Follicular Fluid Levels Research Articles

Serum and follicular fluid thyroid hormone levels and assisted reproductive technology outcomes

ObjectiveThe objective ofthis study was to assess the association between thyroid hormone (TH) levels in follicular fluid (FF) and serum and to determine whether THs impact assisted reproductive technology (ART) outcomes.MethodsThis study enrolled 299 women undergoing ART. Blood samples were drawn on the day of human chorionic gonadotrophin (HCG) administrationand analysed for thyroid-stimulating hormone (TSH), thyroxine(T4), triiodothyronine(T3),free T4 (fT4),free T3(fT3), thyroid peroxidase antibody (TPOAb) and thyroglobulin antibody (TGAb) levels. FF was obtained on the oocyte pick up (OPU) day and analysed forTSH, T4, T3, fT4, fT3, TPOAb, TgAb and estradiol levels.Results(1) There were significant positive correlations between serum and FF TH and thyroid autoantibody levels. Statistically significant differences were discovered in serum and FF levels of TSH (p ≤ 0.001), T4 (p ≤ 0.001), T3 (p ≤ 0.001), TPOAbs (p ≤ 0.001) and TGAbs (p = 0.021).(2) Serum T4 levels [121.9(104.8,140.8) vs 114.1(98.6,130.6) nmol/l, p = 0.026], serum fT4 levels[(19.0(17.7,21.8) vs 18.6(17.0,20.1) pmol/l, p = 0.026], serum T4/T3 ratios [62.5 (55.7, 66.2) vs 59.4 (53.4, 64.9), p = 0.029], FF fT4 levels [19.0(17.5,21.3) vs 18.1(16.8,19.9) pmol/l, p = 0.009] and FF T4/T3 ratios [52.6 (46.4, 57.3) vs 50.0 (43.7, 53.1), p = 0.004] were significantly higher in the successful pregnancy group than the implantation failure group.(3) Spearman’s rank correlation analysis revealed positive associations of both the FF T4/T3 ratio and serum TSH levels with the numbers of retrieved oocytes (total or MII) and embryos (fertilized, cleavage, and good quality).ConclusionsTH levels in FF are strongly correlated with those in serum on the HCG day, and THs on the HCG day may affect ART outcomes.

Increased Expression of KISS1 and KISS1 Receptor in Human Granulosa Lutein Cells-Potential Pathogenesis of Polycystic Ovary Syndrome.

Kisspeptins are a family of neuropeptides that are essential for fertility. Recent experimental data suggest a putative role of kisspeptin signaling in the direct control of ovarian function. To explore the expression of KISS1 and KISS1 receptor (KISS1R) in human granulosa lutein cells and the potential role of KISS1/KISS1R system in the pathogenesis of polycystic ovary syndrome (PCOS), we measured the concentration of KISS1 in follicular fluid, the expression of KISS1 and KISS1R in granulosa lutein cells, and the circulating hormones. The expression levels of KISS1 and KISS1R were significantly upregulated in human granulosa lutein cells obtained from women with PCOS. The expression levels of KISS1 in human granulosa lutein cells highly correlated with those of KISS1R in non-PCOS patients, but not in patients with PCOS, most likely due to the divergent expression patterns in women with PCOS. Additionally, the expression levels of KISS1 highly correlated with the serum levels of anti-Müllerian hormone (AMH). The expression levels of KISS1 and KISS1R, as well as the follicular fluid levels of KISS1, were not significantly different between the pregnant and nonpregnant patients in both PCOS and non-PCOS groups. In conclusion, the increased expression of KISS1 and KISS1R in human granulosa lutein cells may contribute to the pathogenesis of PCOS. The expression levels of KISS1 highly correlated with the serum levels of AMH. The KISS1 and KISS1R system in the ovary may not have a remarkable role in predicting the in vitro fertilization (IVF) outcome.

Melatonin induces progesterone production in human granulosa-lutein cells through upregulation of StAR expression.

Steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in ovarian steroidogenesis and progesterone (P4) synthesis. Melatonin and its receptors are expressed in human granulosa cells, and have been shown to influence basal P4 production. However, previous studies addressing the regulation of StAR expression by melatonin and its impact on P4 secretion yielded contradictory results. Here, we demonstrate that melatonin upregulates StAR expression in primary cultures of human granulosa-lutein (hGL) cells obtained from women undergoing in vitro fertilization (IVF). Using pharmacological inhibitors, we show that the stimulatory effect of melatonin on StAR expression is mediated via both MT1 and MT2 melatonin receptors. Melatonin exposure activates the PI3K/AKT signaling pathway and its inhibition attenuates the stimulatory effect of melatonin on StAR expression. Moreover, siRNA-mediated knockdown of StAR abolishes melatonin-induced P4 production. Importantly, clinical analyses demonstrate that melatonin levels in human follicular fluid are positively correlated with P4 levels in serum. By illustrating the potential physiological role of melatonin in the regulation of StAR expression and P4 production in hGL cells, our results may serve to improve current strategies used to treat clinical infertility.

Ovarian HBV replication following ovulation induction in female hepatitis B carriers undergoing IVF treatment: A prospective observational study.

Hepatitis B virus (HBV) can be found in ovarian tissues. This study compared HBV DNA levels in follicular fluid collected during oocyte retrieval with paired serum samples in HBV carriers after ovarian stimulation during IVF treatment for infertility. Sixty-four HBV carrier women referred to the Assisted Reproductive Units of two Hong Kong hospitals were recruited. At oocyte retrieval, the follicular fluid aspirated from the first follicle was collected for study. In 22 women, the first follicular fluid sample from both ovaries was similarly collected and studied. These women were also tested for liver function test and HBeAg. In 28 (43.8%) women, HBV DNA was detected in follicular fluid and the level correlated with serum levels (Spearman's correlation P<.001). There was concordant detection of HBV DNA in both ovaries, and the levels were significantly correlated (Spearman's correlation P=.029). In 40% of women with FF HBV DNA, the follicular fluid:serum ratio was >1.0, suggesting stimulation of HBV replication. These women also had significantly different liver function test results. Increased HBV replication exists in 40% of women with HBV DNA detected in follicular undergoing ovarian stimulation during IVF treatment.

Steroidogenic, Metabolic, and Immunological Markers in Dairy Cows Diagnosed With Cystic Ovarian Follicles at Early and Mid-Late Lactation.

The etiology of cystic ovarian follicles (COF) remains a conundrum with steroidogenic, immunological, and metabolic dysfunctions linked to its development. Studies suggest that COF development may occur as a result of disruption of the insulin signaling pathway and the severity of a negative energy balance in dairy cows, but mid to late lactation cows diagnosed with COF are unlikely to have issues with energy metabolism. Herein, we characterized the mRNA expression of steroidogenic (LHCGR, StAR, CYP11A1, 3β-HSD, CYP19A), immunological (IL-1β, IL-6, IL-8, TLR-4, TNF), and metabolic markers (IGF-1, IRS1) in follicular fluid; and plasma and follicular fluid levels of E2, IL-1β, glucose, and NEFA in early and mid-late lactation COF cows. Lactating dairy cows were diagnosed as having COF (n = 11, follicle >20 mm persistent for 7 days, absence of corpus luteum, and flaccid uterus) while 11 herdmates cycling with a dominant follicle were classified as the control cows. Cows diagnosed with COF were classified as early lactation (COF-E, n = 5) cows, <35 days in milk (DIM); or mid-late lactation (COF-M/L, n = 6), ≥118 DIM cows. Results revealed that mRNA expression StAR was greater (P < 0.01) in COF-E cows than COF-M/L cows and the control cows. The mRNA expression CYP19A1 was lower (P < 0.01) in COF-E cows and COF-M/L cows than in the control cows. The mRNA expression IL-6 and IRS-1 tended to be greater and lower, respectively, in COF-M/L cows compared to the control cows. The mRNA expression IGF-1 was greater (P < 0.01) in COF-E and COF-M/L cows than in the control cows. The plasma and follicular fluid concentration of NEFA was greater (P < 0.05) in COF-E cows than in COF-M/L and the control cows. Cows with COF-E had disturbances in steroidogenic and metabolic markers, while cows with COF-M/L had steroidogenic, immunological, and metabolic dysregulations, suggesting that COF pathogenesis may vary between early and mid-late lactation dairy cows.

