Follicular Fluid Samples Research Articles

Follicular fluid lipidomic profiling reveals potential biomarkers of polycystic ovary syndrome: A pilot study.

BackgroundPolycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder associated with multiple metabolic conditions including obesity, insulin resistance, and dyslipidemia. PCOS is the most common cause of anovulatory infertility; however, the molecular diversity of the ovarian follicle microenvironment is not fully understood. This study aimed to investigate the follicular fluid (FF) lipidomic profiles in different phenotypes of PCOS and to explore novel lipid biomarkers.MethodsA total of 25 women with PCOS and 12 women without PCOS who underwent in vitro fertilization and embryo transfer were recruited, and their FF samples were collected for the lipidomic study. Liquid chromatography-tandem mass spectrometry was used to compare the differential abundance of FF lipids between patients with different PCOS phenotypes and controls. Subsequently, correlations between specific lipid concentrations in FF and high-quality embryo rate (HQER) were analyzed to further evaluate the potential interferences of lipid levels with oocyte quality in PCOS. Candidate biomarkers were then compared via receiver operating characteristic (ROC) curve analysis.ResultsIn total, 19 lipids were identified in ovarian FF. Of these, the concentrations of ceramide (Cer) and free fatty acids (FFA) in FF were significantly increased, whereas those of lysophosphatidylglycerol (LPG) were reduced in women with PCOS compared to controls, especially in obese and insulin-resistant groups. In addition, six subclasses of ceramide, FFA, and LPG were correlated with oocyte quality. Twenty-three lipid subclasses were identified as potential biomarkers of PCOS, and ROC analysis indicated the prognostic value of Cer,36:1;2, FFA C14:1, and LPG,18:0 on HQER in patients with PCOS.ConclusionsOur study showed the unique lipidomic profiles in FF from women with PCOS. Moreover, it provided metabolic signatures as well as candidate biomarkers that help to better understand the pathogenesis of PCOS.

Assessment of bacterial diversity associated with assisted reproductive technologies through next-generation sequencing

BackgroundTo assess bacterial diversity in infertile couples with their biochemical pregnancy outcomes. Using a retrospective case-control study design, participants were recruited for collection of vaginal swab, follicular fluid, endometrial fluid, and semen samples. The microbial composition was analyzed by 16S ribosomal RNA (rRNA) gene amplification with (MinION) Oxford Nanopore Ltd.ResultsOur findings revealed that age and endometrial thickness had a significant impact on the pregnancy success rate of pregnant (P) and non-pregnant (NP) patients receiving IVF, with high levels of luteinizing hormone, estrogen, and progesterone in the P group. In addition, the partial least squares discriminant analysis (PLS-DA) revealed a difference in microbial composition between the P and NP groups, as well as a higher microbial abundance in non-pregnant patients compared to pregnant patients. After comparison between pregnant patients and non-pregnant patients, pregnant patients had a higher abundance of Firmicutes and Proteobacteria and a lower abundance of Actinobacteria, Fusobacterium, and Bacteroidetes at the phylum level. Non-pregnant patients had a lower abundance of the probiotics lactobacillus and a higher abundance of infections Prevotella and Gardnerella at the genus level. As a result, a disordered microbiota in non-pregnant patients, characterized by a decrease in probiotics and an increase in pathogens, could be used as a possible marker for a higher IVF failure rate.ConclusionAlteration of the microbiota of the reproductive tract or the presence of certain microbes, regardless of the degree of pathogenicity that can affect fertilization, as well as implantation and subsequent embryonic development. This could result in failed fertility treatments and a lower live birth (LBR) rate.

Proteomics and bioinformatics analysis of follicular fluid from patients with polycystic ovary syndrome.

Objectives: Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder with heterogeneous manifestations and complex etiology. We used quantitative proteomics analysis based on mass spectrometry to identify the differences in proteomics profiles for follicular fluid obtained from patients with or without PCOS and explore possible mechanisms underlying PCOS. Methods: Follicular fluid samples were collected from infertile patients with (n = 9) or without (n = 9) PCOS. Total protein was extracted, quantitatively labeled with a tandem mass tag (TMT), and analyzed using liquid chromatography-mass spectrometry (LC‐MS). TMT-based proteomics and bioinformatics analysis were used to determine the differentially expressed proteins (DEPs) and understand the protein networks. The analysis included protein annotation, unsupervised hierarchical clustering, functional classification, functional enrichment and clustering, and protein-protein interaction analysis. Selected DEPs were confirmed by ELISA, and correlation analysis was performed between these DEPs and the clinical characteristics. Results: In this study, we have identified 1,216 proteins, including 70 DEPs (32 upregulated proteins, 38 downregulated proteins). Bioinformatics analysis revealed that the inflammatory response, complement and coagulation cascades, activation of the immune response, lipid transport, and regulation of protein metabolic processes were co-enriched in patients with PCOS. Based on ELISA results, insulin-like growth factor binding protein 1 (IGFBP1) and apolipoprotein C2 (APOC2) were differentially expressed between patients with and without PCOS. Follicular IGFBP1 showed a positive correlation with the serum levels of high-density lipoprotein cholesterol (HDL-C) (r = 0.3046, p = 0.0419), but negatively correlated with the serum levels of anti-Müllerian hormone (AMH) (r = –0.2924, p = 0.0354) and triglycerides (r = –0.3177, p = 0.0246). Follicular APOC2 was negatively correlated with the serum apolipoprotein A1 (APOA1) levels (r = 0.4509, p = 0.0002). Conclusion: Our study identified DEPs in the follicular fluid of patients with PCOS. Inflammatory response, complement and coagulation cascades, activation of the immune response, lipid transport, and regulation of protein metabolic process were deregulated in PCOS, which may play essential roles in the pathogenesis of PCOS.

Markers of ovarian reserve are associated with reproductive age acceleration in granulosa cells from IVF patients.

