AMH is a confirmed quantitative marker of ovarian follicles. Yet, doubts persist on its possible relationship with oocyte competence. Whereas, for some authors, serum AMH levels are an age-independent marker of pregnancy and live birth rates after IVF-ET, for others they are not. These conflicting views may result from the confounding role of the intensity of the ovarian response to COH, which is stronger in patients with increased AMH levels. In addition, scarce data is available on the possible link between AMH and miscarriage rates in IVF-ET, an indirect marker of chromosomal status of embryos and, therefore, oocyte-embryo health. Hence, we decided to investigate whether serum AMH levels are independently related to IVF-ET ouctome, in particular, miscarriage rates. Prospective study. We studied 2,365 infertile women undergoing 2,688 IVF-ET cycles. All of them had a reference, centralized, serum AMH measurement using AMH Gen II ELISA and started COH within the 12 subsequent months. Each AMH determination was paired to a single cycle. Patients had a normal karyotype, no history of genetic disorders, congenital malformation or recurrent pregnancy loss, and no uterine abnormalities. Cycles were sorted into 3 different age groups: ≤33 years (n=1,033), 34-36 years (n=690) and ≥37 years (n=965) and into 3 different AMH groups: Low AMH (0.04-1.60 ng/mL; n=540), Intermediate AMH (1.61-5.59 ng/mL; n=1,608), and High AMH (5.60-35.00 ng/mL; n=540). Binary logistic regression was conducted to verify whether AMH was related to miscarriage rates independently of age and ovarian response to COH. Our data showed that clinical pregnancy and live birth rates increased from the Low to the High AMH groups, independently from age and oocyte yield. It is remarkable that, in the Low AMH group, miscarriage rates were higher than those in the Intermediate or High AMH groups. Differences reached statistical significance in 34-36 years (33%, 15%, and 17.0%, respectively; P<0.02), and ≥37 years (35%, 26%, and 18%, respectively; P<0.04), but not in ≤33 years groups. Yet, binary logistic regression run over the whole population confirmed the association between serum AMH and miscarriage rates, independently of patient’s age and oocyte yield. This extensive investigation showed that, irrespective of age and ovarian response to COH, patients aged >34 years with low AMH levels displayed poorer IVF-ET outcome, in particular, higher miscarriage rates. This supports the hypothesis that AMH is not only a biomarker of oocyte quantity but may be also related to oocyte reproductive potential. Further age- and oocyte yield- controlled studies, possibly including preimplantation genetic screening of embryos, will be contributive to challenge these results.

Circulating or “extracellular” microRNAs (miRNAs) detected in biological fluids, could be used as potential diagnostic and prognostic biomarkers of several disease, such as cancer, gynecological and pregnancy disorders. However, their contributions in female infertility and in vitro fertilization (IVF) remain unknown. This study investigated the expression profiles of five circulating miRNAs (let-7b, miR-29a, miR-30a, miR-140 and miR-320a) in human follicular fluid from 91 women with normal ovarian reserve and 30 with polycystic ovary syndrome (PCOS) and their ability to predict IVF outcomes. The combination of FF miR-30a, miR-140 and let-7b expression levels discriminated between PCOS and normal ovarian reserve with a specificity of 83.8% and a sensitivity of 70% (area under the ROC curve, AUC = 0.83 [0.73–0.92]; p < 0.0001). FF samples related to low number of mature oocytes (≤2) contained significant less miR-320a levels than those related to a number of mature oocytes >2 (p = 0.04). Moreover, FF let-7b predicted the development of expanded blastocysts with 70% sensitivity and 64.3% specificity (AUC = 0.67 [0.54–0.79]; p = 0.02) and FF miR-29a potential to predict clinical pregnancy outcome reached 0.68 [0.55–0.79] with a sensitivity of 83.3% and a specificity of 53.5% (p = 0.01). Therefore, these miRNAs could provide new helpful biomarkers to facilitate personalized medical care during IVF.