Regional difference of metal levels in follicular fluid and serum AMH level between infertile women from Eastern China and Southern China

Regional difference of metal levels in follicular fluid and serum AMH level between infertile women from Eastern China and Southern China

Effect of genistein and daidzein levels on ovarian response and embryo quality in PGT-A cycles

To evaluate if genistein and daidzein (soy-derived phytoestrogens) levels in urine and follicular fluid (FF) could have an impact on the number of antral follicles, MII oocytes, fertilization, embryo quality on day 3, and aneuploidy rates.

Cell-free nucleic acids and melatonin levels in human follicular fluid predict embryo quality in patients undergoing in-vitro fertilization treatment

Despite many advances in assisted reproductive technology (ART), the most viable embryo selection remains a challenge for infertility treatment. This study was designed to investigate whether intra-follicular circulating cell-free DNA (cfDNA) fragments and Melatonin levels predict embryo quality in patients undergoing IVF treatment. A total of eight hundred and ninety-five follicular fluid (ff) samples were collected from 325 infertile patients undergoing IVF treatment. Patients were enrolled from August 2017 to December 2018 in the infertility center of a tertiary care hospital. A clear non-hematic follicular fluid was aspirated after the removal of eggs from the dominant follicles (>18mm) of each patient. Melatonin and E2 levels in each follicular sample were estimated by immune-chemiluminescence using commercially available kits. ALU-qPCR evaluated cfDNA levels in individual follicular fluid samples. Our study presented a significant and negative relationship between intra-follicular cfDNA and melatonin concentration (-0.541: P<0.001). Each individual follicle contains measurable copy number of cfDNA [mean: 1.85±2.98ng/μl (median; 1.86ng/μl (95% Cl: 0.96–2.87)]. In pregnant women cfDNA copy number was significantly decreased in follicular fluid samples(ff) aspirated from matured oocytes than in immature ones [p<0.01; β = -0.42±0.49; median; 1.45ng/ml (95% Cl: 0.36–2.97) vs. 3.57ng/μl (95% Cl: 0.37–4.01) respectively. While melatonin concentration in ff samples corresponding to mature oocytes was significantly higher than in ff samples related to immature oocytes (p<0.001). Moreover, in pregnant women cfDNA level was significantly lower in ff samples related to oocytes which produces top-quality embryos versus low quality embryos [p<0.001; β=1.81±0.91; median; 1.25ng/μl (95% Cl: 0.35–1.97)] vs. [(median; 3.65ng/ml (95% Cl: 1.23–6.36)] respectively. Likewise, in non-pregnant women melatonin levels were significantly decreased in ff samples related to embryos with high fragmentation rate (≥25%) than embryos with low fragmentation rate (<25%; p<0.001). Conclusively, this study indicates that Intra-follicular cfDNA and melatonin concentration possibly a new supplemental tool that supports to establish an advanced non-invasive early prognostic test for the patients undergoing IVF/ICSI procedure.

Ovarian Expression of Adipokines in Polycystic Ovary Syndrome: A Role for Chemerin, Omentin, and Apelin in Follicular Growth Arrest and Ovulatory Dysfunction?

Adipokines are a potential link between reproduction and energy metabolism and could partly explain some infertilities related to some pathophysiology, such as polycystic ovary syndrome (PCOS). However, adipokines were predominantly assessed in blood samples, while very little is known concerning their variations in follicular fluid (FF) and ovarian granulosa cells (GCs) of PCOS women. Thus, the objectives of our study were to investigate adiponectin, chemerin, resistin, visfatin, omentin, and apelin ovarian expression in PCOS women in comparison with controls and women with only a polycystic ovary morphology. In total, 78 women undergoing an in vitro fertilization procedure were divided into three groups: 23 PCOS women, 28 women presenting only ≥12 follicles per ovary (ECHO group), and 27 control women. Each group almost equally included normal weight and obese women. Follicular fluid (FF) concentration and granulosa cells (GCs) mRNA expression of adipokines and their receptors were assessed by ELISA and RT-qPCR, respectively. Omentin levels in FF and GC were higher in PCOS than in ECHO and control women, while apelin expression was increased in both PCOS and ECHO groups. FF chemerin concentration was predominant in normal-weight PCOS women compared to BMI (Body Mass Index)-matched ECHO and control women, while GC mRNA levels were higher in the obese PCOS group than in the ECHO one. Compared to PCOS, ECHO women had increased FF adiponectin concentrations and lower plasma AMH levels. The FF concentration of all adipokines was higher in obese subjects except for adiponectin, predominant in normal-weight women. In conclusion, women with PCOS expressed higher GC chemerin and omentin, whereas the ECHO group presented higher levels of FF adiponectin and apelin and lower plasma AMH and LH concentrations. Chemerin, omentin, and apelin expression was differently regulated in women with PCOS, suggesting their possible role in follicular growth arrest and ovulatory dysfunction characterizing PCOS pathogenesis.

