Endometrial whole metabolome profile at the receptive phase: influence of Mediterranean Diet and infertility

Introduction: Several metabolite classes have been identified in human endometrium, including lipids, nucleotides, amino acids, organic acids, and sugars. The first studies suggest the importance of metabolites in endometrial functions, as imbalance in uterine metabolites has been associated with low implantation rate and endometriosis. Nevertheless, most of studies have put emphasis on specific metabolite classes, and we lack the knowledge of the whole metabolome composition in human uterus. Further, a healthy dietary pattern has been shown to potentially protect against different endometrial dysfunctions and is a potential modulator of metabolomic composition and, consequently, the intrauterine microenvironment. The Mediterranean Diet (MD), characterized by a high intake of fruits, vegetables, cereals, nuts, legumes, fish, and olive oil, and a low consumption of meat, dairy products, and processed foods, has been associated with a wide range of benefits for health. Indeed, the MD pattern has displayed a beneficial role in endometriosis management and fertility; however, the relationship between the MD and the endometrial metabolome is still unknown. In our study, we set out to analyze receptivephase endometrial metabolome profiles among women with infertility and their associations with MD. Methods: The study included women with male factor infertility (n=8), unexplained infertility (n=10), recurrent implantation failure (n=14), and endometriosis (n=13). The endometrial metabolome was analyzed with ultrahigh-performance liquid chromatography-tandem mass spectroscopy (UPLC–MS/MS). The MD adherence of the participants was assessed using the 14-point MEDAS questionnaire of adherence to the MD. Results: We provide the whole metabolome profile of the endometrium, where 925 different metabolites were identified. Among these metabolites, lipids comprised the largest part, where polyunsaturated fatty acids (PUFAs) prevailed. Women with endometriosis and recurrent implantation failure were found to have lower levels of PUFAs compared to women with male factor and unexplained infertility (i.e., no clear endometrial alterations), identifying a metabolome profile associated with infertility diagnoses where altered endometrial functions are suspected. Moreover, MD adherence seemed to be associated with the endometrial metabolomic profile in a manner dependent on the health status of the uterus. Conclusion: The study findings provide insight into the molecular background of female infertility and lead to identification of potential molecular biomarkers and possibilities for modulating the endometrial microenvironment and, thereby, endometrial functions involved in embryo implantation and infertility.MCIN/AEI Endo-Map PID2021-12728OB-100 ENDORE SAF2017-87526-R PRE2018-085440 RYC-2016-21199 FPU19/03745 FPU19/01638 FPU19/01609ERFD A way of making EuropeESF Investing in your futureFEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento ROBIN A-CTS-614-UGR20 IRENE P20_00158University of Granada Plan Propio de Investigacion PPJIB2021-02Plan de Recuperacion, Transformacion y resiliencia, Ayudas para la recualificacion del sistema universitario espanol, Ayudas Margarita Sala

Mammalian spermatozoa and cumulus cells bind to a 3D model generated by recombinant zona pellucida protein-coated beads

The egg is a spherical cell encapsulated by the zona pellucida (ZP) which forms a filamentous matrix composed of several glycoproteins that mediate gamete recognition at fertilization. Studies on molecular mechanisms of sperm-egg binding are limited in many mammalian species by the scarcity of eggs, by ethical concerns in harvesting eggs, and by the high cost of producing genetically modified animals. To address these limitations, we have reproduced a three-dimensional (3D) model mimicking the oocyte’s shape, by means of magnetic sepharose beads coated with recombinant ZP glycoproteins (BZP) and cumulus cells. Three preparations composed of either ZP2 (C and N-termini; BZP2), ZP3 (BZP3) or ZP4 (BZP4) were obtained and characterized by protein SDS-PAGE, immunoblot and imaging with confocal and electron microscopy. The functionality of the model was validated by adhesion of cumulus cells, the ability of the glycoprotein-beads to support spermatozoa binding and induce acrosome exocytosis. Thus, our findings document that ZP-beads provide a novel 3D tool to investigate the role of specific proteins on egg-sperm interactions becoming a relevant tool as a diagnostic predictor of mammalian sperm function once transferred to the industry.Supported by Fundación Seneca-Agencia de Ciencia y Tecnología de la Región de Murcia “Ayudas a la realización de proyectos para el desarrollo de investigación científica y técnica por grupos competitivos 2018” (20887/PI/18), MINECO and FEDER (AGL2015 70159 P), Groups and Units of Scientific Excellence of the Region of Murcia (20040/GERM/16) and the AGL2015–66341‐R from the Ministry of Economy and Competitiveness (Spain)

