Apolipoprotein B is regulated by gonadotropins and constitutes a predictive biomarker of IVF outcomes

International audienceAbstractBackgroundFollicular fluid (FF) is an important micro-environment influencing oocyte growth, its development competence, and embryo viability. The FF content analysis allows to identify new relevant biomarkers, which could be predictive of in vitro fertilization (IVF) outcomes. Inside ovarian follicle, the amount of FF components from granulosa cells (GC) secretion, could be regulated by gonadotropins, which play a major role in follicle development.MethodsThis prospective study included 61 female undergoing IVF or Intra-cytoplasmic sperm injection (ICSI) procedure. Apolipoprotein B (APOB) concentrations in follicular fluid and APOB gene and protein expression in granulosa cells from reproductively aged women undergoing an in vitro fertilization program were measured. The statistical analyses were performed according to a quartile model based on the amount of APOB level found in FF.ResultsAmounts of APOB were detected in human FF samples (mean ± SD: 244.6 ± 185.9 ng/ml). The odds of obtaining an oocyte in the follicle and a fertilized oocyte increased significantly when APOB level in FF was higher than 112 ng/ml [i.e., including in Quartile Q 2, Q3 and Q4] (p = 0.001; p < 0.001, respectively). The probabilities of obtaining an embryo and a top quality embryo on day 2, were significantly higher if APOB levels were within the ranges of 112 and 330 ng/ml (i.e. in Q2 and Q3) or 112 and 230 ng/ml (i.e. in Q2), respectively (p < 0.001; p = 0.047, respectively). In addition, our experiments in vitro indicated that APOB gene and protein expression, along with APOB content into culture were significantly under-expressed in GC upon stimulation with gonadotropins (follicular stimulating hormone: FSH and/or human chorionic gonadotropin: hCG).ConclusionWe are reporting a positive and statistically significant associations between APOB and oocyte retrieval, oocyte fertilization, and embryo quality. Using an experimental study component, the authors report significant reduced APOB expression and content for luteinized granulosa cells cultured in the presence of gonadotropins

Circulating nucleic acids : innovative biomarkers in Assisted Reproductive Technology.

Au cours de ces dernières années, l’utilisation des acides nucléiques circulants comme outils diagnostiques et/ou pronostiques en cancérologie a largement été documentée. Récemment, le développement du diagnostic prénatal non-invasif a également révélé l’intérêt grandissant de ces biomarqueurs en gynécologie-obstétrique. Les acides nucléiques circulants ou extracellulaires ont la particularité d’être facilement détectables dans les fluides biologiques de l’organisme et sont de deux types: (1) l’ADN libre, courts ou longs fragments d’ADN provenant des processus apoptotiques et/ou nécrotiques cellulaires (2) les microARNs, petites séquences d’ARN non codantes, qui régulent l’expression des gènes en interférant avec leurs ARNm cibles. Sachant qu’il a été démontré que le taux d’ADN libre circulant est anormalement élevé dans certaines conditions pathologiques et que les microARNs sont impliqués dans la régulation de nombreux processus biologiques tels que la folliculogénèse et la stéroïdogénèse, ces deux types d’acides nucléiques pourraient alors constituer des biomarqueurs d’intérêt dans le domaine de l’Assistance Médicale à la Procréation. Dans ce travail de thèse, nous avons à la fois mesuré le taux d’ADN libre et analysé les profils d’expression de plusieurs microARNs d’intérêt par PCR quantitative, dans le liquide folliculaire (LF) de patientes prises en charge en fécondation in vitro (FIV). Nous avons observé des taux d’ADN libre significativement élevés ainsi que des profils d’expression de microARNs spécifiques dans le LF de patientes présentant des anomalies de la réserve ovarienne (telles que le syndrome des ovaires micropolykystiques ou une faible réserve ovarienne). Nous avons ensuite évalué le potentiel des acides nucléiques circulants en tant biomarqueurs prédictifs des résultats en FIV. Nous avons démontré que le taux d’ADN libre intra-folliculaire et l’expression de certains microARNs étaient significativement associés à la qualité des embryons obtenus in vitro ainsi qu’à la survenue d’une grossesse clinique. Les acides nucléiques circulants offrent donc de nouvelles perspectives à la fois d’un point de vue diagnostique et pronostique dans la prise en charge de l’infertilité humaine.During the last years, the use of circulating nucleic acids as diagnostic and/or prognostic tools in cancerology was widely documented. The recent development of non-invasive prenatal testing also reveals the growing interest of these biomarkers in obstetrics and gynecology. The circulating or extracellular nucleic acids have for particularity to be easily detectable in the biological fluids of the body and there are two types: (1) Cell-free DNA (cfDNA), short or long DNA fragments resulting from cellular apoptosis and/or necrosis (2) microRNAs (miRNAs), small non-coding RNA sequences, which regulate gene expression by interfering with their mRNA targets. Since it was demonstrated that cfDNA level is abnormally increased in some pathological conditions and miRNAs are involved in the regulation of several biological processes such as folliculogenesis and steroidogenesis, these two types of nucleic acids could constitute new biomarkers of interest in Assisted Reproductive Technology. In this thesis work, we quantified the cfDNA level and analysed the expression profiles of some miRNAs by quantitative PCR, in follicular fluid (FF) samples from women undergoing in vitro fertilization (IVF) procedure. We observed significant high cfDNA levels as well as specific miRNA expression profiles in FF from women with ovarian disorders (such as polycystic ovary syndrome or low ovarian reserve). Next, we investigated the potential of circulating nucleic acids as predictive biomarkers of IVF outcomes. We demonstrated that the intra-follicular cfDNA level and some miRNA expressions were significantly associated with in vitro embryo quality and the clinical pregnancy outcome. Therefore, the circulating nucleic acids offer new diagnostic and prognostic perspectives in human infertility management