The Origin of Follicular Bile Acids in the Human Ovary

Bile acids (BAs) are present in ovarian follicular fluid (FF) and are linked to embryo development. However, information on the source of ovarian BA is scarce. Therefore, we aimed to explore local ovarian synthesis and BA transport from blood into FF. BA levels were determined in matching FF and serum from women undergoing invitro fertilization. Invitro BA production by human mural granulosa cells (MGCs) and cumulus granulosa cells (CGCs) was measured by mass spectrometry. Gene and protein expression were quantified in MGC and CGC and in human ovarian tissue by quantitative PCR and Western blot/immunohistochemistry, respectively. BA levels in blood and FF were significantly correlated (rs=0.186, P=0.027) but were almost twofold higher in FF (P<0.001). Primary BA levels were increased in FF, indicating that, in addition to passive diffusion, other sources of ovarian BA might exist. The key BA synthesis enzyme cytochrome P450 A1 was absent in MGC and CGC; BA production invitro was undetectable. Therefore, local ovarian BA production is unlikely. However, common BA importers (Na+/taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter) and an exporter (ATP-binding cassette subfamily C member 3) were identified in GC, theca cells, and oocyte. In summary, these results suggest that passive and active transport of BAs from blood into FF constitute sources of FF BA.

Cell-free mitochondrial DNA in human follicular fluid: a promising bio-marker of blastocyst developmental potential in women undergoing assisted reproductive technology

BackgroundCell-free mitochondrial DNA (cf-mtDNA) in body fluids has attracted much attention for the purpose of monitoring disease because of the clinical advantages. This study investigated whether the cf-mtDNA content in human follicular fluid samples was associated with oocyte and embryo developmental competence.MethodsWe collected 225 individual follicular fluid samples from 92 patients undergoing conventional in vitro fertilization (n = 53) or intracytoplasmic sperm injection (n = 39). cf-mtDNA and cell-free nuclear DNA (cf-nDNA) were measured using real-time quantitative PCR for the ND1 and β-globin genes. Multivariate logistic regression and linear regression were used to analyze data.ResultsThe relative cf-mtDNA content (cf-ND1/cf-β-globin ratio) in follicular fluid was significantly lower in the group showing blastocyst development than in the non-blastocyst group (P = 0.030). Additionally, the relative cf-mtDNA content was significantly and positively correlated with the age of the female patient (P = 0.009), while the relative cf-mtDNA content for older women (≥38 years old) with anti-Müllerian hormone (AMH) ≤1.1 ng/ml was significantly higher than in those with AMH > 1.1 ng/ml (P <0.05). The cf-nDNA content was significantly positively correlated with the antral follicle count (P = 0.012), and significantly negatively correlated with both the number of days of stimulation and the total dose of gonadotropin administration (P = 0.039 and P = 0.015, respectively). Neither cf-mtDNA nor cf-nDNA levels in follicular fluid were associated with oocyte maturation, fertilization, or Day 3 embryo morphological scoring.ConclusionsThe relative cf-mtDNA content in human follicular fluid was negatively correlated with blastulation and positively correlated with the patient age, indicating that it is a promising bio-marker to evaluate oocyte developmental competence.

