Follicular Fluid Samples Research Articles

Validation of the French IVF guidelines during the COVID-19 pandemic

Research questionIs a symptom questionnaire as per the French IVF guidelines adequate for screening patients during the COVID-19 pandemic? DesignPatients planning IVF from June 2020 to February 2021 were included in the study. In compliance with French IVF guidelines, all patients fever-free on the day of oocyte retrieval were screened for risk of COVID-19 by completing a symptom questionnaire after being counselled regarding the importance of a COVID-19-free medical practice. Patients with IVF planned between June and September 2020 only completed the questionnaire (group 1), while those planning IVF after September 2020 also underwent the RT-PCR test for SARS-CoV-2 RNA (group 2). Cycle cancellation rates between groups were compared. Group 1 patients consented for follicular fluid testing for SARS-CoV-2 and an interview after cycle completion to determine COVID-19 exposure during the 6 months before and after retrieval. ResultsCycle cancellation rates for groups 1 and 2 were 0% (0/214) versus 1.4% (8/577), respectively, (P = 0.116). All 183 follicular fluid samples from group 1 were negative for SARS-CoV-2 RNA. Of 171 patients interviewed post-IVF, 16 (93.4%) developed COVID-19 symptoms or a positive real-time PCR (RT-PCR) RT-PCR test, but none within 2 months pre- or post-retrieval. ConclusionsThese results provide reassurance that, consistent with the COVID-19 French IVF guidelines, use of a symptom questionnaire is effective in screening patients planning to undergo IVF. Failure to detect viral RNA in any follicular fluid sample does not negate the possibility that follicular fluid is a viral reservoir. However, the findings provide reassurance that the follicular environment in this study's carefully screened population was COVID-free.

Metal elements associate with in vitro fertilization (IVF) outcomes in 195 couples

PurposeEnvironmental factors may affecting reproductive function reduction and embryonic development. Couples who are exposed to heavy metals for a long time may affect the outcome of in vitro fertilization (IVF). To evaluate the effect of elements on IVF outcomes, a total of 195 couples undergoing IVF were included in this study. MethodsElements including V, Cr, Mn, Ni, Cu, Zn, As, Mo, Cd, Ba, Hg, Tl, Pb were measured in serum and follicular fluid (FF) samples of female and semen samples of male by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Multiple linear regression were applied to evaluate the association between metal elements and semen quality parameters and the number of oocytes in MII stage. Poisson regression and the robust variance estimation of the generalized estimation equation were used to evaluate the association between elements and IVF outcomes. ResultsThe statistical results showed that Cr had a significant negative correlation with total sperm concentration (TSC) and total motile sperm count (TMC), the correlation coefficients were -0.52 (-0.27∼1.43) and -0.4(-1.24, 0.45), respectively. At the same time, Ba was significantly correlated with TSC and TMC, the correlation coefficients were 0.1(-0.15∼0.34) and 0.12(-0.13, 0.36), respectively. Cr, Ba and Pb in follicular fluid (FF) had a significant positive correlation with the number of oocytes in MII stage. The correlation coefficients were 3.15 (0.79, 5.52), 1.54 (-0.27, 3.36), 12.27 (7.49, 17.04). The Tl level of FF was significantly associated with the high probability of blastocyst formation and high-quality blastocysts (RR: 2.83, 95 % CI: 0.92∼7.95; RR: 3.12, 95 % CI: 0.64, 12.84). The Hg level (RR: 3.98, 95 % CI: 0.78∼14.77) and the Ba level in serum (RR: 12.75 95 % CI: 1.31∼89.71) were significantly correlated with high-quality blastocysts. The levels of Ni, Cu, Mo in seminal plasma of men were significantly correlated with blastocyst formation and high-quality blastocysts (RR values were all greater than 1.5). In addition, the level of Ba was significantly correlated with the high probability of blastocyst formation (RR: 1.7, 95 % CI: 1.14∼2.52). ConclusionOur results reveal that Cr, Ba and Pb may affect TSC, TMC and MII oocytes. Moreover, Ba, Cr, As, Hg and Tl in serum and Mo in seminal plasma were related to fertilization results, good embryos, blastocyst formation, high-quality embryos, and pregnancy and live birth rates. Tl in FF may related to the quality of embryonic development, Ba was an important risk factor which closely related to the outcomes of IVF in both male and female. Through our detection and statistical analysis of clinical samples, it is shown that although not all elements will affect the outcome of IVF the key elements we have selected need to arouse our attention, which benifit to the diagnosis and prevention of clinical infertility.