The aim of this study is to investigate if cell-free DNA (cfDNA) level in serum at day 3 of menstrual cycle could be clinically useful for prediction of patient's ovarian response to stimulation during in vitro fertilization (IVF) procedure. This prospective study included 32 serum samples collected at day 3 of menstrual cycle from patients undergoing IVF/ICSI procedure. Anti-Müllerian hormone (AMH) and cfDNA levels were measured in each serum sample in order to compare their predictive value for patient's ovarian response to stimulation. Serum samples were prepared in Proteinase K buffer and the total cfDNA was quantified by qPCR, using ALU 115 primers. CfDNA concentration was determined based on a standard curve obtained by successive dilution of genomic DNA. The P-values were calculated by using the unpaired t-test, Spearman correlation, multiple and logistic regressions and ROC curveanalysis on GraphPad and Medcalc softwares. Cell-free DNA concentrations (mean ± SD=152.02 ± 160.48 ng/m, median=92 ng/ml) were significantly and positively correlated with patient's age (r=0.18; p=0.02). Indeed, cfDNA levels were significantly higher in serum from older women (≥38 years) than those from young patients (<38 years) (p=0.035). Very interestingly, cell-free DNA level was significantly related to the number of oocyte collected at oocyte retrieval (p=0.031). Furthermore, cfDNA levels predicted significantly a low ovarian response (<6 retrieved oocytes), independently of AMH concentrations [Adjusted Odd Ratio=1.01 (1.0-1.02); p=0.02]. The area under the ROC curve (AUC), which quantifies the low ovarian response prediction of cfDNA level was 0.79 [0.61-0.91] with higher sensitivity (73.33%) and specificity (82.35%) (p<0.001) than AMH level (AUC=0.72 [0.54-0.87]; sensitivity=60%; specificity=88.2%, p=0.02). Cell-free DNA levels in serum at day 3 of menstrual cycle can predict significantly the number of retrieved oocyte, independently of AMH concentrations. High cfDNA levels in serum could be significantly related to a low ovarian response. Cell-free DNA quantification at day 3 could be used as a supplemental tool to identify more accurately women with a risk of poor ovarian response, in order to develop a personalized care program for these patients.

Sperm DNA methylation abnormalities have been detected in oligozoospermic men. However, the association between sperm DNA methylation defects, sperm parameters and sperm DNA, and chromatin integrity remains poorly understood. This study was designed to clarify this issue. We recruited a cohort of 92 men (62 normozoospermic and 30 oligoasthenozoospermic) presenting for infertility evaluation during a 1-year period. Sperm global DNA methylation was evaluated by an ELISA-like method, DNA fragmentation was evaluated by flow cytometry-based terminal transferase dUTP nick end-labeling (TUNEL) assay (reported as DNA fragmentation index or DFI), and sperm denaturation was evaluated by aniline blue staining (reported as sperm denaturation index or SDI, a marker of chromatin compaction). We found a significant positive association between sperm global DNA methylation level and conventional sperm parameters (sperm concentration and motility), supported by the results of methylation analysis on H19-DMR. We also identified significant inverse relationships between sperm global DNA methylation, and, both DFI and SDI. However, sperm global DNA methylation level was not related to sperm vitality or morphology. Our findings suggest that global sperm DNA methylation levels are related to conventional sperm parameters, as well as, sperm chromatin and DNA integrity.

Circulating nucleic acids (cell-free DNA and microRNAs) have for particularity to be easily detectable in the biological fluids of the body. Therefore, they constitute biomarkers of interest in female and male infertility care. Indeed, in female, they can be used to detect ovarian reserve disorders (polycystic ovary syndrome and low functional ovarian reserve) as well as to assess follicular microenvironment quality. Moreover, in men, their expression levels can vary in case of spermatogenesis abnormalities. Finally, circulating nucleic acids have also the ability to predict successfully the quality of in vitro embryo development. Their multiple contributions during assisted reproductive technology (ART) make of them biomarkers of interest, for the development of new diagnostic and/or prognostic tests, applied to our specialty. Circulating nucleic acids would so offer the possibility of personalized medical care for infertile couples in ART.