Acupuncture for polycystic ovarian syndrome

Background Polycystic ovarian syndrome (PCOS) is characterised by the clinical signs of oligo-amenorrhoea, infertility and hirsutism. Conventional treatment of PCOS includes a range of oral pharmacological agents, lifestyle changes and surgical modalities. Beta-endorphin presents in the follicular fluid of both normal and polycystic ovaries. It was demonstrated that the beta-endorphin levels in ovarian follicular fluid of otherwise healthy women who were undergoing ovulation were much higher than the levels measured in plasma. Given that acupuncture has an impact on beta-endorphin production, which may affect gonadotropin-releasing hormone (GnRH) secretion, it is postulated that acupuncture may have a role in ovulation induction and fertility. Objectives To assess the effectiveness and safety of acupuncture treatment of oligo/anovulatory women with polycystic ovarian syndrome (PCOS). Search methods We identified relevant studies from databases including the Cochrane Central Register of Controlled Trials (CENTRAL), Ovid MEDLINE, EMBASE, PsycINFO, CNKI and trial registries. The data are current to 19 October 2015. Selection criteria We included randomised controlled trials (RCTs) that studied the efficacy of acupuncture treatment for oligo/anovulatory women with PCOS. We excluded quasi- or pseudo-RCTs. Primary outcomes were live birth and ovulation (primary outcomes), and secondary outcomes were clinical pregnancy, restoration of menstruation, multiple pregnancy, miscarriage and adverse events. We assessed the quality of the evidence using GRADE methods. Data collection and analysis Two review authors independently selected the studies, extracted data and assessed risk of bias. We calculated Mantel-Haenszel odds ratios (ORs) and mean difference (MD) and 95% confidence intervals (CIs). Main results We included five RCTs with 413 women. They compared true acupuncture versus sham acupuncture (two RCTs), true acupuncture versus relaxation (one RCT), true acupuncture versus clomiphene (one RCT) and electroacupuncture versus physical exercise (one RCT). Four of the studies were at high risk of bias in at least one domain. No study reported live birth rate. Two studies reported clinical pregnancy and found no evidence of a difference between true acupuncture and sham acupuncture (OR 2.72, 95% CI 0.69 to 10.77, two RCTs, 191 women, very low quality evidence). Three studies reported ovulation. One RCT reported number of women who had three ovulations during three months of treatment but not ovulation rate. One RCT found no evidence of a difference in mean ovulation rate between true and sham acupuncture (MD -0.03, 95% CI -0.14 to 0.08, one RCT, 84 women, very low quality evidence). However, one other RCT reported very low quality evidence to suggest that true acupuncture might be associated with higher ovulation frequency than relaxation (MD 0.35, 95% CI 0.14 to 0.56, one RCT, 28 women). Two studies reported menstrual frequency. One RCT reported true acupuncture reduced days between menstruation more than sham acupuncture (MD 220.35, 95% CI 252.85 to 187.85, 146 women). One RCT reported electroacupuncture increased menstrual frequency more than no intervention (0.37, 95% CI 0.21 to 0.53, 31 women). There was no evidence of a difference between the groups in adverse events. Evidence was very low quality with very wide CIs and very low event rates. Overall evidence was low or very low quality. The main limitations were failure to report important clinical outcomes and very serious imprecision. Authors' conclusions Thus far, only a limited number of RCTs have been reported. At present, there is insufficient evidence to support the use of acupuncture for treatment of ovulation disorders in women with PCOS.

Effect of exogenous gonadotropin on the transcriptome of human granulosa cells and follicular fluid hormone profiles