Vitamin D and anti-Müllerian hormone concentration in human follicular fluid individually aspirated from all patient follicles

Objective The main aim of this prospective study was to investigate the relationship between intrafollicular vitamin D and anti-Müllerian hormone (AMH) concentration and its impact on oocyte quality and developmental competence. Methods The analysis was performed on 208 follicular fluid (FF) samples obtained from 33 patients undergoing ovarian stimulation as part of in vitro fertilization (IVF) treatment that included intracytoplasmic sperm injection. Results Our study shows that vitamin D concentration in FF varies according to the developmental stage of the oocyte and corelates with embryo development status on day 3, while AMH concentration in FF is not correlated with the developmental potential of an oocyte. We demonstrated that the levels of vitamin D and AMH were higher in FF than in serum. Moreover we showed that AMH and vitamin D levels were positively correlated in FF but not in serum. Conclusion FF-AMH levels do not appear to be a suitable as noninvasive test of the developmental potential of an oocyte, while FF-vitamin D level can be used to evaluate whether embryos obtained from particular oocytes have potential of reaching the third day of culture. However, our results encourage further research to be carried out on a larger number of patients and testing additional components found in FF such as androgens.

Resistin and chemerin levels in follicular fluid of infertile women with endometriosis undergoing ICSI

Chemerin and resistin are two adipocytokines involved in inflammatory processes that may paly a role in the development of endometriosis. The purpose of the current study was to examine the levels of chemerin and resistin in the follicular fluid (FF) of endometriosis patients and additionally, assess the association of FF chemerin and resistin with the severity of endometriosis and the number of mature oocyte and embryos. A total of 80 reproductive-aged women who underwent intracytoplasmic sperm injection and embryo transfer were evaluated in this study. FF samples were obtained from subjects with (n = 40) and without endometriosis (n = 40). The concentrations of chemerin and resistin were examined using ELISA. The Resistin FF level was significantly (p-value=.03) higher in women with endometriosis than women without endometriosis, while the effect size was medium (d = 0.47). There was no significant difference in Chemerin concentration between the two groups of this study. Results also showed a tendency towards the increase of FF values of resistin and chemerin in endometriosis patients with stage III-IV than stage I-II; however, this difference was not significant. No significant correlations were found between FF levels of resistin and chemerin with the number of mature oocyte and embryos. Thus, elevated FF resistin levels may be associated with endometriosis. IMPACT STATEMENT What is already known on this subject? To our knowledge, no studies have investigated chemerin levels in follicular fluid of infertile women with endometriosis undergoing ICSI. Although follicular fluid levels of resistin have been previously studied in endometriosis, it has not been studied yet whether its level is associated with the number of oocyte and embryos. What do the results of this study add? Our results indicate that significantly increased resistin levels in follicular fluid may be associated with the presence of endometriosis. What are the implications of these findings for clinical practice and/or further research? The findings are promising in that significantly increased resistin levels may add to the knowledge of the pathophysiology of endometriosis.

Oxidative stress and DNA damage status in couples undergoing in vitro fertilization treatment.

SummaryThis study examined the status of oxidative stress in 599 couples undertaking in vitro fertilization (IVF) treatment and its association with reproductive hormones, smoking, and outcomes. Oxidative stress biomarkers such as malondialdehyde, 8-hydroxy-2-deoxyguanosine, hydrogen peroxide (H2O2), catalase (CAT), and total antioxidant capacity (TAC) were determined in follicular fluid and seminal plasma. Tail moment (TM) was used to evaluate DNA damage in the sperm and granulosa cells. Reproductive hormones in serum and cotinine (COT) in urine, follicular fluid, and seminal plasma samples were determined. Separate multivariate linear regression was used to assess associations between levels of each oxidative stress biomarker and each hormone and smoking parameter (modeled as natural log-transformed). The findings indicate that some oxidative stress and DNA damage biomarkers played a role in disrupting certain reproductive hormones in women and their male partners either by overproducing reactive oxygen species or reducing antioxidant defense capacity. Although women were nonsmokers, COT levels > 50 and 10 µg/L in urine and follicular were observed in 5.7 and 1.7%, respectively. Levels of follicular fluid COT were positively associated with H2O2 and TM. We used log-binomial multivariate regression to estimate relative risks for the association between oxidative stress/DNA damage and IVF binary outcomes (fertilization rate > 50%, biochemical pregnancy, clinical pregnancy, and live birth). An increase in the CAT levels of follicular fluid was associated with a 48 and 41% decrease in the risk of poor fertilization rate (≤50%) and unsuccessful live birth, respectively. After the models were adjusted for hormonal factors, the associations remained the same, except that the elevated TAC in follicular fluid became significantly associated with a decrease of 42% in the risk of poor fertilization rate (≤50%). The higher antioxidant activity (CAT and TAC) in follicular fluid might positively impact specific IVF outcomes.Lay summaryOxidative stress occurs when antioxidant molecules are insufficient in the body to destroy free radicals that can damage the cells, proteins and DNA, causing different health conditions, including infertility. The role of oxidative stress in female infertility has not received as much attention as male infertility, and research is still limited. This study explored whether the overproduction of free radicals can impact the success of in vitro fertilization (IVF) treatment using several biological markers such as hydrogen peroxide, catalase, and total antioxidant capacity. Our findings revealed that the high antioxidant levels in the fluid surrounding the egg were linked with a high fertilization rate. Additionally, oxidative stress status in couples was associated negatively with several reproductive hormones and smoking status. Biomarkers of oxidative stress and DNA damage might have potential applications in evaluating IVF patients’ clinical characteristics such as causes of infertility, hormonal profile, fertilization rate, implantation and live birth.