Is reproductive aging in granulosa cells associated with markers of ovarian reserve? Age acceleration was associated with anti-Mullerian hormone (AMH) levels, antral follicle count (AFC), oocyte yield and maturity, and the number of successfully fertilized embryos. The rate of reproductive aging varies among women of the same age. DNA methylation can be used to predict epigenetic age in a variety of tissues. This was a cross-sectional study of 70 women at the time of oocyte retrieval. The 70 participants were recruited for this study at an academic medical center and they provided follicular fluid samples at the time of oocyte retrieval. Granulosa cells were isolated and assessed on the MethylationEPIC array. Linear regression was used to evaluate the associations between DNA methylation-based age predictions from granulosa cells and chronological age. Age acceleration was calculated as the residual of regressing DNA methylation-based age on chronological age. Linear regressions were used to determine the associations between age acceleration and markers of ovarian reserve and IVF cycle outcomes. Participants were a mean of 36.7 ± 3.9 years old. In regards to race, 54% were white, 19% were African American and 27% were of another background. Age acceleration was normally distributed and not associated with chronological age. Age acceleration was negatively associated with AMH levels (t = -3.1, P = 0.003) and AFC (t = -4.0, P = 0.0001), such that women with a higher age acceleration had a lower ovarian reserve. Age acceleration was also negatively correlated with the total number of oocytes retrieved (t = -3.9, P = 0.0002), the number of mature oocytes (t = -3.8, P = 0.0003) and the number of fertilized oocytes or two-pronuclear oocytes (t = -2.8, P = 0.008) in the main analysis. This study used pooled follicular fluid, which does not allow for the investigation of individual follicles. Infertility patients may also be different from the general population, but, as we used granulosa cells, the participants had to be from an IVF population. This study demonstrated that epigenetic age and age acceleration can be calculated from granulosa cells collected at the time of oocyte retrieval. GrimAge most strongly predicted chronological age, and GrimAge acceleration was associated with baseline and cycle characteristics as well as cycle outcomes, which indicates its potential clinical relevance in evaluating both oocyte quantity and quality. This study was supported by the National Institutes of Health (UL1TR002378) and the Building Interdisciplinary Research Careers in Women's Health Program (K12HD085850) to A.K.K. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funding source had no role in any aspect of this study. J.B.S. serves as Vice Chair for the American Society for Reproductive Medicine Education Committee, is a Medical Committee Advisor for the Jewish Fertility Foundation and works with Jscreen. J.B.S. has received funding from Georgia Clinical Translational Research Alliance. H.S.H., J.B.S. and A.K.S. have received NIH funding for other projects. A.K.K., S.A.G., S.G., Q.S.K., L.J.M. and W.S. have no conflicts of interest. N/A.

Role and mechanism of miR-335-5p in the pathogenesis and treatment of polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a common endocrine disorder of unknown etiology that occurs in women of reproductive age. Despite being considered to affect up to one-fifth of women in this cohort, the condition lacks generally accepted diagnostic biomarkers and options for targeted therapy. Hereby, we analyzed the diagnostic, therapeutic, and functional potential of a recently discovered miR-335-5p that was observed to be reduced in the follicular fluid (FF) of PCOS patients as compared with healthy women. We found miR-335-5p to be significantly decreased in the serum and FF samples of PCOS patients (n=40) vs healthy women (n=30), as well as in primary human granulosa cells (hGCs), and in 3 different hormonally induced PCOS-like murine models vs. wild-type (WT) mice. The level of circulating miR-335-5p was found to significantly correlate with the impaired endocrine and clinical features associated with PCOS in human patients. Ovarian intrabursal injection of the miR-335-5p antagomir in WT mice ovaries induced a PCOS-like reproductive phenotype. Treatment with the miR-335-5p agomir rescued the dihydrotestosterone-induced PCOS-phenotype in mice, thereby providing a functional link between miR-335-5p and PCOS. We identified SP1 as a miR-335-5p target gene by using the dual-luciferase reporter assay. Both the luciferase reporter assay and chromatin immunoprecipitation assay showed that SP1 bound to the promoter region of human CYP19A1 and inhibited its transcription. miR-335-5p increased the production of estradiol via the SP1/CYP19A1 axis in hGCs, thereby suggesting its mechanistic pathway of action. In conclusion, these results provide evidence that miR-335-5p may function as a mediator in the etiopathogenesis of PCOS, as well as has the potential as both a novel diagnostic biomarker and therapeutic target for PCOS.

Уровень андрогенов в крови и фолликулярной жидкости у женщин с бесплодием и сниженным овариальным резервом в программах ВРТ

Androgen concentrations in follicular fluid samples collected from patients undergoing in vitro fertilization (IVF) may provide useful clinical indicators. This study aimed to analyze possible associations of the androgen levels in follicular fluid and blood plasma in patients with diminished ovarian reserve (POR) in IVF programs. Cross-sectional study with a parallel group design, conducted in 2019–2021, enrolled 300 patients with infertility, aged 18–42 years, applying for assisted reproduction involving IVF/intracytoplasmic sperm injection and embryo transfer. The androgen profiles of blood plasma and follicular fluid were determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Androgen concentrations in blood plasma and follicular fluid, particularly those of dehydroepiandrosterone (DHEA-S), androstenedione and total testosterone, significantly correlated. The results implicate androgen levels in blood plasma and follicular fluid as early markers of POR in patients with infertility.

Metabolic signatures in human follicular fluid identify lysophosphatidylcholine as a predictor of follicular development

In order to investigate the metabolic characteristics of human follicular fluid (FF) and to reveal potential metabolic predictors of follicular development (FD) with clinical implications, we analyzed a total of 452 samples based on a two-stage study design. In the first stage, FF samples from both large follicles (LFs) and matched-small follicles (SFs) of 26 participants were analyzed with wide-spectrum targeted metabolomics. The metabolic signatures were described by multi-omics integration technology including metabolomic data and transcriptomic data. In the second stage, the potential biomarkers of FD were verified using enzyme-linked immunoassay with FF and blood serum from an independent 200 participants. We describe the FF metabolic signatures from ovarian follicles of different developmental stages. Lysophosphatidylcholine (LPC) can be used as a biomarker of FD and ovarian sensitivity, advancing the knowledge of metabolic regulation during FD and offering potential detection and therapeutic targets for follicle and oocyte health improvements in humans.

IL-6 of follicular fluid and outcome of in vitro fertilization.

The quality of an oocyte is influenced by its microenvironment, which includes cumulus cells and follicular fluid, as well as cells of the immune system and their products. The ovarian interleukins, which are secreted by the granulosa cells and other immune cells within the ovaries and follicles, regulate various functions between the cells. IL-6 is a cytokine that is present in the follicular fluid and may affect the quality of oocytes. There are some inconsistencies in the literature regarding the concentration of interleukin 6 in the follicular fluid.The main objective of this study was to examine whether the concentration of interleukin 6 in the follicular fluid affects the outcome of IVF.This study involved 83 patients who underwent IVF. Follicular fluid was used as the biological material for the analysis. Examination of the obtained follicular fluid and collection of oocytes under a stereomicroscope was performed in the embryological laboratory. The concentration of IL-6 in the follicular fluid was analyzed. IVF and ICSI methods were used as the fertilization methods. Pregnancy was confirmed by the positive serum β-hCG level. The software package SPSS 20 was used for statistical data processing.Analysis of the follicular fluid samples showed a correlation between the concentration of IL-6 in the follicular fluid and the outcome of IVF. The concentration of IL-6 in the follicular fluid was higher in patients with confirmed pregnancy (9.55 ± 7.47 ng/ml).Based on our results, we conclude that the concentration of IL-6 affects the outcome of IVF. If the range of IL-6 concentration is between 3,67 ng/ml and 10 ng/ml, we can expect good IVF outcome with vital pregnancy.