BackgroundSuperovulation treatment had some adverse effects on maturity and development of oocytes. Can superovulation dose of gonadotropins (Gns) affect the transcriptome of granulosa cells and follicular fluid (FF) hormone levels?MethodsOne leading pre-ovulatory follicle per subject was used from three natural-cycle and four Gn-stimulated patients. Granulosa cells and FF samples were collected from the same leading follicle of each patient. RNA was extracted from granulosa cells and subjected to deep sequencing and analysis. Follicle-stimulating hormone (FSH), estradiol (E2), androstenedione (AND), testosterone (T), luteinizing hormone (LH), and progesterone (P4) levels in FF were measured by immunoassays. Student’s t test was used for statistical analysis.ResultsA total of 715 genes were up-regulated, and 287 genes were down-regulated, in the Gn-stimulated group relative to the control group. Gene Ontology analysis revealed that the down-regulated genes were enriched in cell cycle and meiosis pathways, primarily those associated with follicle or oocyte maturation and quality. On the other hand, the up-regulated genes were enriched in functions related to immunity and cytokine–cytokine receptor interactions. Compared to the follicles of natural cycle, the E2 and LH concentrations were significantly reduced (P < 0.001), the P4 concentration was significantly increased (P = 0.003), and the concentrations of FSH, T and AND had no difference in the follicles of Gn-stimulated cycle.ConclusionsCell cycle– and meiosis-associated genes were down-regulated by Gns stimulation, whereas immune- and cytokine-associated genes were up-regulated. Hormone levels were also affected by Gns stimulation. Compared with natural-cycle follicles,putative markers associated with oocyte quality and follicle maturation were significantly different from those in Gn-stimulated follicles. Hormone levels in follicles were compatible with the steroidogenic patterns of granulosa cell, which reflects the follicle maturation and oocyte quality.

A shift in follicular fluid from triacylglycerols to membrane lipids is associated with positive pregnancy outcome.

Follicular fluid (FF) is a liquid that surrounds the ovum. Its metabolite and, specifically, its lipid content have been associated with oocyte development. To characterize possible association between the lipid composition of FF and the outcome of pregnancy, we carried out a lipidomics study and compared the abundance of lipids from FF of patients with positive and negative outcomes. We found a differential lipid network wiring in positive-outcome FF, with a significant decrease (∼2 fold; P < 0.001) in triacylglycerol levels and higher accumulation (10-50%; P < 0.001) of membrane lipids groups (phospholipids and sphingolipids). In addition to this major metabolic alteration, other lipid groups such as cholesteryl esters showed lower levels in positive-outcome patients, whereas derivatives of vitamin D were highly accumulated in positive-outcome FF, supporting previous studies that associate vitamin D levels in FF to pregnancy outcome. Our data also point to specific lipid species with a differential accumulation pattern in positive-outcome FF that predicted pregnancy in a receiver operating characteristic analysis. Altogether, our results suggest that FF lipid network is associated with the oocyte development, with possible implications in diagnostics and treatment.-Shehadeh, A., Bruck-Haimson, R., Saidemberg, D., Zacharia. A., Herzberg, S., Ben-Meir, A., Moussaieff, A. A shift in follicular fluid from triacylglycerols to membrane lipids is associated with positive pregnancy outcome.

Anti-M\xfcllerian hormone and progesterone levels in human follicular fluid are predictors of embryonic development

BackgroundHuman follicular fluid is an intricate biological fluid contributing to the developing oocyte microenvironment. Accumulating evidence suggests that sex hormones present in follicular fluid (FF) may play an important role in regulating oocyte developmental potential. The aim of this study was to determine if anti-Müllerian hormone (AMH) and progesterone (P4) levels in FF are correlated with oocyte quality as defined by subsequent embryonic development.MethodsThis was a prospective cohort study of 88 women undergoing IVF/ICSI at a university associated fertility clinic. Follicular fluid was collected from the first follicle aspirated at the time of oocyte retrieval. The corresponding oocyte was individually cultured in order to track its developmental outcome. FF-AMH and P4 concentrations from follicles where the oocyte fertilised normally and developed into a blastocyst on day 5 (Group 1: BLAST, n = 23) were compared with FF from follicles where the oocyte fertilised normally but failed to reach blastocyst stage by day 5 (Group 2: FERT, n = 19). No significant differences were observed between the two groups in terms of maternal age, body mass index, previous live births, previous pregnancy loss, number of antral follicles, number of oocytes recovered, IVF:ICSI ratio or percentage of recovered oocytes that fertilised.ResultsFF-AMH and P4 levels were significantly increased in Group 1: BLAST compared to Group 2: FERT (P = 0.007 and P = 0.013 respectively). Twenty-one FF samples had an AMH level > 15 pmol/L, of which 17 related to oocytes that progressed to blastocyst stage, providing a positive prediction value (PPV) of 76.96%. Eleven FF samples had a P4 level > 60 mg/ml, of which 10 progressed to blastocyst stage, providing a PPV of 90.99%. Six samples had an AMH level > 15 pmol/L and a P4 level > 60 mg/ml, of which 100% progressed to blastocyst stage, providing a PPV of 96.83%.ConclusionsFF-AMH and P4 levels from individual follicles can accurately predetermine subsequent embryonic development. Combining follicular fluid analysis with routine morphological assessment, could allow for a more accurate and sensitive method of determining embryonic developmental competence.