Extracellular microRNAs: key players to explore the outcomes of in vitro fertilization

BackgroundMicroRNAs (miRNAs) are small RNA molecules that modulate post-transcriptional gene regulation. They are often used as promising non-invasive biomarkers for the early diagnosis of cancer. However, their roles in assisted reproduction are still unknown.MethodsThis prospective study was designed to evaluate the expression profiles of seven extracellular miRNAs (miR-7-5p, miR-202-5p, miR-378-3p, miR-224, miR-320a, miR-212-3p, and miR-21-5p) in human follicular fluid (FF) to explore the outcomes of in vitro fertilization (IVF). Of 255 women, 145 were without polycystic ovary syndrome (PCOS), and their ovarian assets were normal (NOR), while 110 were with normo-androgenic PCOS.ResultsThe combination of six FF miRNAs expression profile discriminated between PCOS and NOR women with a sensitivity of 79.2% and a specificity of 87.32% (AUC = 0.881 [0.61; 0.92], p = 0.001). MiR-202-5p significantly had a lower abundance level, and miR-378-3p had a high abundance level in pooled FF samples from patients treated with human menopausal gonadotropin (hMG) than those treated with recombinant follicle-stimulating hormone (rFSH) (p < 0.001). Our results showed that miRNA-320a was significantly different in top-quality embryos versus non-top-quality embryos on day 3 in NOR patients with a sensitivity of 80% and specificity of 71%, (AUC = [0.753 (0.651; 0.855)], p = 0.001). For clinical pregnancy outcome prediction, FF miRNA-21 exhibited high sensitivity (74.8%) and specificity (83.7%) with the AUC value of 0.774 (0.682; 0.865).ConclusionConclusively, our results provide evidence that miR-7-5p, miR-378-3p, miR-224, miR-212-3p were a differentially high expression in normo-androgenic PCOS patients than NOR patients. While miRNA-320a was significantly different in top-quality embryos versus non-top-quality embryos on day 3 (p = 0.001). The expression level of FF miR-212-3p was significantly related to the probability of embryos to develop into a high-quality blastocyst in patients with normal ovarian reserve.

Roles of miR-10a-5p and miR-103a-3p, Regulators of BDNF Expression in Follicular Fluid, in the Outcomes of IVF-ET.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, plays critical roles in the physiological process of oocyte mature and IVF outcomes of patients with infertility. However, the regulation of BDNF expression in the microenvironment surrounding the oocyte is still unknown. We initially predicted some microRNA (miRNA) candidates targeting bdnf with a series of bioinformatics analysis tools to determine the underlying regulatory mechanisms of BDNF, particularly the effect of miRNAs on BDNF expression. Then, we assessed whether the expression of these 14 selected miRNAs was negatively associated with BDNF expression in follicular fluid (FF) samples obtained from mature (>18 mm) or immature (<15 mm) follicles. Finally, we used the candidate miRNAs, miR-103a-3p and miR-10a-5p, to further investigate the relationship between their expression in FF and the outcomes of infertile patients undergoing IVF–ET treatment. The results of the bioinformatics analysis revealed 14 miRNAs that might directly regulate BDNF expression and might have a close relationship with oocyte development. BDNF was expressed at significantly lower levels in FF from immature follicles than in FF from mature follicles, and only the expression of miR-103a-3p and miR-10a-5p was negatively correlated with BDNF expression in FF. Moreover, in another cohort of 106 infertile women undergoing IVF-ET treatment, miR-103a-3p or miR-10a-5p expression predicted the developmental status of the corresponding oocytes in which high expression of miR-103a-3p or miR-10a-5p resulted in a poor quality of embryo on days 3 and 5 during the IVF-ET treatment. In conclusion, our study is the first to show that miR-103a-3p or miR-10a-5p negatively affects the maturation of oocytes by regulating the expression of BDNF in human FF. Additionally, the expression levels of miR-103a-3p or miR-10a-5p in FF may predict the outcomes of IVF, which are helpful for improving embryo selection and consequently the IVF success rate in the clinic.