Quantitative label-free proteomic analysis of human follicle fluid to identify novel candidate protein biomarker for endometriosis-associated infertility

BackgroundEndometriosis (EM) leads to a decline in fertility, which is characterized by a decrease in the number and quality of follicles, and thus has a negative impact on in vitro fertilization (IVF) outcomes. However, the mechanism of how EM affects oocytes and leads to infertility remains unclear. As a potentially available sample directly related to oocyte growth, follicular fluid (FF) has important research value. Evaluating the association of FF content and EM-associated infertility through proteomics may helpful to explore the possible pathogenesis of EM-associated infertility. MethodsIn the present experimental study, from August 2019 to June 2020, FF samples were obtained as control group (CON-G; n = 10) from women with no one female factor of infertility and were undergoing IVF due to other reasons, 20 women with EM-associated infertility undergoing IVF with no other female factors were distributed into the EM group according to the time for IVF: (i) EM-group 1 (EM-G1, Stage I to Stage III, n = 10); (ii) EM-group 2 (EM-G2, Stage I to Stage III, n = 10). label-free quantitative proteomics (LFQP) technology and parallel reaction monitoring (PRM) approach were combined to aid in identifying and validating FF protein biomarkers for EM-associated infertility. In PRM analysis, another 20 subjects were enrolled as EM-associated infertility group (EM,Stage I to Stage III, n = 10) and controls (CON, n = 10) within the same time and inclusion criteria are the same as previously described. Finally, a potential protein biomarker panel of FF differential expressed proteins to EM-associated infertility was also evaluated by t-test and receiver operating characteristic (ROC) curve and binary Logistic regression models. Results7 significant differential expressed proteins which closely related to EM-associated infertility were found by LFQP technology, among which immunoglobulin lambda variable 7–46 (IGLV7–46), Immunoglobulin heavy constant gamma 2 (IGHG2), glia-derived nexin (GDN) and Inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) were significantly up-regulated (p < 0.05), while corticosteroid-binding globulin (CBG), angiotensinogen (AGT) and Fetuin-B (FETUB) were significantly down regulated (p < 0.05). Additionally, GDN and AGT was identified as a potential protein biomarker by further PRM analysis for EM-associated infertility according to ROC curve analysis and t-test (p < 0.05), the area under the curve (AUC) for GDN and AGT was 0.78 and 0.69 with optimum sensitivity of 50%, 70% and specificity of 100%, 90%, respectively. According to binary logistic regression and evaluated ROC analysis, the AUC for the combination of GDN and AGT was 0.80. ConclusionsTo the best of our knowledge, this is the first time that elevated GDN protein levels have been found in the FF of patients with EM-associated infertility. Combining LFQP technology and PRM method we found the abnormal of GDN and AGT in FF may be the potential cause of EM-associated infertility which may help to better understand the physiological and pathological mechanism of EM-associated infertility. Further experimental studies are required to confirm their mechanism in EM-associated infertility. The results of this study are also consistent with the previous conclusion that EM is a chronic inflammatory disease. SignificanceTo the best of our knowledge, this is the first time that elevated GDN protein levels have been found in the follicular fluid of patients with EM-associated infertility. Combining LFQP technology and PRM methods we found the abnormal of GDN and AGT protein in FF may be the potential cause of EM-associated infertility which may help to better understand the physiological and pathological mechanism of EM-associated infertility.Clinically, it has been recognized that EM is related to infertility, but the mechanism remains unclear. Our study combines label-free quantitative proteomics technology and parallel reaction monitoring methods to identify and verify the FF protein biomarkers of EM-associated infertility, which provides a good research method for follow-up research.

Preovulatory follicular fluid and serum metabolome profiles in lactating beef cows with thin, moderate, and obese body condition.

Extremes in body condition reduce fertility and overall productivity in beef cattle herds, due in part to altered systemic metabolic conditions that influence the intrafollicular and uterine environment. Follicular fluid and serum metabolome profiles are influenced by body composition in women and dairy cattle; however, such information is lacking in beef cattle. We hypothesized that body condition score (BCS)-related alterations in the metabolome of preovulatory follicular fluid and serum may influence oocyte maturation while impacting the oviductal or uterine environment. Therefore, we performed a study with the objective to determine the relationship between BCS and the metabolome of follicular fluid and serum in lactating beef cattle. We synchronized the development of a preovulatory follicle in 130 cows of varying BCS. We collected blood and performed transvaginal follicle aspirations to collect follicular fluid from the preovulatory follicle ~18 h after gonadotropin-releasing hormone administration to stimulate the preovulatory gonadotropin surge. We then selected follicular fluid and serum samples from cows with BCS 4 (Thin; n = 14), BCS 6 (Moderate; n = 18), or BCS >8 (Obese; n = 14) for ultra-high performance liquid chromatography-high resolution mass spectrometry. We identified differences in the follicular fluid or serum of thin, moderate, and obese animals based on multiple linear regression. MetaboAnalyst 5.0 was used for enrichment analysis of significant metabolites. We identified 38 metabolites in follicular fluid and 49 metabolites in serum. There were no significant differences in follicular fluid metabolite content among BCS classifications. There were 5, 22, and 1 serum metabolites differentially abundant between thin-obese, moderate-thin, and moderate-obese classifications, respectively (false discovery rate [FDR] < 0.10). These metabolites were enriched in multiple processes including "arginine biosynthesis," "arginine/proline metabolism," and "D-glutamine/D-glutamate metabolism" (FDR < 0.04). Pathways enriched with serum metabolites associated with BCS indicate potentially increased reactive oxygen species (ROS) in serum of thin cows. ROS crossing the blood follicular barrier may negatively impact the oocyte during oocyte maturation and contribute to the reduced pregnancy rates observed in thin beef cows.

Preovulatory serum estradiol concentration is positively associated with oocyte ATP and follicular fluid metabolite abundance in lactating beef cattle.

