Belén Lledó

Improving human embryos selection in IVF: non-invasive metabolomic and chemometric approach

We present here a new metabolomic methodology to predict embryo implantation ability in in vitro fertilization (IVF). In the present study we have included a total of 23 patients scheduled for IVF. Embryos were selected to be transferred by using morphological criteria on day 3 of in vitro culture. The relative amino acid concentrations in the embryo culture media were analyzed by HPLC–MS and HPLC–MS/MS. 1H NMR metabolomic profiles were also obtained for the embryo culture media. Chemometric models were performed with SIMCA (soft independent modeling of class analogy) for samples from both, non-pregnancy and pregnancy cycles. The metabolic differences between the embryos, with pregnancy and non-pregnancy outcome, can be correlated with the relative amino acid concentrations and with 1H NMR profiles. We used interval partial least square (iPLS) in order to identify the higher correlation between regions in the 1H NMR spectra and the embryo implantation capability. The 1H NMR regions with higher correlation are between 1.2 and 0.5xa0ppm, that included the signals for cholesterol backbone –C(18)H3, –CH3 and CH2 groups of triglycerides, cholesterol compounds and phospholipids. Our results can allow building a quick, non invasive, useful and feasible chemometric models in order to identify embryos with a high pregnancy rate and embryos unable to achieve successful pregnancies.

A crucial step in assisted reproduction technology: human embryo selection using metabolomic evaluation.

We present a new methodology to predict embryo viability in assisted reproductive technology (ART) treatments by determining the relative amino acid concentrations in human embryo culture medium on day 3, using high-performance liquid chromatography with mass spectroscopy analysis without derivatization. The model was performed with soft independent modeling of class analogy for the samples from nonpregnancy and pregnancy cases.

Effect of follicle-stimulating hormone receptor N680S polymorphism on the efficacy of follicle-stimulating hormone stimulation on donor ovarian response.

Objective The aim of this study was to investigate whether N680S FSHR polymorphism has a predictive value for the ovarian response to stimulation with gonadotropins and cycle outcome in our egg donor program. Methods The oocyte donor candidates were selected according to the Instituto Bernabeu egg donation program requirements and ASRM and ESHRE guidelines for oocyte donation. The FSHR polymorphism N680S was studied in 145 oocyte donors. All donors underwent controlled ovarian hyperstimulation (COH) (n=355) using urinary follicle-stimulating hormone in a GnRH antagonist protocol and receiving a GnRH agonist triggering. The main outcome measures were oocyte yield, days of stimulation, gonadotropin doses, biochemical pregnancy, ongoing pregnancy, and miscarriage rates. Results Significant differences were reported in the antral follicle count (16.5±5.0 for NN, 14.5±4.7 for NS, and 14.1±3.8 for SS), number of eggs retrieved (21.5±9.2 for NN, 18.5±8.2 for NS, and 19.8±8.9 for SS), and gonadotropin doses (2098.5±639.4 IU for NN, 2023 ±490.1 IU for NS, and 2149.5±552.3 IU for SS) between the genotypes. The clinical outcome was not affected by the N680S polymorphism of the FSHR gene in the egg donors. Conclusion In a population of fertile egg donors, the FSHR gene polymorphism at position 680 is associated with different ovarian responses to COH. The genotype of the FSHR gene is an important factor for determining the prognosis of the COH cycles in normo-ovulatory fertile women.

The effect of ammonium on assimilatory nitrate reduction in the haloarchaeon Haloferax mediterranei

Physiology, regulation and biochemical aspects of the nitrogen assimilation are well known in Prokarya or Eukarya but they are poorly described in Archaea domain. The haloarchaeon Haloferax mediterranei can use different nitrogen inorganic sources (NO3−, NO2− or NH4+) for growth. Different approaches were considered to study the effect of NH4+ on nitrogen assimilation in Hfx. mediterranei cells grown in KNO3 medium. The NH4+ addition to KNO3 medium caused a decrease of assimilatory nitrate (Nas) and nitrite reductases (NiR) activities. Similar effects were observed when nitrate-growing cells were transferred to NH4+ media. Both activities increased when NH4+ was removed from culture, showing that the negative effect of NH4+ on this pathway is reversible. These results suggest that ammonium causes the inhibition of the assimilatory nitrate pathway, while nitrate exerts a positive effect. This pattern has been confirmed by RT-PCR. In the presence of both NO3− and NH4+, NH4+ was preferentially consumed, but NO3− uptake was not completely inhibited by NH4+ at prolonged time scale. The addition of MSX to NH4+ or NO3− cultures results in an increase of Nas and NiR activities, suggesting that NH4+ assimilation, rather than NH4+ per se, has a negative effect on assimilatory nitrate reduction in Hfx. mediterranei.

