental competence of bovine oocytes. Reproduction 133(1):95\u0026ndash;106\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalvert ME et al (2022) Serum and urine profiles of TGF-β superfamily members in reproductive aged women. Clin Chim Acta 524:96\u0026ndash;100\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWen X et al (2006) Follicular fluid levels of inhibin A, inhibin B, and activin A levels reflect changes in follicle size but are not independent markers of the oocyte's ability to fertilize. Fertil Steril 85(6):1723\u0026ndash;1729\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang NF et al (2025) Impact of female age on concentrations of reproductive hormones and oocyte-specific growth factors in follicular fluid from human small antral follicles. Hum Reprod 40(4):707\u0026ndash;716\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDani C (2013) Activins in adipogenesis and obesity. Int J Obes (Lond) 37(2):163\u0026ndash;166\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOngaro L, Bernard DJ (2025) Activin Actions in Adipocytes. J Clin Endocrinol Metab 110(7):1803\u0026ndash;1810\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"hormones","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"HORM","sideBox":"Learn more about [Hormones](https://www.springer.com/journal/42000)","snPcode":"42000","submissionUrl":"https://www.editorialmanager.com/horm/default2.aspx","title":"Hormones","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Activin A, Inhibin B, Follistatin, Follicular fluid, In vitro Fertilization","lastPublishedDoi":"10.21203/rs.3.rs-8907446/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8907446/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eFollicular microenvironment influence soocyte and embryo quality and therefore plays a critical role in assisted reproduction therapy outcomes. This study prospectively evaluated the association between follicular fluid (FF) concentrations of activin-A, follistatin,and inhibin-B and \u003cem\u003ein vitro\u003c/em\u003e fertilization (IVF) outcome parameters.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eCouples with primary infertility attending an Assisted Conception Unit in Athens, Greece, were recruited as part of an ongoing prospective cohort study evaluating the impact of dietary and lifestyle patterns on fertility. FF levels of activin-A, follistatin, and inhibin-B were measured in 86 women undergoing IVF/ICSI cycles, along with assessments of oocyte yield and quality, embryo number and quality, implantation success, clinical pregnancy, and live birth rates. FF samples were collected at oocyte retrieval from follicles\u0026thinsp;\u0026ge;\u0026thinsp;18 mm in diameter and analyzed using enzyme-linked immunosorbent assay. Associations between FF biomarkers and IVF outcomes were assessed using correlation analyses and multivariable-adjusted regression models.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFF activin-A concentrations were positively correlated with both follistatin and inhibin-B levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while FF follistatin and inhibin-B levels were significantly associated with oocyte and embryo number and quality (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Multivariable-adjusted relative risk analyses demonstrated that higher FF activin-A levels were independently associated with improved reproductive outcomes. Specifically, each 10 pg/mL increase in FF activin-A was associated with a 58% higher likelihood of successful implantation [relative risk (95% confidence intervals)\u0026thinsp;=\u0026thinsp;1.58 (1.03\u0026ndash;2.45)] and a 69% higher likelihood of achieving a clinical pregnancy [1.69 (1.08\u0026ndash;2.62)].\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn conclusion, FF activin-A, follistatin and inhibin-B levels may serve as predictive biomarkers of IVF outcomes in women undergoing assisted reproductive treatment.\u003c/p\u003e","manuscriptTitle":"Intrafollicular Activin-A, Inhibin-B, and Follistatin may predict invitro fertilization outcomesin subfertile women.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-27 13:13:15","doi":"10.21203/rs.3.rs-8907446/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2026-03-19T07:34:57+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-02-28T16:34:07+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-25T11:08:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-19T09:39:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Hormones","date":"2026-02-18T05:42:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"hormones","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"HORM","sideBox":"Learn more about [Hormones](https://www.springer.com/journal/42000)","snPcode":"42000","submissionUrl":"https://www.editorialmanager.com/horm/default2.aspx","title":"Hormones","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1fb7032c-8658-4f76-ae69-dc50b6f671ac","owner":[],"postedDate":"February 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-06-17T14:39:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-27 13:13:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8907446","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8907446","identity":"rs-8907446","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}