Cattle induced to ovulate a small, physiologically immature preovulatory follicle had reduced oocyte developmental competence that resulted in decreased embryo cleavage and day 7 embryo quality compared with animals induced to ovulate a more advanced follicle. RNA-sequencing was performed on oocytes and their corresponding cumulus cells approximately 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the preovulatory gonadotropin surge suggested reduced capacity for glucose metabolism and oxidative phosphorylation in the cumulus cells and oocytes from follicles ≤11.7 mm, respectively. We hypothesized that induced ovulation of a small, physiologically immature preovulatory follicle results in a suboptimal follicular microenvironment and reduced oocyte metabolic capacity. We performed a study with the objective to determine the impact of preovulatory follicle diameter and serum estradiol concentration at GnRH administration on oocyte metabolic competence and follicular fluid metabolome profiles. We synchronized the development of a preovulatory follicle and collected the follicle contents via transvaginal aspiration approximately 19 h after GnRH administration in lactating beef cows (n = 319). We determined ATP levels and mitochondrial DNA (mtDNA) copy number in 110 oocytes and performed ultra-high-performance liquid chromatography–high resolution mass spectrometry metabolomic studies on 45 follicular fluid samples. Intraoocyte ATP and the amount of ATP produced per mtDNA copy number were associated with serum estradiol concentration at GnRH and time from GnRH administration to follicle aspiration (P < 0.05). mtDNA copy number was not related to follicle diameter at GnRH, serum estradiol concentration at GnRH, or any potential covariates (P > 0.10). We detected 90 metabolites in the aspirated follicular fluid. We identified 22 metabolites associated with serum estradiol concentration at GnRH and 63 metabolites associated with follicular fluid progesterone concentration at the time of follicle aspiration (FDR < 0.10). Pathway enrichment analysis of significant metabolites suggested altered proteinogenesis, citric acid cycle, and pyrimidine metabolism in follicles of reduced estrogenic capacity pre-gonadotropin surge or reduced progesterone production by the time of follicle aspiration.

Follicular fluid 25-hydroxyvitamin D levels determine fertility outcome in patients with polycystic ovary syndrome

ObjectiveTo determine the possible relationship between follicular fluid 25-hydroxyvitamin D [25(OH)D] levels and fertility outcome of women who underwent IVF/ICSI with the diagnosis of lean polycystic ovary syndrome. Materials and methodsThirty patients who were diagnosed with PCOS according to the Rotterdam criteria and decided on IVF/ICSI were included in the study. Thirty patients who were scheduled for IVF/ICSI for reasons other than PCOS and matched in terms of age and BMI were taken as the control group (non-PCOS). According to BMI values, patients in both PCOS and non-PCOS groups were lean. Women in both groups were aged 21–35 years with a normal BMI (18.5–24.9 kg/m2) and first IVF/ICSI attempt. Both groups of patients were followed up using the antagonist protocol. Vit D levels were measured in serum and follicular fluid (FF) samples taken on the day of oocyte collection. The correlation between FF vit D levels, the number of total oocytes, MII oocytes and 2 PN zygotes, HOMA-IR, hormonal and demographic parameters, clinical pregnancy rate (CPR), live birth rate (LBR), and miscarriage rate were evaluated. ResultsAt the time of oocyte retrieval women with PCOS had similar serum Vitamin D compared to non-PCOS women (21.8 (12.6–24.8) ng/ml vs 22.3 (11.5–25.1) ng/ml, p < 0.54). In FF, assessed on the day of oocyte retrieval, the concentration of Vitamin D was similar in women with PCOS when compared to non-PCOS women (11.2 (9.2–14.4) ng/ml vs 13.3 (11.1–17.4) ng/ml, p < 0.06). For both groups, Vitamin D levels were lower in FF compared to serum vit D. A positive correlation was found between serum and FF Vitamin D concentrations in the full cohort. A positive and significant correlation was found between FF-vit D levels and the number of total oocyte (r = 0.344, p < 0.04) and MII oocyte (r = 0.404, p < 0.02) in the PCOS group. The number of total oocyte, MII oocyte and 2 PN zygotes of the PCOS group were significantly higher than the non-PCOS group. Positive pregnancy test rate, clinical pregnancy and live birth rates were similar in both groups. The miscarriage rates in the non-PCOS group were significantly higher than in the PCOS group. A positive and significant correlation was also found between FF vit D levels and positive pregnancy test (r = 0.566, p < 0.03) and CPR (r = 0.605, p < 0.02) in PCOS group. There was no correlation between FF-vit D levels and live birth and miscarriage rates in neither the PCOS nor the non-PCOS group. ConclusionsBoth serum and FF 25-hydroxyvitamin D level of women with PCOS at the time of oocyte retrieval are similar to non-PCOS controls. While FF 25-hydroxyvitamin D levels correlate with total and MII oocyte counts, positive pregnancy test and CPR, it does not correlate with miscarriage and live birth rates.

P-571 The newly developed highly sensitive reagent GL13 indicates extensively increased cellular senescence in the follicular fluid of poor responder patients: A prospective observational study