Cannabis alters epigenetic integrity and endocannabinoid signalling in the human follicular niche.

STUDY QUESTIONDo phytocannabinoids (PCs) affect follicular endocannabinoid signalling and the epigenome in the surrounding granulosa cells (GCs)?SUMMARY ANSWERExposure to PCs increases the expression of endocannabinoid receptors and reduces DNA methylation enzyme expression and global DNA methylation in naïve GCs.WHAT IS KNOWN ALREADYCannabis plant derivatives, known as PCs, are used for medicinal and recreational purposes. The main PC, tetrahydrocannabinol (THC), is the third most commonly used substance by women of childbearing age, hence knowledge of the effect it has on reproduction is of utmost importance. THC exerts its effects via receptors of the endocannabinoid system (ECS) and can interfere with folliculogenesis, oocyte development and ovulation. Endocannabinoids have been measured in follicular fluid (FF) obtained during oocyte retrieval and are implicated in controlling folliculogenesis. It has been established that in the placenta, PCs disrupt endocannabinoid homeostasis via impairment of the synthetic and degrading enzymes, leading to a net increase of endocannabinoid levels. Finally, previous studies have shown that THC alters methylation and histone modifications in sperm, brain and blood cells.STUDY DESIGN, SIZE, DURATIONThis study included an in vivo cohort assessment of cannabis exposure and its effects on the follicle and in vitro assays conducted to validate the in vivo findings and to explore possible mechanisms of action.PARTICIPANTS/MATERIALS, SETTING, METHODSA total of 318 FF samples, from 261 patients undergoing IVF treatment at a private fertility clinic who consented for biobanking biological waste material between January 2018 and July 2019, were included in this study. Concentrations of PCs and endocannabinoids were assessed in FF by liquid chromatography-mass spectrometry (LC-MS/MS). Exposure to PCs was determined based on these measured levels. Levels of both endocannabinoid receptors (CB1R, CB2R) and the de novo DNA methylating enzyme, DNMT3b, in GCs were assessed by flow cytometry both in vitro and in vivo and global DNA methylation was assessed in vitro by ELISA. In vivo effects were assessed by comparing samples positive for at least one PC, with samples negative for all measured PCs. In vitro effects were determined in naive GCs, obtained concurrently with FF samples that had tested negative for all PCs. These GCs were treated with different combinations of the main three PCs.MAIN RESULTS AND THE ROLE OF CHANCEOverall, 17 patients (6.4%) were positive for cannabis consumption. Furthermore, the prevalence of cannabis positivity in the FF increased from 4% of the tested samples that were collected prior to national legalisation in October 2018 to 12% of those collected following legalisation. Of note, 59% of patients who tested positive for PCs (10 of 17) reported previous or ongoing exposure to cannabis upon their initial intake. Endocannabinoid levels were not affected by the presence of PCs. CB2R was more prevalent than CB1R in GCs and its expression increased following acute and chronic in vitro exposure to PCs. The expression of DNMT3b and global methylation decreased following exposure, suggesting that cannabis may affect the epigenome in the follicular niche. The acute changes were sustained throughout chronic treatment.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONOur study is limited by lack of details regarding mode, frequency and timing of PC consumption. Moreover, we were not able to adequately assess the effect of PCs on immediate or long-term clinical outcomes, due to the small sample size and the lack of follow up. Future, large-scale studies should focus on assess the clinical implications of cannabis exposure, validate our findings, and determine to what extent cannabis affects the epigenome ovarian follicle and the developing oocyte.WIDER IMPLICATIONS OF THE FINDINGSTo our knowledge, this is the first study measuring PCs in FF by LC-MS/MS. We show that consuming cannabis alters the ECS in the developing follicle, and directly affects DNMT expression and global DNA methylation levels. Cannabis legalisation and use is increasing worldwide, therefore further understanding its role in female fertility and folliculogenesis is critical.STUDY FUNDING/COMPETING INTEREST(S)All funding was provided by CReATe Fertility Centre through the reinvestment of clinical earnings. The authors declare no competing interests.