ObjectiveOur aim was to investigate if circulating microRNAs (miRNAs) in human follicular fluid (FF) could be used as helpful biomarkers for predicting IVF/ICSI outcomes.DesignIn this prospective study, a pool of FF was retrieved for each patient during IVF/ICSI procedure. A total of 91 FF samples from women with normal ovarian reserve (n=91) were collected at oocyte retrieval day.Materials and MethodsFor each patient, all follicles were aspirated and all FF samples were pooled. MicroRNAs were extracted from each FF pool and quantified by RT-qPCR, using TaqMan technology. The expressions of miR-320a, let-7b and miR-29a were analyzed in FF and related to IVF/ICSI outcomes.ResultsFF pools related to low number of mature oocytes (≤2) contained significant lower miR-320a expression levels than those related to high number of mature oocytes (>2), respectively (p=0.03). Moreover, significant high let-7b levels were found in FF pools related to embryo cohorts with a high total blastomere number/total embryo number ratio at day 3 (>8, ie accelerated development) than in those with normal developmental kinetics (ratio between 6 and 8) (p=0.02). Interestingly, we found a significant and negative correlation between FF let-7b expression levels and blastulation rate (r=-0.33, p=0.003). The Receiving Operator Curve (ROC) analysis indicated that the performance of FF let-7b in predicting the expanded blastocyst development was 0.67 (0.54-0.79), with 70% sensitivity and 64.3% specificity (p=0.02). In addition, the area under the ROC curve (AUC), evaluating the potential of FF miR-29a in predicting clinical pregnancy outcome reached 0.68 [0.55-0.79] with a high sensitivity of 83.3% and a specificity of 53.5% (p=0.01).ConclusionsOur results suggest that circulating miRNAs constitute non-invasive powerful tools in IVF process to predict embryo development and clinical pregnancy outcomes, in order to promote personalized IVF strategy. ObjectiveOur aim was to investigate if circulating microRNAs (miRNAs) in human follicular fluid (FF) could be used as helpful biomarkers for predicting IVF/ICSI outcomes. Our aim was to investigate if circulating microRNAs (miRNAs) in human follicular fluid (FF) could be used as helpful biomarkers for predicting IVF/ICSI outcomes. DesignIn this prospective study, a pool of FF was retrieved for each patient during IVF/ICSI procedure. A total of 91 FF samples from women with normal ovarian reserve (n=91) were collected at oocyte retrieval day. In this prospective study, a pool of FF was retrieved for each patient during IVF/ICSI procedure. A total of 91 FF samples from women with normal ovarian reserve (n=91) were collected at oocyte retrieval day. Materials and MethodsFor each patient, all follicles were aspirated and all FF samples were pooled. MicroRNAs were extracted from each FF pool and quantified by RT-qPCR, using TaqMan technology. The expressions of miR-320a, let-7b and miR-29a were analyzed in FF and related to IVF/ICSI outcomes. For each patient, all follicles were aspirated and all FF samples were pooled. MicroRNAs were extracted from each FF pool and quantified by RT-qPCR, using TaqMan technology. The expressions of miR-320a, let-7b and miR-29a were analyzed in FF and related to IVF/ICSI outcomes. ResultsFF pools related to low number of mature oocytes (≤2) contained significant lower miR-320a expression levels than those related to high number of mature oocytes (>2), respectively (p=0.03). Moreover, significant high let-7b levels were found in FF pools related to embryo cohorts with a high total blastomere number/total embryo number ratio at day 3 (>8, ie accelerated development) than in those with normal developmental kinetics (ratio between 6 and 8) (p=0.02). Interestingly, we found a significant and negative correlation between FF let-7b expression levels and blastulation rate (r=-0.33, p=0.003). The Receiving Operator Curve (ROC) analysis indicated that the performance of FF let-7b in predicting the expanded blastocyst development was 0.67 (0.54-0.79), with 70% sensitivity and 64.3% specificity (p=0.02). In addition, the area under the ROC curve (AUC), evaluating the potential of FF miR-29a in predicting clinical pregnancy outcome reached 0.68 [0.55-0.79] with a high sensitivity of 83.3% and a specificity of 53.5% (p=0.01). FF pools related to low number of mature oocytes (≤2) contained significant lower miR-320a expression levels than those related to high number of mature oocytes (>2), respectively (p=0.03). Moreover, significant high let-7b levels were found in FF pools related to embryo cohorts with a high total blastomere number/total embryo number ratio at day 3 (>8, ie accelerated development) than in those with normal developmental kinetics (ratio between 6 and 8) (p=0.02). Interestingly, we found a significant and negative correlation between FF let-7b expression levels and blastulation rate (r=-0.33, p=0.003). The Receiving Operator Curve (ROC) analysis indicated that the performance of FF let-7b in predicting the expanded blastocyst development was 0.67 (0.54-0.79), with 70% sensitivity and 64.3% specificity (p=0.02). In addition, the area under the ROC curve (AUC), evaluating the potential of FF miR-29a in predicting clinical pregnancy outcome reached 0.68 [0.55-0.79] with a high sensitivity of 83.3% and a specificity of 53.5% (p=0.01). ConclusionsOur results suggest that circulating miRNAs constitute non-invasive powerful tools in IVF process to predict embryo development and clinical pregnancy outcomes, in order to promote personalized IVF strategy. Our results suggest that circulating miRNAs constitute non-invasive powerful tools in IVF process to predict embryo development and clinical pregnancy outcomes, in order to promote personalized IVF strategy.