Abstract Study question Do women of diminished ovarian reserve and poor ovarian response (POR) present with increased lipofuscin follicular fluid (FF) levels, indicating increased ovarian cellular senescence? Summary answer Poor responders present with a six-fold increased lipofuscin FF levels indicating extensive senescence in POR ovaries. Lipofuscin may be a new sensitive biomarker for POR. What is known already Lipofuscin is a nondegradable substrate of metabolism accumulating in cells due to impaired mitochondrial/lysosome/proteasome function, upon stress or damage. It is well-documented that lipofuscin constitutes a highly sensitive biomarker of cellular aging and senescence. Recently, a novel reagent (GL13) coupled by a hybrid histochemical–immunohistochemical method were developed for detecting and measuring soluble lipofuscin levels in biological fluids, ascertaining high sensitivity and specificity. Data indicate that lipofuscin levels are associated with oocyte competence and age-related infertility. However, hitherto no data has been published indicating the value of lipofuscin FF levels as a biomarker towards accurately predicting ovarian reserve and response. Study design, size, duration This prospective observational study was collaboratively conducted between November 2020 and September 2021 at the Athens University Medical School and at Genesis Athens Clinic. A total of 32 patients undergoing IVF treatment were enrolled. The study group comprised of 16 POR patients defined according to the Bologna criteria. The control group consisted of 16 normal responder women undergoing IVF due to tubal factor or/and mild male factor infertility. Patients with other infertility aetiologies were excluded. Participants/materials, setting, methods Participants in both groups received the standard short GnRH-antagonist protocol. The FF samples were collected as part of the oocyte retrieval process. Lipid and protein parts of lipofuscin were isolated from FF samples and stained with GL13. The complex of lipofuscin-GL13 was labeled with an anti-biotin HRP conjugated antibody and detected employing a chemiluminescence reaction. Luminescence was measured and signal intensity was corresponding to lipofuscin concentration. Statistical analysis was performed employing R Programming Language. Main results and the role of chance Poor responders presented with a statistically significant six-fold higher lipofuscin FF levels in comparison to the normal responder group (869.21 ± 501.87 vs 146.6 ± 107.64 RLU; P-value &amp;lt;0.0001). Lipofuscin levels were negatively correlated with AFC (Spearman’s Rho: -0.68; P-value &amp;lt;0.0001), AMH levels (Rho: -0.61; P-value =0.0002), estradiol levels on triggering day (Rho: -0.68; P-value &amp;lt;0.0001), number of oocytes retrieved (Rho: -0.54; P-value =0.001), number of mature metaphase II (MII) oocytes obtained (Rho: -0.58; P-value =0.0008), number of normally fertilized (2PN) zygotes (Rho: -0.51; P-value =0.003), number of cleavage stage embryos (Rho: -0.48; P-value =0.005) and number of blastocyst stage embryos (Rho: -0.41; P-value =0.02). These correlations remained statistically significant when adjusting for ovarian stimulation response status. No association was established between Lipofuscin levels and embryo quality neither on Day 3 nor on Day 5. Lipofuscin levels, with a cut-off value at 294, were able to predict ovarian stimulation response status with an area under the curve (AUC) at 0.96. The sensitivity was 0.875, the specificity was 0.938 and the accuracy was 0.906. The positive predictive value was 88.24% and the negative predictive value was 93.33%. Limitations, reasons for caution Limitations of our study refer to the limited size of the studied population, as well as to the lack of data referring to pregnancy outcomes. Moreover, molecular data with regards to the possible mechanisms leading to the observed increased senescence are required. Future studies are needed to verify these findings. Wider implications of the findings Data presented herein indicates, for the first time in literature, that lipofuscin FF levels measured via the GL13 method may be a promising and sensitive tool for predicting POR and stimulation outcome. Lipofuscin could further serve as a valuable novel biomarker indicating ovarian senescence, ovarian reserve status and oocyte competence. Trial registration number Not Applicable

O-191 Endometriosis is associated with a lowered cumulative live birth rate by alters the macrophage polarization and the cytokine concentrations in follicular fluid

Abstract Study question The impact of endometriosis on the outcomes of ART is still ambiguous and the mechanism by which endometriosis impacts fertility has not been fully elucidated Summary answer Endometriosis was associated with low CLBR in IVF, which might be due to the change of follicular microenvironment and compromising the quality of embryos. What is known already Despite the great advantages of IVF treatment in endometriosis-linked infertility, the impact of endometriosis on outcomes of infertility management with ART is controversial and few studies focused on the association between endometriosis and CLBR. Numerous mechanisms have been proposed in an effort to delineate the multifaceted pathophysiology that induces impairment of reproductive dynamics in patients with endometriosis. Reactive oxygen species, dysregulation of the immune system and cellular architectural disruption constitute the crucial mechanisms that detrimentally affect oocyte and embryo developmental potential. Study design, size, duration This study retrospectively included 433 patients with endometriosis and 1299 patients with tuber factor infertility to evaluate the impact of endometriosis on IVF pregnancy outcome between January 2016 and December 2018.The basic study prospectively recruited 30 patients with ovarian endometriosis and 35 controls with tubal factor infertility to analyze polarization stage of macrophages in their follicular fluid, and another 20 females with ovarian endometriosis and 30 controls to detect cytokines in their FF. Participants/materials, setting, methods Data on all women undergoing fresh or frozen IVF treatment cycles were analysed to compare the CLBR between endometriosis and tuber factor infertility patients. A cytometry panel of 4 antibodies (CD45, CD3, CD80, CD163) was designed to enumerate the numbers of MI and MII macrophages from the cell sediment from FF samples. Quantibody® array was utilized to determine the concentration of 10 cytokines in FF, including IFN-γ, IL-1α, IL-1β, IL-10,IL-13,IL-4, IL-6, IL-8, MCP-1 and TNF-α. Main results and the role of chance The results showed that patients with endometriosis were associated with noticeably fewer retrievable oocytes, a lower oocyte maturity rate, decreased numbers of available and high-quality embryos (all p &amp;lt; 0.001) in comparison with the control group. The clinical pregnancy and live birth rate of the endometriosis group were lower in the frozen-thawed embryo transfer cycles (p = 0.028 and p = 0.008, respectively), which leading to a declined cumulative live birth rate (CLBR) (p = 0.001). Logistic regression analysis indicated a close association between endometriosis and low CLBR (p = 0.002). Furthermore, the numbers of type I and type II macrophages in follicular fluid (FF) of patients with ovarian endometriosis were significantly increased compared with the control group (p &amp;lt; 0.001). The expressions of IL-1α, IL-1β, TNF-α, IL-6, IL-13, and IL-10 in FF were higher in endometrioma group(p &amp;lt; 0.05). The increased expression of cytokines was negatively correlated with embryo outcomes, including the numbers of total oocytes retrieved, mature oocytes, and fertilized oocytes, and the numbers of high-quality blastocysts and embryos. Additionally, IL-6 and IL-8 were positively correlated with AMH, rate of available blastocysts, number of blastocysts formed and available blastocysts, but were negatively associated with rate of MII oocytes. Limitations, reasons for caution The present study being of a retrospective and monocentric study, may not come to a very convincing conclusion, and some unknown biases might still exist due to possible underestimation of some confounders. Wider implications of the findings Collectively, our results indicate that endometriosis does adversely affect pregnancy outcomes of ART, as women with endometriosis produce a lower quantity of oocytes and embryos. Moreover, endometriosis might play a role in oocyte or embryo outcomes via regulating the production of cytokines or the number of immune cells in FF. Trial registration number no

P-228 Higher oxidation levels in follicular fluid correlate with better outcome in ICSI treatments