The Effects of Adiponectin on Infertile Women Undergoing IVF/ICSI Treatment and on Human Granulosa Cells

Adiponectin, one of the most abundant adipocyte-secreted protein, has been involved in female reproductive regulation. This study aimed to 1) compare serum adiponectin levels in various phases of in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment including Phase I (the beginning of gonadotropin stimulation), Phase II (around 8 days after gonadotropin stimulation), and Phase III (on the day of ovum pick-up) between success and unsuccess subjects; 2) compare follicular fluid (FF) adiponectin levels between success and unsuccess groups; 3) compare serum adiponectin levels among different phases of IVF/ICSI treatment in success and unsuccess groups; 4) compare the levels of adiponectin between serum and FF; and 5) investigate the effects of adiponectin on mRNA expressions of follicle stimulating hormone receptor (FSHR) and CYP19A1 (aromatase) in human granulosa-like tumor cell line (KGN) (n=3). In the human study, recruited participants (n=30) with age of 26-40 years were enrolled between April 2018 - May 2019. Blood samples were collected at Phases I, II, and III while FF samples were collected at Phase III. Adiponectin levels were comparable between success and unsuccess subjects in both serum (all phases) and FF (Phase III). Furthermore, serum adiponectin levels were comparable among Phase I, II, and III in success and unsuccess groups. In Phase III, serum adiponectin showed positive correlations with serum adiponectin in Phase I and II and serum FSH in Phase I. Interestingly, adiponectin levels in FF were significantly lower than serum at Phase III in unsuccessful pregnancies but were comparable in successful pregnancies. Moreover, FF adiponectin had a negative correlation with serum LH at Phase III in success subjects. In the KGN cell study, adiponectin had no effects on FSHR and CYP19A1 (aromatase) mRNA expression compared with control. In conclusion, high adiponectin levels in serum compared to FF might contribute to unsuccessful IVF/ICSI treatment.

Antioxidant activities and lipid peroxidation status in human follicular fluid: age-dependent change

Maternal age is a significant factor influencing in vitro fertilization (IVF) outcomes. Oxidative stress (OS) is one of the major causes of age-related cellular and molecular damage. The purpose of this work was to investigate the correlation between maternal age with intrafollicular antioxidants and OS markers in follicular fluid (FF), and also to determine the OS status in patients of advanced age. This study was a prospective study including 201 women undergoing IVF whose age was between 24 and 45 years old. FF samples were obtained from mature follicles at the time of oocyte retrieval. After treatment of FF, lipid peroxidation levels (MDA) and enzyme activities such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione (GSH) level were evaluated using spectrophotometry. The results indicated that the age cutoff point for increasing the MDA level was fixed at 37 years, allowing the study to be differentiated into two age groups. Group I included patients whose age was less than 37 years, and group II included patients whose age was greater than or equal 37 years. Statistical analysis revealed that MDA and GSH levels and GR activity were significantly higher in group II compared with group I. The SOD and CAT activities were significantly less in group II compared with group I. We concluded that from 37 years old a reproductive ageing was accompanied by a change in the antioxidant pattern in FF that impaired reactive oxygen species scavenging efficiency.

The effect of plastic bottled water consumption on outcomes of ICSI cycles undertaken for unexplained infertility

Research questionDo bisphenol A (BPA) levels in maternal urine, serum and follicular fluid affect embryo quality and intracytoplasmic sperm hinjection (ICSI) cycle outcomes in women with unexplained infertility? DesignProspective study conducted between 1 April 2019 and 30 September 2019. The study cohort consisted of 82 women aged between 23 and 33 years who underwent intracytoplasmic sperm injection owing to unexplained infertility and provided urine, blood and follicular fluid samples on the day of oocyte retrieval. Consumption of drinking water from plastic carboys or bottles at home were considered as chronic BPA exposure. Demographic features and IVF outcomes of the patients were collected. ResultsAmong the 82 women with unexplained infertility, clinical pregnancy was achieved in 22 (26.8%) patients after the IVF and embryo transfer cycle. The patients who consumed tap water had statistically significantly lower BPA values in three body fluids compared with patients who consumed plastic bottled water (all P < 0.001). Women who had grade 1 embryos transferred had lower serum BPA values than women who had grade 2 embryos transferred (10.8 ± 5.2 versus 26.9 ± 22 ng/ml, P = 0.003). Serum and follicular fluid BPA levels were statistically significantly higher in women who failed to achieve clinical pregnancy (P < 0.001, P = 0.006, respectively) and obtain a live birth (both P = 0.007). ConclusionsA negative relationship was found between serum and follicular fluid BPA levels and embryo quality, clinical pregnancy and live birth in these women. In addition, the BPA levels of women who consume tap water at home were lower than those who use plastic bottled water.

Association between follicular fluid phthalate concentrations and extracellular vesicle microRNAs expression.