Circulating nucleic acids (cell-free DNA and microRNAs) have for particularity to be easily detectable in the biological fluids of the body. Therefore, they constitute biomarkers of interest in female and male infertility care. Indeed, in female, they can be used to detect ovarian reserve disorders (polycystic ovary syndrome and low functional ovarian reserve) as well as to assess follicular microenvironment quality. Moreover, in men, their expression levels can vary in case of spermatogenesis abnormalities. Finally, circulating nucleic acids have also the ability to predict successfully the quality of in vitro embryo development. Their multiple contributions during assisted reproductive technology (ART) make of them biomarkers of interest, for the development of new diagnostic and/or prognostic tests, applied to our specialty. Circulating nucleic acids would so offer the possibility of personalized medical care for infertile couples in ART. (C) 2015 Elsevier Masson SAS. All rights reserved.

Our aim is to determine if circulating nucleic acids can be related to ovarian reserve status and thus, be used as non-invasive biomarkers for infertile patients undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) procedure. In this prospective study, cell-free DNA (cfDNA) and microRNAs (miRNAS) levels such as Let-7b, miR-30d, miR-320a, miR-125a and miR-191 levels were quantified in 60 serum samples (n= 60 patients) on day 3 of their natural cycle. Circulating nucleic acid content in serum was compared with classic ovarian biomarkers such as FSH, LH, AMH and estradiol levels. MiRNAs were extracted with the QIAamp kit from QIAGEN and quantified by RT-qPCR using the TaqMan technology. CfDNA was prepared in a Proteinase K buffer and quantified by ALU sequence qPCR. Means ± SEM are presented. The P-values were calculated by using the unpaired t-test, on GraphPad software. On day 3 of natural menstrual cycle, circulating Let-7b was significantly increased (P=0.05) in serum samples from patients with AMH<2ng/ml, whereas miR-30d and miR-320a were drastically reduced (P=0.02; P=0.01, respectively) in serum samples from patients with high LH rates (>5UI/l), suggesting a potential role of these microRNAs in folliculogenesis. Moreover, miR-125a and miR-191 were both also significantly decreased in serum samples from patients with a BMI>25 compared to those with a BMI≤25 (P=0.001) since obesity represent a bad prognosis parameter for Assisted Reproductive Technology success. Interestingly, circulating cfDNA level is significantly higher in serum from patients over 37 years (P=0.02), suggesting a role of cfDNA in natural decline of ovarian reserve status with age. Our results suggest that circulating nucleic acids could represent new clinical biomarkers of ovarian reserve status in women included in IVF/ICSI cycles. Their profiling in bloodstream opens new perspectives for the development of diagnosis/prognosis tests for ovarian disorders detection and for the prediction of IVF/ICSI successful.