Abstract Study question Can the oxidative status of the follicular fluid be a valid biomarker of the quality of the oocyte cohort and clinical outcome in ICSI treatments? Summary answer The follicular fluid (FF) of oocyte cohorts that achieved at least one pregnancy, ongoing pregnancy and life birth have higher oxidative stress levels. What is known already The role of oxidative stress in female infertility is yet unclear, due to the numerous factors that participate in the production and activity of reactive oxygen species (ROS), and the different techniques used for its detection. The Fertissimo TCL AnalyzerTM (Carmel Diagnostics, Israel) uses thermochemiluminiscence to assess the oxidative status (OS) of biological samples by counting the photons emitted per second (cps) after a heat-catalyzed oxidative reaction. Using this technique, a negative correlation was found between ROS levels in FF and patient age (Wiener-Megnazi et al, 2004), but its potential as a biomarker of clinical outcome is unclear. Study design, size, duration Prospective observational study performed in a single IVF center during one year. 173 women were subjected to controlled ovarian stimulation as part of an autologous (103 patients) or ovum donation ICSI treatment (70 donors). The OS of the follicular fluid (FF) obtained in the oocyte retrieval was assessed and compared with the characteristics of the treatment and clinical outcome. Participants/materials, setting, methods The first tube of follicular fluid obtained in the oocyte retrieval was centrifuged (10 minutes at 600g) and the supernatant was analyzed in the Fertissimo TCL AnalyzerTM. OS results were normalized per volume and summarized as TCL-Score (average photon counting from 50-280s) and Ratio (slope of the reaction time curve). TCL parameters were compared with the demographics, number of oocytes retrieved and clinical outcome of the cohort using Pearsons Correlation and Anova tests Main results and the role of chance FF from donors had higher TCL-Score (44.27±14.41cps vs 40.59±11.65cps, P = 0.066) and Ratio (367.38±89.47 vs 329.88±78.12, P = 0.004) than patients. A significant inverse correlation was found between TCL Ratio and the age of the women (Pearsons Correlation Coefficient (PCC)=-0.207; P = 0.006). A direct significant correlation was found between the body mass index (BMI) and the TCL-Score (PCC=0.186, P = 0.014) and Ratio (PCC=0.186, P = 0.014). TCL-Score (PCC=0.173, P = 0.023) and Ratio (PCC= 0.275, P &amp;lt;0.001) also had a significant positive correlation with the number of mature oocytes retrieved. In donors, TCL Ratio was negatively correlated with the fertilization rate (PCC=-0.350, P = 0.009) and positively correlated with the blastocyst formation rate at day 5 (PCC=0.289, P = 0.032), but said correlation was not found in FF of patients (PCC(Ratio-Fertilization rate)=0.066, P = 0.554; PCC(Ratio- Blastocyst rate)=-0.005, P = 0.965). However, in patients, a statistically significant negative correlation was found between TCL-Score and the good quality blastocyst rate (PCC=-0.234, P = 0.038), which was not statistically significant in donors (PCC=-0.131, P = 0.342). Finally, TCL Ratio levels resulted significantly higher in FF of oocyte cohorts that resulted in at least one pregnancy (362.77±86.60 vs 321.90±77.60, P = 0.005), one ongoing pregnancy (365.99±84.10 vs 322.66±80.94, P = 0.003) and one life birth (364.93±83.74 vs 330.07±84.17, P = 0.017). Limitations, reasons for caution Some miscalibration issues were met in the course of the analysis, which may hinder its reproducibility. Stimulation protocol or sperm quality was not considered in the analysis. A larger scale, controlled trial should be performed to validate results. Wider implications of the findings Presented results are in tune with previously published data using similar and different techniques for assessing oxidative and antioxidative capacity in FF samples. The analysis of the oxidative status of the FF could be a valid biomarker of clinical outcome of ICSI treatments and as indicator for antioxidant therapy. Trial registration number APOTIP/2020/015 Generalitat Valenciana.

P-570 Prokineticin-1 follicular fluid levels are strongly associated with diminished ovarian reserve and poor ovarian response: A prospective observational study

Abstract Study question Are Prokineticin-1 (PROK1) follicular fluid (FF) levels associated with poor ovarian response (POR) as well as with other well-known biomarkers associated with ovarian reserve status? Summary answer Poor responders present with increased PROK1 FF levels. Also, PROK1 FF levels are negatively correlated with anti-müllerian hormone (AMH) levels and number of oocytes retrieved. What is known already Prokineticin-1 is an angiogenic factor with pleiotropic properties. Considering its role in reproduction, PROK1 mediates significant angiogenic functions in the fetal-maternal interface. Thus, alterations in PROK1 expression and/or function lead to placenta-derived pregnancy complications. Recently published studies also indicate that PROK1 is associated with ovarian function and oocyte competence. However, limited data associating PROK1 with POR are published. This study uniquely investigates possible associations between PROK1 FF levels in POR cases, with stimulation outcome, as well as with FF levels of other factors related to ovarian functionality, namely VEGF, bone morphogenetic protein 15 (BMP-15) and Pigment Epithelium Derived Factor (PEDF). Study design, size, duration This prospective-observational study was collaboratively conducted between November 2020 and July 2021 at Genesis Athens Clinic and at the University of Athens Medical School. A total of 64 patients undergoing IVF treatment were enrolled. The study group comprised of 32 POR patients defined according to the Bologna criteria. The control group consisted of 32 normal responder women undergoing IVF due to tubal factor and/or mild male factor infertility. Patients with other infertility aetiologies were excluded. Participants/materials, setting, methods Participants in both groups received the standard short GnRH-antagonist protocol. Prior to ovarian stimulation, participants were subjected to basic infertility investigation, including antral follicle count and AMH levels evaluation. The FF samples were collected as part of the oocyte retrieval process, then centrifuged and stored at -80 °C till analysis. Follicular fluid levels of PROK1, VEGF, BMP-15 and PEDF were evaluated via ELISA employing commercially available kits. Statistical analysis was performed employing R Programming Language. Main results and the role of chance Significantly higher PROK1 (3229.63 ± 2372.66 vs 1945.95 ± 1408.01 pg/ml; P-value&amp;lt;0.0001), VEGF (2309.63 ± 412.49 vs 2013.22 ± 330.16 pg/ml; P-value=0.006) and lower BMP-15 (434.13 ± 106.38 vs 532.5 ± 108.26 pg/ml; P-value=0.001) levels were recorded in the POR group. No difference was observed regarding PEDF levels (7.43 ± 1.95 vs 8.23 ± 2.54 ng/ml; P-value=0.2). However, the POR group presented with lower PEDF/VEGF ratio (3.33 ± 1.08 vs 4.18 ± 1.41; P-value=0.02), indicating a reduced antioxidant capacity. PROK1 levels were negatively correlated with AMH (P-value=0.04), number of oocytes retrieved (P-value=0.001) and number of MII oocytes (P-value=0.005). BMP-15 was positively correlated with number of oocytes retrieved (P-value&amp;lt;0.001), number of MII oocytes (P-value=0.002), number of normally fertilized zygotes (P-value=0.007), number of cleavage stage embryos (P-value=0.03) and number of blastocysts (P-value=0.04). VEGF was negatively correlated with AMH (P-value=0.004) and number of oocytes retrieved (P-value=0.03). These correlations remained significant when adjusting for ovarian response status. PROK1 levels, with a cut-off value of 2854.25 pg/ml, were able to predict ovarian response status with an area under the curve at 0.64. Sensitivity was 0.55, specificity was 0.88, and accuracy was 0.71. The positive and negative predictive values were 81.82% and 66.67%, respectively. Limitations, reasons for caution Limitations of our study refer to the limited size of the studied population, as well as to the lack of data considering pregnancy outcomes. Moreover, molecular data with regards to the possible role of PROK1 on POR pathogenesis are required. Future studies are needed to verify the results presented herein. Wider implications of the findings Data presented herein indicate that PROK1 FF levels are strongly associated with diminished ovarian reserve and POR. Considering that FF-PROK1 presents with a similar profile with FF-VEGF, we can form the hypothesis that the compromised angiogenesis observed in POR patients leads to PROK1 and VEGF increase via a negative-feedback-loop. Trial registration number Not applicable