Genes expression and phenotypic differences in corpus luteum and cumulus cells of commercial line and piau breed gilts

We aimed to characterize the expression of angiogenesis-related genes in corpus luteum (CL) and cumulus cells (CC) during estrous cycle in gilts of different genetic groups, as well as to study the relation between gene expression and phenotypic data. Forty five gilts were used as follows: L1, Commercial Line 1 (Large White x Landrace x Duroc) (n = 15); L2, Commercial Line 2 (Large White x Landrace x Pietrain) (n = 15); and Piau, Piau breed gilts (n = 15). Estrus observation started from 120 days of age. After the second observed estrus females were slaughtered (n = 3) on days 3, 5, 10, 14 and 18 of estrous cycle (first day of estrous cycle as Day 0). CL sampling was performed on days 3, 5, 10 and 14 and collection of CC and follicular fluid on days 14 and 18. Follicular fluid was used for analysis of estradiol levels and CC and CL samples for analysis of angiogenesis-related genes expression, ANGPT-1/-2 and TEK in CC and MMP-2, VEGFA, VEGFR-1/-2, ANGPT-1/-2 and TEK in CL. Piau gilts showed lower ovulation rate than both L1 and L2 gilts (P < 0.05), lower number of large antral follicles (>6 mm) at 18 days than L2 gilts (P < 0.05), and smaller diameter of the largest follicles at 14 days than L1 gilts (P < 0.05). Piau and L2 gilts showed higher estradiol levels in follicular fluid on day 18. Expression of ANGPT-1 and -2 genes in CC did not differ among genetic groups neither among days of the estrous cycle, but TEK gene expression was higher in L1 than L2 gilts on day 18. Expression of VEGFA, VEGFR-2 and MMP-2 genes in CL did not differ among genetic groups and days of cycle, but VEGFR-1 expression was higher in Piau than L2 gilts on days 10 and 14, and it was higher in L1 than L2 gilts on day 14. The ANGPT-1/-2 and TEK genes expression in CL were significantly higher in Piau than L1 gilts on day 10. The ANGPT-2/ANGPT-1 gene expression ratio in CL was higher in L1 than Piau and L2 gilts at 14 days, suggesting a shorter luteal phase for L1 gilts. Results indicated differences among genetic groups for the pattern of the angiogenesis-related genes expression in CL along estrous cycles, which may be reflected in phenotypic traits such as ovulation rate, estradiol levels in follicular fluid and number and diameter of antral follicles.

High concentration of chemerin caused by ovarian hyperandrogenism may lead to poor IVF outcome in polycystic ovary syndrome: a pilot study

A chronic low-grade inflammation state accounts for an important part of the pathogenesis of polycystic ovary syndrome (PCOS). The adipose tissue derived cytokine chemerin has recently been proven to be a proinflammatory chemokine, but its mechanism involved in the pathogenesis of PCOS remains largely unresolved. From non-obese patients with and without PCOS, follicular fluid and granulosa cells were retrieved. The effect of testosterone on the expression of chemerin and its receptors was explored in granulosa cells. IVF outcomes in different groups based on FF-chemerin (chemerin in the follicular fluid) level were further compared. The concentration of FF-chemerin, and the mRNA expression of chemerin and its receptors in granulosa cells from PCOS were significantly higher than those from non-PCOS. FF-chemerin was positively correlative to total testosterone (TT) and luteinizing hormone (LH) in the follicular fluid. Furthermore, testosterone upregulated the expression of chemerin and its receptors in vitro. The oocyte utilization rate and high-quality embryo rate were significantly decreased in the high FF-chemerin group. The upregulated chemerin levels in the ovary of PCOS patients, which may be caused by ovarian hyperandrogenism, may be a risk factor for oocyte maturation and embryo development. These findings may provide a basis for novel interventions to improve IVF outcomes.