Are phthalate metabolite concentrations in follicular fluid (FF) associated with the expression of extracellular vesicle microRNAs (EV-miRNAs)? Phthalate metabolite concentrations are associated with the expression of EV-miRNA and their associated pathways in FFs. Phthalate metabolites were recently detected in FF. Urinary phthalate metabolite concentrations alter the expression of EV-miRNAs in FF. Prospective study including 105 women recruited between January 2014 and August 2016 in a tertiary university-affiliated hospital. We assessed FF concentrations of 12 phthalate metabolites. EV-miRNAs were isolated from aliquots of the same FF, and their expression profiles were measured using a human miRNA panel. Associations between EV-miRNAs that were present in >50% of the samples and phthalate metabolites that were measured in >74% of the FF samples were tested. Genes regulated by EV-miRNAs that were found to be significantly (false discovery rate q-value < 0.1) correlated with FF-phthalates were analyzed for pathways linked with female fertility using miRWalk2.0 Targetscan database, DAVID Bioinformatics Resources and Kyoto Encyclopedia of Genes and Genomes (KEGG). Of 12 phthalate metabolites, 11 were measured in at least one FF sample. Mono (6-COOH-2-methylheptyl) phthalate (MCOMHP), mono-2-ethyl-5-carboxypentyl phthalate (mECPP), mono-n-butyl phthalate (MnBP), monobenzyl phthalate (MBzP), mono-isobutyl phthalate (MiBP), monoethyl phthalate (MEP) and mono (7-COOH-2-methyloctyl) phthalate (MCOMOP) were detected in more than 74% of the samples. Of 754 EV-miRNAs tested, 39 were significantly associated either with MEP, MBzP, MCOMOP, MCOMHP and/or with mECPP, after adjusting for multiple testing (P < 0.05). KEGG-based pathway enrichment analysis of the genes regulated by these miRNAs showed that these EV-miRNAs may be involved in pathways related to ovary or oocyte development, maturation and fertilization. The use of miRNA panel array limits the number of potential relevant miRNAs. Moreover, several of the phthalate metabolites examined may be biased due to internal (enzymatic activity) or external (contamination in medical interventions) causes. Phthalate metabolites may alter follicular EV-miRNAs profile and thus impair pathways that are involved with oocyte development, maturation and fertilization. Our results contribute to understanding of possible mechanism(s) in which endocrine disruptor chemicals interfere with female fertility. This work was supported by the National Institutes of Environmental Health Sciences [Grant R21-ES024236]; and Environmental Health Fund, Israel [Grant 1301], no competing interests. N/A.

Nitrite and Nitrate Levels in Follicular Fluid From Human Oocyte Donors Are Related to Ovarian Response and Embryo Quality.

Nitric oxide, a key regulatory molecule in the follicular fluid, has been suggested as a possible biomarker to predict ovarian response in stimulated cycles and the potential of the retrieved oocytes for developing high-quality embryos. Nevertheless, a consensus on whether or not nitric oxide can help in this context has not been reached. We simultaneously measured the oxidation products of nitric oxide, nitrite, and nitrate, via high-performance liquid chromatography (HPLC)-UV in follicular fluid samples from 72 oocyte donors. We found no associations of follicular fluid nitrite, nitrate, total nitric oxide, or nitrate/nitrite ratio with total or metaphase II (MII) oocyte yield. However, nitrite and nitrate levels were related to the yield of MII oocytes when this outcome was expressed as a proportion of all oocytes retrieved. The adjusted MII proportion in the lowest and highest nitrite levels were 68% (58–77%) and 79% (70–85%), respectively (p, linear trend = 0.02), whereas the adjusted MII proportion in extreme tertiles of nitrate levels were 79% (70–85%) and 68% (57–77%) (p, linear trend = 0.03). In addition, nitrate levels showed a suggestive inverse correlation with embryos with maximum or high potential of implantation (p = 0.07). These results suggest that the follicular fluid concentrations of nitrite and nitrate may be a useful tool in predicting how healthy oocyte donors respond to superovulation and the implantation potential of the embryos produced from their oocytes.

Steroid Hormones of Follicular Fluid and the Outcome of in Vitro Fertilization

Abstract One of the succes factors of biomedically assisted fertilization is the regular maturation of one or more oocytes. The quality of the oocytes is significantly influenced by the environment in which it is located, the so-called „microenvironment” that includes cumulus cells, follicular fluid in which hormones and growth factors involved in its growth and development are secreted. The main aim was to examine whether the concentration of steroid hormones in the follicular fluid affects the rate of fertilization and the outcome of the in vitro fertilization process itself. The study included 31 patients who were included in vitro fertilization procedure at the Department for Biomedically Assisted Fertilization, Clinic for Gynecology and Obstetrics, Clinical Center Kragujevac. We used follicular fluid as biological material for analysis. Examination of the obtained follicular fluid and collection of oocytes under a stereomicroscope was done in the embryological laboratory at the Department. Biochemical parameters of follicular fluid were analyzed in the Department for Laboratory Diagnostics, Clinical Center Kragujevac. In vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) methods were used as the fertilization method. The criteria of the Istanbul Consensus of Clinical Embryologists were used as a reference framework for embryo quality assessment. Pregnancy was confirmed by a positive serum level of the hormone β-hCG 14 days after embryo transfer. A software package SPSS 20 was used for statistical data processing. The results of the analysis of follicular fluid samples show that there was no statistically significant difference in the concentration of estradiol, progesterone and testosterone in follicular fluid in relation to fertilization rate and the outcome of in vitro fertilization. Based on our results, it can be concluded that the concentration of steroid hormones did not affect fertilization rate and the outcome of in vitro fertilization.