P-670 Phytoestrogen concentration in women’s urine and follicular fluid are associated with better IVF outcomes in euploid single embryo transfer cycles

Abstract Study question What is phytoestrogen exposure impact on the reproductive outcomes of women undergoing IVF treatment? Summary answer Increased urinary and follicular fluid phytoestrogen concentration were associated with better IVF outcomes. What is known already The impact and safety of phytoestrogens, plant-derived isoflavones with estrogenic activity predominantly present in soy, on female reproductive health and on IVF outcomes continues to be hotly debated. Numerous studies suggest healthy benefits of phytoestrogen consumption: protection against reprotoxic substances, reductions in breast and other cancers, fibroids, menopausal symptoms, cardiovascular disease, inflammation, and metabolic syndrome and obesity. However, many studies suggest a harmful effect due to the endocrine-disruptor properties of phytoestrogens in both male and female reproductive tissues. Its impact on IVF treatments success is still understudied. Study design, size, duration 60 women attending IVI-RMA New Jersey undergoing IVF with single-embryo transfer (SET) of euploid good-quality blastocyst after PGT analysis were recruited. Patients underwent PGT-SET cycles following standard protocols. Concentrations of two phytoestrogens (daidzein and genistein) were measured in follicular fluid (FF) and urine collected at oocyte pick-up (UOP) and urine collected at transfer day (UT). These measurements were correlated with IVF clinical outcomes. Participants/materials, setting, methods Participants were aged 18-42 years old 18.5-29.9 kg/m2 BMI. Measure of both phytoestrogens’ concentration in follicular fluid and urine samples collected at two different time-points was performed by UPLC-MSMS, with internal standards. Urine phytoestrogen concentrations were normalized by creatinine, measured by Jaffe reaction. Generalized linear models were employed to explore IVF outcomes association, estimated as percentile 20th to 80th increase (95% confidence intervals), among log-transformed phytoestrogen concentration. Both unadjusted and age-BMI-adjusted models were applied. Main results and the role of chance In fully adjusted models, higher sum of phytoestrogen concentration in follicular fluid (FF) were significantly associated with higher estradiol at hCG [1.31 (1.04, 1.65), p = 0.023]; whereas the sum of creatinine-corrected phytoestrogens measured in oocyte pick-up urine (UOP) were significantly associated with higher MII oocytes number [1.29 (1.07, 1.56), p = 0.008] and higher fertilized oocytes number [1.34 (1.10, 1.64), p = 0. 004]. Regarding clinical IVF outcomes, in fully adjusted models a higher follicular fluid and oocyte pick-up urine phytoestrogens concentration was significantly associated with higher implantation ratio [8.60 (1.09, 68.03), p = 0. 023] for FF, and [5.01 (0.96, 26.21), p = 0. 05] for UOP; higher clinical pregnancies ratio [5.58 (1.31, 23.73), p = 0.017] for FF, and [3.25 (0.99, 10.63), p = 0.045] for UOP; higher live newborns ratio [5.58 (1.31, 23.73), p = 0.017] for FF, and [3.25 (0.99, 10.63), p = 0.045] for UOP; and higher number of women with live newborns among those who started treatment [4.29 (1.28, 14.38), p = 0.016] for FF, and [2.83 (0.99, 8.07), p = 0.047] for UOP. Limitations, reasons for caution Further studies are needed to confirm this association in other populations, including the measurement of other estrogenic isoflavones and its metabolites. Wider implications of the findings This is the first work evaluating the association between phytoestrogens concentrations in different biofluids and IVF reproductive outcomes. Our results, using an euploid SET, suggest a beneficial effect of phytoestrogen consumption in women undergoing IVF treatment, further studies are needed to assess the benefit of phytoestrogen supplementation on IVF outcomes. Trial registration number -

Lipid Analysis of Follicular Fluids by UHPLC-ESI-HRMS Discovers Potential Biomarkers for Ovarian Hyperstimulation Syndrome.

Ovarian hyperstimulation syndrome (OHSS) is a serious iatrogenic complication during ovarian stimulation. Even though the incidence of OHSS was relatively low in clinical practice, the consequence can be potentially devastating and life-threatening. Abnormal lipid metabolism may relate to the pathological development of OHSS, but there is still a research gap in the lipidomic research. So here in our study, an ultra-high-performance liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry (UHPLC-ESI-HRMS) based lipidomic analysis was performed using follicular fluid samples obtained from 17 patients undergoing OHSS. The lipid profiles of OHSS patients were characterized by increased cholesterol ester (ChE) and decreased lysophosphatidylcholine (LPC), phosphatidylinositol (PI), sphingomyelin (SM), dimethylphosphatidylethanolamine (dMePE) and lysodimethylphosphatidylethanolamine (LdMePE). Totally 10 lipids including LPC(18:0), SM(d18:1/16:0), PC(18:0/18:1), PC(20:2/20:5), PC(16:0/18:1), TG(16:0/18:1/18:1), TG(16:0/18:2/18:2), TG(16:0/16:1/18:1), ChE(20:4) and TG(8:0/8:0/10:0) were selected as differential lipids. In conclusion, this study demonstrated the alteration of various lipids in OHSS patients, which suggested the key role of lipids during the development of OHSS and shed light on the further pathophysiological research of OHSS.