Male infertility: an obstacle to sexuality?

International audienceInteractions between infertility and sexuality are numerous and complex. Infertile men may suffer from sexual dysfunction (SD) when undergoing an assisted reproductive technology programme. We undertook a review both in French and English of the available data on male SD when being diagnosed with a fertility problem with a specific focus on azoospermic men. The review was performed over a 30-year time period using PubMed/Medline. The sexual concerns and needs of infertile/sterile men for whom potential parenting can be compromised were evaluated. When diagnosed with infertility, men usually go through a crisis that can have a deleterious effect on their sexuality with sometimes a feeling of sexual inadequacy. Infertile men will feel stigmatized because they are perceived as being deficient in a specific component of their masculinity. Hence, subsequent SD may occur that can impact the couple sexuality and the infertility management. However, little is known on how the announcement of azoospermia may affect male on a sexual and psychological point of view. The present review suggests that a global management through a healthcare network (biologist, andrologist, sexologist and psychologist) is required which will allow to consider infertility and its subsequent sexual disorders as a whole and not as dichotomized issues

Cell-free DNA in Human Follicular Microenvironment: New Prognostic Biomarker to Predict in vitro Fertilization Outcomes

Article number: e0136172International audienceCell-free DNA (cfDNA) fragments, detected in blood and in other biological fluids, are released from apoptotic and/or necrotic cells. CfDNA is currently used as biomarker for the detection of many diseases such as some cancers and gynecological and obstetrics disorders. In this study, we investigated if cfDNA levels in follicular fluid (FF) samples from in vitro fertilization (IVF) patients, could be related to their ovarian reserve status, controlled ovarian stimulation (COS) protocols and IVF outcomes. Therefore, 117 FF samples were collected from women (n = 117) undergoing IVF/Intra-cytoplasmic sperm injection (ICSI) procedure and cfDNA concentration was quantified by ALU-quantitative PCR. We found that cfDNA level was significantly higher in FF samples from patients with ovarian reserve disorders (low functional ovarian reserve or polycystic ovary syndrome) than from patients with normal ovarian reserve (2.7 ± 2.7 ng/μl versus 1.7 ± 2.3 ng/μl, respectively, p = 0.03). Likewise, FF cfDNA levels were significant more elevated in women who received long ovarian stimulation (> 10 days) or high total dose of gonadotropins (≥ 3000 IU/l) than in women who received short stimulation duration (7-10 days) or total dose of gonadotropins < 3000 IU/l (2.4 ± 2.8 ng/μl versus 1.5 ± 1.9 ng/μl, p = 0.008; 2.2 ± 2.3 ng/μl versus 1.5 ± 2.1 ng/μl, p = 0.01, respectively). Finally, FF cfDNA level was an independent and significant predictive factor for pregnancy outcome (adjusted odds ratio = 0.69 [0.5; 0.96], p = 0.03). In multivariate analysis, the Receiving Operator Curve (ROC) analysis showed that the performance of FF cfDNA in predicting clinical pregnancy reached 0.73 [0.66-0.87] with 88% specificity and 60% sensitivity. CfDNA might constitute a promising biomarker of follicular micro-environment quality which could be used to predict IVF prognosis and to enhance female infertility management

[Comparison of frozen embryo transfer outcomes at blastocyst stage according to freezing method and type of endometrial preparation].