BACKGROUND:Proper folliculogenesis is fundamental to obtain a competent oocyte that, once fertilized, can support the acquisition of embryo developmental competence and pregnancy. MicroRNAs (miRNAs) are crucial regulators of folliculogenesis, which are expressed in the cumulus-oocyte complex and in granulosa cells and some can also be found in the bloodstream. These circulating miRNAs are intensively studied and used as diagnostic/prognostic markers of many diseases, including gynecological and pregnancy disorders. In addition, serum contains small amounts of cell-free DNA (cfDNA), presumably resulting from the release of genetic material from apoptotic/necrotic cells. The quantification of nucleic acids in serum samples could be used as a diagnostic tool for female infertility.METHODS:An overview of the published literature on miRNAs, and particularly on the use of circulating miRNAs and cfDNA as non-invasive biomarkers of gynecological diseases, was performed (up to January 2014).RESULTS:In the past decade, cell-free nucleic acids have been studied for potential use as biomarkers in many diseases, particularly in gynecological cancers, ovarian and endometrial disorders, as well as in pregnancy-related pathologies and fetal aneuploidy. The data strongly suggest that the concentration of cell-free nucleic acids in serum from IVF patients or in embryo culture medium could be related to the ovarian hormone status and embryo quality, respectively, and be used as a non-invasive biomarker of IVF outcome.CONCLUSIONS:The profiling of circulating nucleic acids, such as miRNAs and cfDNA, opens new perspectives for the diagnosis/prognosis of ovarian disorders and for the prediction of IVF outcomes, namely (embryo quality and pregnancy).

Cigarette smoking is a very well accepted risk factor for female infertility. However, mechanisms are still discussed, and the impact on ovarian reserve (OR), has not been established, on large samples and using AMH as marker. The objective of the study was to investigate, in a population of women addressed for an OR determination whether smoking has a deleterious effect on OR. Transversal epidemiological study on 1963 women 18-45 years recruited in August-December 2012 in a single private laboratory with OR testing between cycle day 1 and 5. AMH was measured using AMH/MIS enzyme-linked immunosorbent assay kit (Beckman Coulter), FSH and E2 by Chemiluminescent Microparticle (Abbott). Cigarette smoking (Yes/No), and the number of cigarettes were recorded. Analysis considered the main confounders: age, BMI, cycle day and previous cycle duration. Women with current hormonal treatment were discarded (n=59). In total, 20.9% of women were smokers (8.3 ± 6.3 cigarettes / day). Smokers had not a decreased AMH (4.1 ± 4.0 vs. 3.7 ± 3.1 pg/ml), or increased FSH (8.2 ± 5.1 vs. 8.8 ± 6.5 UI/l) and the percentage of low OR (AMH<1.5pg/ml) was not increased (17.5% vs. 22.4%). The results were similar when entering women's age and other confounders (BMI, cycle day, previous cycle duration) in the model and when considering the number of cigarettes. Only age was related to low OR assessed by AMH. We found no impact of cigarette smoking on OR assessed by AMH. Potential bias cannot totally be ruled out (infertile women, only current exposition measured). However, the large number, the consideration of major confounders reinforce the results. The absence of effect means that the deleterious role of smoking may be explained by other factors. In utero exposition to smoking has been hypothesized to play a detrimental role on the ovary, but this may be not the case in adult women. More research is needed.