Von Willebrand factor in follicular fluid in women undergoing IVF/ICSI: a “ghost” associated with oocyte and embryo quality

Introduction: The purpose of the present study to determine the presence of Von Willebrand factor (vWF) in the follicular fluid of women undergoing IVF/ICSI and to investigate the association of its concentration with oocyte and embryo quality. Methods: We conducted a prospective observational study based in women undergoing in vitro fertilization treatment. vWF concentrations in follicular fluid samples collected during transvaginal oocyte retrieval were measured using an enzyme-linked immunosorbent assay (Elisa). They were also analyzed by the VIDAS® vWF method for confirmation of the results. The latter is an automated quantitative test for the measurement of vWF in human plasma, using the ELFA technique (enzyme-linked fluorescent assay). vWF concentrations were correlated to oocyte and embryo quality. Results: Contrary to previous reports, vWF was determined in follicular fluid. A higher percentage of good quality oocytes were found in the presence of lower vWF follicular fluid levels. Similarly, better quality embryos were derived from oocytes retrieved from follicles with lower vWF follicular fluid levels. Conclusions: Contrary to existing evidence to date, vWF was found in the follicular fluid of women undergoing IVF/ICSI treatment. In addition, the quality of oocytes and embryos was adversely related to vWF follicular fluid levels. In fact, a better outcome of IVF/ICSI treatment was suggested when the levels of vWF in follicular fluid were low. In this context, vWF follicular fluid levels could be used as a prognostic factor for oocyte and embryo quality.

Associations between medication use and phthalate metabolites in urine and follicular fluid among women undergoing in vitro fertilization

BackgroundPhthalates, which are used as excipients of drugs, have been related to adverse reproductive outcomes. However, the relationships between medication use and phthalate exposure among women undergoing in vitro fertilization (IVF) have not been studied. ObjectiveTo investigate the associations between the medication intake and phthalate metabolites in urine and follicular fluid (FF). MethodEight phthalate metabolites were measured in urine and FF samples from 274 women undergoing IVF using liquid chromatography-tandem mass spectrometry. Information on recent medication intake was obtained via interview by trained staff. We constructed generalized linear regression models to examine the associations of medication intake with phthalate metabolite concentrations and dose-response relationships between the number of medicines used and metabolite concentrations in two matrices. ResultsFour of 10 drugs were used by more than 10% of the participants, including vitamins (23.0%), traditional Chinese medicine (TCM, 22.3%), antioxidants (12.4%) and amoxicillin (10.2%). Participants who had used TCM had 26.0% (95% CI: 0.0, 58.8%), 32.6% (95% CI: 4.2, 68.8%) and 32.3% (95% CI: 2.6, 70.6%) higher urinary mono-n-butyl phthalate (MBP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) concentrations, respectively, than those who had not. Antioxidant intake was associated with a 30.6% (95% CI: −48.5, −6.6%) decrease in the urinary MBP concentration. Compared with non-users, women who reported the use of medicines had 53.2% (95% CI: 2.7, 128.5%) higher concentrations of MMP and a 37.7% (95% CI: −60.7, −1.5%) lower level of MBP in FF, respectively. ConclusionOur data suggest that the intake of some medications may increase phthalate exposure among women undergoing IVF.

Circulating MMP-7 and VEGF as potential predictive biomarkers for recurrent implantation failures.

Recurrent implantation failure (RIF) is considered to be one of the major limiting factors of assisted reproductive technology (ART) programme success. The current study focused on the investigation of matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), cytokines and cell adhesion molecules in peripheral blood (PB) and follicular fluid (FF) obtained from 44 women aged between 25 and 39 years old and undergoing intracytoplasmic sperm injection (ICSI). These women were divided into two groups: 22 RIF women with embryo implantation failures after the transfer of at least four fresh or frozen-thawed good quality embryos in a minimum of three ICSI cycles, and 22 ICSI success women (controls) who achieved a clinical pregnancy at their first ICSI attempt. The PB and FF samples were obtained from each patient on the day of oocyte retrieval. MMP-1, -2, -3, -7, -9, TIMP-1, -2, vascular endothelial growth factor (VEGF), leukaemia inhibitory factor (LIF), vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecules 1 (ICAM1) were analyzed using enzyme-linked immunosorbent assay of PB and FF. Our results showed significant decreases in PB MMP-7 and PB VEGF in the RIF group compared with controls [281.11 (33-614) pg/ml vs 119.92 (27-441) pg/ml; P-value = 0.030] and [82.54 (25.94-210.20) pg/ml vs 30.93 (13.62-193.33) pg/ml; P-value = 0.022; respectively]. Receiver operating characteristic (ROC) curve analysis showed informative area under the curve values for PB MMP-7, as well as for PB VEGF, making them able to be proposed as biomarkers of the RIF. Therefore, circulating MMP-7 and VEGF seem to play an interesting role in embryo implantation in in vitro fertilization (IVF)/ICSI cycles and could be proposed as circulating biomarkers of the RIF. These results could be helpful for clinicians and patients to choose the best rescue strategy and treatment to minimize implantation failure in women undergoing IVF/ICSI procedures after the first attempt.