Can follicular Emmprin and BMP 4 levels predict ICSI outcome?

To evaluate the relationship of clinical pregnancy rates with bone morphogenetic proteins 2-4-7 (BMP 2, 4, 7), growth differentiation factor 9 (GDF 9), and Emmprin levels in follicular fluid of intracytoplasmic sperm injection patients. Follicular fluid of 77 patients who underwent ICSI procedure was collected during the oocyte retrieval procedure. And follicular fluid levels of BMP 2, BMP 4, BMP 7, GDF 9, and Emmprin (Basigin) were measured and compared for clinical pregnancy rates. Follicular levels of BMP 4 was significantly higher whereas Emmprin levels were lower in patients who had achieved clinically diagnosed pregnancy compared with those who did not achieve clinical pregnancy after ICSI procedure (P= 0.007 and P= 0.035, respectively). BMP 2, BMP 7, and GDF 9 levels were comparable for both groups. Clinical pregnancy rates after ICSI may be associated with follicular fluid levels of Emmprin and BMP 4. Follicular levels of Emmprin and BMP 4 can be used as a marker (as markers for predicting ICSI outcomes) for a better ICSI outcome.

Serum and follicular fluid Stem Cell Factor assay in IVF poor responder and normal responder patients: a predictive biomarker of oocyte retrieval.

The aim of the study is to investigate serum stem cell factor (SCF) concentrations as potential biomarker for oocyte retrieval efficiency in IVF patients with poor prognosis. A pilot case-control study was performed on 30 poor and 30 normal responders that were stimulated with antagonist protocol. SCF concentrations were evaluated in samples of serum and follicular fluid obtained by all patients on the day of oocyte retrieval. At the time of oocyte retrieval, follicular fluid from at least two follicles ≥ 14mm and two follicles < 14mm was collected for SCF determination. We did not find any statistical difference when comparing serum and follicular fluid levels of SCF in both poor- and normal-responder patients, the same results were achieved when poor-responder patients were stratified according to the number of MII oocytes retrieved. Moreover, levels of SCF (OR 1.000, 0.994-1.006) or in follicular fluid from ovarian follicles ≥ 14mm (OR 0.995, CI 0.989-1.001) or from ovarian follicles < 14mm (OR 1.003, CI 0.999-1.0069), were not significantly associated with the chances of ongoing pregnancies in poor-responder patients. SCF was unable to predict oocyte retrieval efficiency or the chances of reaching embryo transfer.

Insulin treatment does not affect follicular development but alters granulosa cell gene expression in dairy cows

The use of strategies to stimulate follicular growth are important, especially for use in timed artificial insemination (TAI) protocols, aiming to increase dairy cow's fertility. The aim of this study was to investigate the effect of insulin on follicular growth, steroid production and expression of genes related to follicular development. For this, cows were submitted to a progesterone (P4) and estradiol (E2) based synchronization protocol. In study 1, eleven primiparous lactating Holstein cows, received a single s.c. application of 0.25 IU/kg human insulin or no treatment (control) on D8 of the protocol. Blood samples were collected, and the dominant follicle diameter was assessed daily via transrectal ultrasonography, from D8 to D12. In study 2, eight multiparous non-pregnant and non-lactating Jersey cows, received a single s.c. application of 0.25 IU/kg human insulin, whereas cows from the control group received a single s.c. injection (1 mL) of saline solution (NaCl 0.9%). Blood samples were collected, and the dominant follicle diameter was assessed daily via transrectal ultrasonography from D6 to D9 of the protocol. Sixteen hours after insulin injection, follicular aspiration was performed. In study 1, insulin treatment decreased systemic glucose levels, but did not affect follicular growth. In study 2, the glucose decrease induced by insulin treatment was accompanied by a tendency of decreased progesterone levels in follicular fluid, along with a decrease in steroidogenic acute regulatory protein (STAR) and insulin like growth factor binding protein 2 (IGFBP2) mRNA abundance in granulosa cells. In conclusion, insulin treatment does not increase follicle growth and estradiol secretion in dairy cows, but decreases IGFBP2 and tends to increase pappalysin (PAPPA) mRNA abundance in granulosa cells, suggesting a positive effect on follicle development.