Negative effect of P72 polymorphism on p53 gene in IVF outcome in patients with repeated implantation failure and pregnancy loss

PurposeInvestigate whether R72P on p53 gene polymorphism has a higher prevalence among women with a history of recurrent implantation failure (RIF) and pregnancy loss (RPL) and its influence in their IVF cycle outcome.Material and methodsp53 polymorphism R72P has been studied in 181 women. The control group included 83 oocyte donors. In the study group 98 women were included: 44 with RIF and 54 with RPL. From the study group, 76 patients underwent IVF-cycles (55 RPL and 21 RIF).ResultsThe frequency of PP genotypes on p53 among RIF was 11.4xa0% compared with 18.5xa0% for RPL and 6xa0% in controls (pu2009<u20090.01). There were no significant differences with respect to patient characteristics. Significant differences were reported in pregnancy rate (69.4xa0% for RR/RP and 33.3xa0% for PP; pu2009<u20090.05), embryo implantation rate (33.3xa0% for RR/RP and 7.3xa0% for PP; pu2009<u20090.05) and ongoing pregnancy rate (53.1xa0% for RR/RP and 14.3xa0% for PP; pu2009<u20090.05) among RIF and RPL.ConclusionsThis investigation reveals that in RIF and RPL patients R72P on p53 gene is more prevalent than fertile population. Moreover, patients carrying a PP genotype on p53 codon 72 will have less chance to achieve an ongoing pregnancy. This information together with some additional markers will allow development of diagnostic tests for detects risk for RIF and RPL before infertility treatment is initiated.

NO3−/NO2− assimilation in halophilic archaea: physiological analysis, nasA and nasD expressions

The haloarchaeon Haloferax mediterranei is able to assimilate nitrate or nitrite using the assimilatory nitrate pathway. An assimilatory nitrate reductase (Nas) and an assimilatory nitrite reductase (NiR) catalyze the first and second reactions, respectively. The genes involved in this process are transcribed as two messengers, one polycistronic (nasABC; nasA encodes Nas) and one monocistronic (nasD; codes for NiR). Here we report the Hfx mediterranei growth as well as the Nas and NiR activities in presence of high nitrate, nitrite and salt concentrations, using different approaches such as physiological experiments and enzymatic activities assays. The nasA and nasD expression profiles are also analysed by real-time quantitative PCR. The results presented reveal that the assimilatory nitrate/nitrite pathway in Hfx mediterranei takes place even if the salt concentration is higher than those usually present in the environments where this microorganism inhabits. This haloarchaeon grows in presence of 2xa0M nitrate or 50xa0mM nitrite, which are the highest nitrate and nitrite concentrations described from a prokaryotic microorganism. Therefore, it could be attractive for bioremediation applications in sewage plants where high salt, nitrate and nitrite concentrations are detected in wastewaters and brines.

Chromosomal polymorphic variants increase aneuploidies in male gametes and embryos