International audienceOBJECTIVE:This study intended to compare frozen embryo transfer (FET) outcomes at blastocyst stage according to freezing methods, slow freezing versus vitrification and according to the type of endometrial preparation.PATIENTS AND METHODS:A total of 172 FET at blastocyst stage (day 5 or 6) were included retrospectively from April, 2007 to December, 2012. The FET outcomes from slow freezing (group 1, n=86) were compared with those from vitrification (group 2, n=86). More particularly, the survival rate after thawing, as well as implantation and pregnancy rates (clinical and ongoing pregnancy rates) were compared respectively between these two groups, after matching on women's age at freezing day, embryo number and embryo development stage for transfer. Furthermore, for each freezing method, FET outcomes were compared according to the type of endometrial preparation, i.e. natural cycle (group N) versus stimulated cycle (group S).RESULTS:The survival rate as well as implantation and clinical pregnancy rates were significantly higher for FET after vitrification compared to FET after slow freezing (97% vs 85%, P<0.0001; 32% vs 20%, P=0.02; 43% vs 28%, P=0.04, respectively). By taking into account the number of transferred embryos for each group, the multiple pregnancy rate was three-fold higher in the group of FET after vitrification compared to the group of FET after slow freezing but not significantly (27.3% vs 8.3%, NS). However, FET outcomes were not affected significantly by the type of endometrial preparation whatever freezing methods. Nevertheless, the early spontaneous abortion (ESA) rate was lower in the case of embryos that were frozen by vitrification and transferred in natural cycle (group N2 vs group S2: 20% vs 47%, NS).DISCUSSION AND CONCLUSION:Our study confirms that the survival rate after thawing at blastocyst stage (day 5 or 6) is significantly improved after freezing by vitrification compared to slow freezing method. Likewise, implantation and clinical pregnancy rates are significantly increased in the case of FET at blastocyst stage when these embryos were frozen by vitrification. The results obtained by vitrification are very satisfactory but are also associated with an increased multiple pregnancy rate. Moreover, FET associated with natural or stimulated cycle does not modify significantly the outcomes of attempts, whatever the freezing method. However, the risk of ESA is reduced in the case of FET with natural cycle and after embryo vitrification

Cell-free DNA level in follicular fluid pools according to the patients’ ovarian reserve status, ovarian reserve parameters and infertility length.

<p><b>A</b>, Follicular fluid cfDNA content in patients with normal ovarian reserve versus patients with ovarian reserve disorders (ovarian insufficiency and polycystic ovary syndrome); *p = 0.03. <b>B</b>, Follicular fluid cfDNA content according to the ovarian reserve parameters; left panel: AFC (<10 versus ≥ 10, *p = 0.04); right panel: AMH (≤ 1 versus > 1 ng/ml, *p = 0.06). <b>C</b>, Follicular fluid cfDNA levels according to the infertility length (1 versus ≥ 5 years, *p = 0.049).</p

ROC curve to evaluate the predictive value of follicular fluid cfDNA level for clinical pregnancy outcome in a multivariate model (including the rank of IVF/ICSI attempts and the number of embryos): area under the curve = 0.73 [0.66–0.87], sensitivity = 60%, specificity = 88%.

<p>ROC curve to evaluate the predictive value of follicular fluid cfDNA level for clinical pregnancy outcome in a multivariate model (including the rank of IVF/ICSI attempts and the number of embryos): area under the curve = 0.73 [0.66–0.87], sensitivity = 60%, specificity = 88%.</p

CfDNA level in follicular fluid pools according to the ovarian stimulation protocol and ovarian response.

<p><b>A</b>, Follicular fluid cfDNA content according to the length of ovarian stimulation (≤ 10 versus > 10 days, *p = 0.008). <b>B</b>, Follicular fluid cfDNA content according to the total dose of gonadotropins (<3000 versus ≥3000 IU/l, *p = 0.01). <b>C</b>, Follicular fluid cfDNA content according to the number of retrieved oocytes (≤ 6 versus > 6 oocytes, *p = 0.045).</p

CfDNA level in follicular fluid pools according to the embryo outcome at day 2 and 3.

<p><b>A</b>, Follicular Fluid cfDNA content according to the total number of embryos at day 2 (≤ 2 versus > 2, *p = 0.03). <b>B</b>, Follicular Fluid cfDNA content according to, left panel: the number of top quality (grade 1–2) embryos per patient (0 versus ≥ 1, *p = 0.002) at day 2, right panel: ratio between number of top quality embryos and total number of embryos (< 0.2 versus ≥ 0.2, *p = 0.04) at day 2. <b>C</b>, Follicular Fluid cfDNA content according to, left panel: number of top quality (grade 1–2) embryos per patient (0 versus ≥ 1, *p = 0.006) at day 3, right panel: ratio between number of top quality embryo and total number of embryos (< 0.2 versus ≥ 0.2, *p = 0.02) at day 3. <b>D</b>, Follicular Fluid cfDNA content according to, left panel: fragmentation rate at day 3 (<20% versus ≥ 20%, p = 0.18) and right panel: ratio between blastomere number and total embryo number at day 3 (<6 versus 6–8, *p = 0.02).</p