To assess the ART prognosis in young healthy women with high circulating levels of basal antimüllerian hormone (AMH). Retrospective cohort study on all ART cycles performed in a single unit in 2006-2010 with Day 3 AMH evaluation. 2101 IVF/ICSI cycles with AMH levels >5ng/ml were compared with 5026 cycles with normal AMH (group 4: 2.0-4.9 ng/ml). Elevated AMH was divided in three groups (5.0 – 7.4: group1; 7.5 – 9.9 group 2 and ≥10 ng/ml : group 3). One or 2 embryos were transferred on day 3 or day 5. Clinical pregnancy rate (CPR) and Delivery rate (DR) were the primary end points. (33.8 ± 4.3 vs. 35.5 ± 34.3, p<0.001), were more susceptible to receive GnRH antagonists, and had a lower total dose of gonadotropins. Mean estradiol on hCG day was significantly higher (2449± 1520 vs. 1885± 1055 p<0.001)), as were the numbers of oocytes (12.3 ± 6.9 vs. 9.5 ± 5.1, p<0.0014), and of day2 embryos (6.3 ± 4.5 vs. 5.0 ± 3.6, p<0,001), and blastocyst culture use (p<0.001). CPR and DR were higher in groups 1-3 (30.6% vs. 27.1% and 23.9% vs. 20,3%, p<0.001 for both). The differences were lower and NS when only elective single and double embryos transfers were considered. There was no difference for PR and DR among the 3 high AMH level groups (PR: 30.7%, 30.5% and 30.3%; DR: 24.3%, 22.7%, 23.2%). Finally, in the multivariate models including age, and protocol, high AMH was associated to higher odds ratio (OR) for PR (group1: OR=1.25, 95% confidence interval=1.08-1.45; group2: 1.31: 1.05-1.64; group3: 1.22: 0.97-1.55) as for DR, even if lower (1.27: 1.07-1.49; 1.19:0.93-1.52; 1.18: 0.91-1.53). Despite a higher level of E2 on triggering day, these findings suggest that elevated basal AMH is not detrimental for ART outcome.

A recent study (Jensen TK et al. 2010) showed that high caffeine intake was associated with a non significant decrease of sperm concentration and total sperm count. The objective of our study was to investigate, in a population of men belonging to infertile couples, if caffeine intake has a deleterious effect on semen characteristics and sperm DNA damage. Transversal epidemiological study on 4474 men recruited from August 1st to December 31st 2012 in a single private laboratory. The following parameters were analyzed: volume, concentration, total sperm count, motility, vitality, morphology, DNA fragmentation and chromatin decondensation. Sperm morphology was assessed according to David's modified classification (Auger and Eustache, 2000). Sperm DNA fragmentation was assessed using TUNEL assay and chromatin decondensation by aniline blue staining. In total, 75.6% of men were coffee consumers (3.0 ± 1.8 cup/day). Among caffeine consumers, semen volume was slightly higher (3.2 ± 1.6 vs. 3.1 ± 1.6 ml, p<0.01) as pH (p<0.01), but concentration was lower (60.0 ± 90.7 vs. 69.6 ± 124.9 millions/ml, p<0.01). azoospermia less frequent (2.7 vs. 4.4%, p<0.01). No relationship was observed for motility and morphology, nor for DNA fragmentation and chromatin decondensation. In a multivariate model including age, results were confirmed for volume (p<0.01), but not for concentration. Caffeine intake was associated with a lower risk of elevated fragmentation (OR=0.92, 95% CI 0.92-0.99). We did not find a detrimental effect of caffeine consumption on semen characteristics. Potential bias cannot totally be ruled out (incomplete participation and selection of infertile couples, incomplete measurement of caffeine consumption), but the large number, the consideration of major confounders (age, abstinence, BMI) reinforce the results.

between in granulosa cells of day4 and day6(P>0.05).Immunofluorescence illustrated the expression of SCF in cytoplasm and the protein level was significantly decreased (P<0.05) in the treated GCs.CONCLUSION: In human GCs, rhAMH shows a down-regulatory effects on the level of the oocyte-derived SCF.Investigation of the underlying mechanisms may lead to better understanding of the follicular development in human ovaries.