ABSTRACT Chromosomal polymorphisms involve heterochromatic regions and occur in the general population. However, previous studies have reported a higher incidence of these variants in infertile patients. The aim of this study was to examine the relationship between polymorphic variants and infertility and their association with aneuploidies in male gametes and embryos. We retrospectively considered 1,551 cytogenetic studies involving infertile patients (study group; n=866) and oocyte/sperm donors as the control group (n=685). We had detected 168 polymorphisms in the study group and 92 in the control group. An increase in the frequency of polymorphic variants was observed among infertile patients (19.4% study group vs. 13.4% control group; P < 0.01). Sperm aneuploidies among 145 infertile men were evaluated by fluorescent in situ hybridization (FISH). The frequency of infertile men with increased rates of sperm aneuploidy was higher among polymorphism carriers. Twenty men showed an abnormal rate of sperm aneuploidy in the carrier group (n=53) vs. 15 in the non-carrier group (n=92) (37.7% vs. 16.3%, respectively; P < 0.01). Finally, aneuploidies in blastocysts (n=301) resulting from donated oocytes were also examined by array comparative genomic hybridization (array-CGH). Significant differences were reported in the embryo aneuploidy rate between female carriers and non-carriers in oocyte donation cycles (50.0% vs. 27.6%; P < 0.001). This study suggests that polymorphic variants have an impact on fertility. Moreover, our results show a relationship between polymorphisms and aneuploidy in spermatozoa and embryos. Abbreviations: FISH: fluorescent in situ hybridization; CGH: comparative genomic hybridization; ESHRE: European Society of Human Reproduction and Embryology; ASRM: American Society for Reproductive Medicine; RPL: recurrent pregnancy loss; WHO: World Health Organization; ISCN: International System for Human Cytogenetic Nomenclature guidelines; WGA: whole genome amplification; SPSS: Statistical Package for Social Sciences

Pharmacogenetics of ovarian response

Effective controlled ovarian stimulation (COS) is crucial for IVF outcome. Ovarian response to follicle-stimulating hormone, however, varies widely among women undergoing ovarian stimulation. Advance identification of patients who will elicit a poor or high response to standard treatment would be of great clinical benefit for such patients. Application of pharmacogenetics to ovarian response may predict stimulation success but also help in the adjustment and design of doses prior to treatment. Different studies have examined the impact of variations in follicle-stimulating hormone receptor, biochemical pathways involved in estrogen production and action, folliculogenesis and other aspects. Recently, gene-association studies have tried to identify a number of genetic variations affecting interindividual variability in COS.

Implantation potential of mosaic embryos

ABSTRACT Chromosomal mosaicism is a relatively common finding in human IVF embryos. However, the association between mosaicism in trophoectoderm and inner mass cells, the mechanisms involved, and its effects on implantation are far from established. We retrospectively reanalyzed array-CGH results from 1,362 trophoectoderm biopsies. We detected chromosomal mosaicism in 183 blastocysts (13.4%). A decrease in the clinical pregnancy rate when we compared the cycles where only mosaic embryos were transferred (26.9%) vs. euploid embryos were transferred (40.2%) was not statistically different (p = 0.127). Also a tendency to increase the biochemical miscarriage was reported (21.2% mosaic group vs. 12.3% euploid group; p=0.102). Our data suggests that the transfer of some mosaic embryos achieve full term pregnancies. Additional studies are needed to clarify how embryo mosaicism affects the outcomes of the IVF cycles.

Characterization of a balanced complex chromosomal rearrangement carrier ascertained through a fetus with dup15q26.3 and del5p15.33: case report

Abstract Complex chromosomal rearrangements (CCRs) are structural aberrations involving more than two chromosomes which rarely appear in individuals with normal phenotypes. These individuals report fertility problems, recurrent miscarriages, or congenital anomalies in newborn offspring as a consequence of either meiotic failure or imbalanced chromosome segregation. A CCR involving chromosomes 5, 15, and 18 was discovered in a phenotypically normal man through a fetus with congenital malformations and partial trisomy of chromosome 15 and monosomy of chromosome 5. Ultrasound examination at 20 weeks of gestation showed severe oligoamnios and hydrothorax. Prenatal cytogenetic analysis and array comparative genomic hybridization (array-CGH) revealed a female fetus with dup15q26.3 and del5p15.33. We diagnosed the CCR using three-color fluorescence in situ hybridization (three-color FISH), and a balanced CCR using array-CGH and FISH was diagnosed in the paternal karyotype. The father is a carrier of a balanced translocation 46,XY,t(5;15;18)(p15.31;q26.3;p11.2). Due to the complexity of these rearrangements the diagnosis is difficult and the reproductive outcome uncertain. Reporting such rare cases is important to enable such information to be used for genetic counseling in similar situations and help estimate the risk of miscarriage or of newborns with congenital abnormalities.