P-655 Insulin like growth factor-1 levels in follicular fluids of patients with poor ovarian response undergoing in vitro fertilization

Abstract Study question Does follicular fluid insulin like growth factor level (IGF-1) in poor responders (PR) differ from normoresponders? Is there a correlation between IGF-1 and IVF outcomes? Summary answer Follicular IGF-1 levels are decreased in poor-responders A positive correlation exists between IGF-1 and number of metaphase II (MII) oocytes, fertilized oocytes and transferred embryos What is known already IGFs are mainly synthesized in liver with growth hormone stimulation. IGF-1 component in follicular fluid has been reported to play a crucial role in follicular and embryonic development. In addition to previously reported positive correlation between follicular IGF-I levels and number of high quality embryos, a significant decrease in IGF-1 levels with PR and advanced maternal age has also been reported. GH supplementation in poor responders undergoing IVF has been suggested to improve IVF outcomes. A recent Cochrane analysis reported that GH slightly increases the number of oocytes retrieved and there is an uncertain effect on live birth rates (LBR). Study design, size, duration Study was designed prospectively. A power analysis, of 91% determined a need for 186 patients. Therefore 186 women undergoing IVF between October 2019 and December 2020 were included. 72 (%38,7) patients were assigned to PRs group according to Bologna criteria (BC) and 114 patients (%61.3) were recruited as controls. Antagonist protocol was used for controlled ovarian hyperstimulation. hCG was administered for ovulation trigger. Participants/materials, setting, methods Following the ovarian hyperstimulation follicular fluid samples were collected during oocyte retrieval. When the total desired number of patients was reached, frozen and preserved follicle fluids were thawed at room temperature. IGF-1 levels in samples were measured using hIGF-1 ELISA kit (BT®, China). Follicular IGF-1 levels in PRs were compared to normoresponders and correlation between IGF-1 levels and AMH levels, number of retrieved oocytes, MII and fertilized oocytes, transferred embriyos and LBR were evaluated. Main results and the role of chance Mean IGF-1 levels of normoresponders and Bologna subgroups (B1, B2, B3, B4) were 13.79±14.50 ng/ml vs 5.75±1.86, 5.47±2.83, 5.96±2.16 and 5.03±1.85 ng/ml respectively (p &amp;lt; 0.001). There was no statistically significant difference in clinical pregnancy (CP) per transfer and LBR per transfer between normoresponders and PRs (CPR/ET: 55.1% vs 43.7%, p:0.375; 26.5% vs 12.5% , p: 0.257). In the ROC analysis for the prediction of collection of fewer than 4 oocytes at retrieval (according to BC definition of PR), statistically determined IGF-1 value was 7.02 with a sensitivity of 83% and a specificity of 38.6%. Patients with an IGF-1 value below 7.02 had a higher risk of having an oocyte number less than 4According to the results of Spearman's correlation analysis, IGF-1 levels were negatively correlated with age and number of previous failed cycles (r:-0.250, p:0,001; r:-0.153, p:0,039 respectively). IGF-1 levels were positively correlated with AMH levels, oocyte, MII oocyte, fertilized oocyte numbers and number of transferred embriyos (r: 0.221, p: 0.007; r: 0.283, p &amp;lt; 0.001; r: 0.293, p &amp;lt; 0.001; r: 0.282, p &amp;lt; 0.001; r: 0.194, p: 0.008 respectively). There was a positive correlation between IGF-1 levels and cycle cancellation rates (r: 0.637, p &amp;lt; 0,001). There was no correlation between IGF-1 levels and LBR (p:0.118). Limitations, reasons for caution Small number of patients was the main limitation of study.Results were not interpreted as LBR in all cases, as 21% (n:24) of the cases had still ongoing pregnancies Wider implications of the findings Detecting lower follicular levels of IGF-1 which plays a crucial role in GH activity may highligt the effect of GH supplementation in poor responders undergoing IVF in terms of improved outcomes. However, there is need for more solid data to employ GH in IVF treatment modalities of poor-responders. Trial registration number not applicable

The Comparison of the Effect of Recombinant FSH in Antagonist Protocol on Serum and Follicular Fluid Kisspeptin between PCOS and non-PCOS Infertile Women during ICSI

Background: Reproduction is a highly delicate organized process accomplished by the controlled interaction of the higher centers in the brain, the gonads, and different body parts. Kisspeptin is a neuropeptide that acts as a key regulator controlling the hypothalamic-pituitary-gonadal axis and organizing the reproduction process. Gonadotropins treatment used in controlled ovarian hyperstimulation increases the follicular count in an attempt to improve the pregnancy rate. However, one of its drawbacks is the increase in ovarian hyperstimulation syndrome risk. Published data proved that kisspeptin has a fundamental role in regulating follicular growth and maturation at central and peripheral levels highlighting its possible role in diagnosing and treating infertility problems. Objective: To study the influence of gonadotropin (recombinant FSH medication) used in ovarian hyperstimulation program on the level serum and follicular fluid kisspeptin level in PCOS and non-PCOS patients. Subject, Materials, and Method: This is a cross-sectional study performed in the High Institute for Infertility Diagnosis and Assisted Reproductive Technologies, Al-Naharin University, from September 2020 to July 2021. The study includes a sample of (80) infertile Iraqi women aged (18–40 years) scheduled for an intracytoplasmic sperm injection program after being stimulated by GnRH antagonist protocol. On the day of oocytes retrieval, blood and follicular fluid samples were drawn from each participant to measure kisspeptin level using ELIZA technique. Results: In PCOS patients, significant positive correlations were found between recombinant FSH medication with serum and follicular fluid kisspeptin level (p is less than 0.05). Serum kisspeptin was insignificantly higher in the PCOS patient (p = 0.838) while follicular fluid kisspeptin was insignificantly lower in the PCOS women (p = 0.651). There were significant correlations between serum and follicular kisspeptin in the PCOS and the control groups (p = 0.000) and (p = 0.002), respectively. Conclusions: Serum and follicular fluid kisspeptin are positively correlated with the cumulative recombinant FSH medication used by infertile polycystic ovarian syndrome women undergoing ICSI, addressing the possible role of kisspeptin in improving the pregnancy rate in PCOS patients with a lower incidence of ovarian hyperstimulation syndrome.