Impact of Nonylphenols and Polyhalogenated Compounds in Follicular Fluid on the Outcome of Intracytoplasmic Sperm Injection.

Endocrine-disrupting chemicals (EDCs) interfere with the mammalian hormone system and alter its endo- and paracrine regulation. The goal of the present study was to examine the presence of 14 EDCs, including the technical mixture of nonylphenols and Mirex, in human follicular fluid (FF) and to find a potential correlation between endocrine active substances and a possible impact on female fertility. Furthermore, potential sources of EDC exposition regarding patients' lifestyle and socioeconomic factors were investigated. Human FF was collected from a total of 210 women undergoing intracytoplasmic sperm injection-treatment cycles because of male subfertility. The presence of EDCs was analyzed using gas chromatography coupled with mass spectrometry. Thirteen of the 14 investigated EDCs were present in every FF sample; compounds with the highest concentrations in FF were nonylphenol and Mirex. Nearly all kinds of EDCs led to significantly reduced maturation and fertilization rate. No significant influence of EDC concentration on the clinical pregnancy rate was observed for neither of the analyzed EDCs. Patients who obtained their clothes and textiles at fashion discounters displayed a higher amount of EDCs in their FF. In contrast, patients' residential area, source of food products, and nicotine or caffeine consumed were not associated with EDC accumulation. Clinicaltrials.gov NCT01385605 (11 July 2011).

Characterization of leptospiral DNA in the follicular fluid of non-pregnant cows.

Bovine leptospirosis is mainly characterized by reproductive disorders. Contamination of the oocyte was previously demonstrated in vitro, resulting in some apparent damage. However, it is not clear whether it occurs under natural conditions. The present study aimed to characterize the presence of pathogenic Leptospira DNA in the ovarian follicles of non-pregnant cows. Follicular fluid samples were collected from 65 animals and subjected to lipL32 PCR and secY sequencing. In total, seven of 65 (10.8%) were positive, indicating a possible early infection of the oocyte. Moreover, secY sequencing identified L. interrogans and L. santarosai, both very closely related to bovine strains from the Sejroe serogroup (Hardjoprjitno and Guaricura). We demonstrated that ovarian follicles can also be infected. It was hypothesised that ovarian infection can contribute to embryonic death, causing reproductive failure and estrus repetition. In the present study, we show that the organism identified in the follicle is closely related to one that is known to be associated with reproductive disorders.

Elevated ovarian pentraxin 3 in polycystic ovary syndrome

PurposePentraxin 3 (PTX3) plays a crucial role in cumulus expansion and fertilization. The ovarian PTX3 level in polycystic ovary syndrome (PCOS) remains uncertain. In the present study, we investigated the follicular PTX3 levels and found the influence of reproductive hormones on ovarian PTX3 concentration.MethodsThis study was based on 204 healthy-weight women (102 PCOS and 102 normal ovulating subjects) undergoing in vitro fertilization (IVF). Follicular fluid (FF) was collected during oocyte retrieval. The PTX3 levels and other hormone levels in FF samples were analyzed by enzyme-linked immunosorbent assay (ELISA).ResultsThe PTX3 level in the follicle was significantly higher in the healthy-weight PCOS women than controls. Positive correlations were found between ovarian PTX3 level and the existence of PCOS, cycle length, basal LH to FSH ratio and TT in serum, antral follicle count, and ovarian insulin and androgen level, and inverse correlations with the basal serum PRL and ovarian SHBG. In multivariant linear regression analysis, the presence of PCOS diagnosis, participants’ basal LH to FSH ratio, and ovarian androstenedione level were the main predictors of ovarian PTX3 level among the enrolled subjects.ConclusionElevated ovarian PTX3 level supports the low-grade chronic inflammatory state in the follicles of PCOS. The existence of PCOS, disturbed pituitary gland, and ovarian hyperandrogenism might also be related to this state of low-grade chronic inflammation and could be a subject of further study.