Female upper reproductive tract harbors endogenous microbial profiles

The vaginal milieu is known to have an active microbiome (>90% of Lactobacillus), but the microbial composition of the upper reproductive tract is not well established, especially in the Fallopian tubes. The first studies on the Fallopian tubes from women diagnosed with a benign disease or for prophylaxis suggest that this site supports an endogenous microbiome. However, today we lack the knowledge of the microbial composition in Fallopian tubes in the non-diseased conditions (as collecting samples from these sites may hamper the tissue and future fertility). Our study includes 24 fertile women with benign uterine pathology submitted to abdominal hysterectomy or tubal ligation at Hospital Universitario Virgen de Arrixaca Murcia, which endometrial and Fallopian tube samples were collected between January and July 2019. After DNA extraction, “Ion 16S Metagenomics Kit” (Ion S5™ System) was used to exploit the V5 to V9 regions of the 16S rRNA gene. Primary data analysis was performed with Torrent Suite™ Software v5.12.1 and advanced analysis using Ion Reporter™ software v5.18.0.2. In our study, distinct microbial community profiles in the Fallopian tubes confirm that this genital tract site harbors an endogenous microbiome and in big part is shared with the endometrial microbial profile (69% of the detected taxa). Nevertheless, 17 bacterial taxa were exclusively detected in the Fallopian tubes that included Enhydrobacter, Granulicatella, Haemophilus, Rhizobium, Alistipes y Paracoccus, among others, while 10 were found only in the endometrium, including Klebsiella, Olsenella, Oscillibacter and Veillonella (FDR <0.05). Regarding the endometrium samples, our study shows that method collection has an influence in results, where there is a Lactobacillus-dominance in fertile women with samples obtained transcervically while Acinetobacter, Arthrobacter, Coprococcus, Methylobacterium, Prevotella, Roseburia, Staphylococcus, Streptococcus were less abundant in patients which samples are collected by methods with lower vaginal and cervical contamination. Although upper reproductive tract is a low microbial biomass site, our results suggest that this upper reproductive site supports an endogenous microbiome that could be characteristic of each individual

Year-Long Phenotypical Study of Calves Derived From Different Assisted-Reproduction Technologies

Assisted reproductive technologies play a major role in the cattle industry. An increase in the use of in vitro-derived embryos is currently being seen around the globe. But the efficiency and quality of the in vitro-derived embryos are substandard when compared to the in vivo production. Different protocols have been designed to overcome this issue, one of those being the use of reproductive fluids as supplementation to embryo culture media. In this study, in vitro-derived calves produced with reproductive fluids added to their embryo production protocol were followed for the first year of life pairwise with their in vivo control, produced by artificial insemination (AI), and their in vitro control, produced with standard supplementation in embryo production. The objective was to assess if any differences could be found in terms of growth and development as well as hematological and biochemical analytes between the different systems. All the analysed variables (physical, hematological, and biochemical) were within physiological range and very similar between calves throughout the entire experiment. However, differences were more evident between calves derived from standard in vitro production and AI. We concluded that the use of reproductive fluids as a supplementation to the embryo culture media results in calves with closer growth and development patterns to those born by AI than the use of bovine serum albumin as supplementation

Reproductive fluids, added to the culture media, contribute to minimizing phenotypical differences between in vitro-derived and artificial insemination-derived piglets

The addition of reproductive fluids (RF) to the culture media has shown benefits in different embryonic traits but its long-term effects on the offspring phenotype are still unknown. We aimed to describe such effects in pigs. Blood samples and growth parameters were collected from piglets derived from in vitro-produced embryos (IVP) with or without RF added in the culture media versus those artificially inseminated (AI), from day 0 to month 6 of life. An oral glucose tolerance test was performed on day 45 of life. We show here the first comparative data of the growth of animals produced through different assisted reproductive techniques, demonstrating differences between groups. Overall, there was a tendency to have a larger size at birth and faster growth in animals derived from in vitro fertilization and embryo culture versus AI, although this trend was diminished by the addition of RFs to the culture media. Similarly, small differences in hematological indices and glucose tolerance between animals derived from AI and those derived from IVP, with a sex-dependent effect, tended to fade in the presence of RF. The addition of RF to the culture media could contribute to minimizing the phenotypical differences between the in vitro-derived and AI offspring, particularly in males

Recogida, almacenamiento y caracterización de fluido oviductal y uterino humano para su futuro uso en tecnologías de reproducción asistida

La literatura actual, revisada en el capítulo I, proporciona evidencias crecientes sobre los beneficios potenciales de los fluidos reproductivos femeninos como suplementos para los medios de cultivo de embriones en la especie humana. A partir de esas evidencias, planteamos la hipótesis de que es posible recoger, caracterizar y validar el uso de estos fluidos en la especie humana y almacenarlos en un biobanco que cumpla todas las condiciones sanitarias y los requisitos legales para su uso futuro en investigación biomédica o en ensayos clínicos. Para evaluar la viabilidad de esta hipótesis, se definieron cuatro objetivos principales en esta tesis descritos en el capítulo II. El primero fue el desarrollo de un procedimiento para la recolección de fluido oviductal (FO) y FU humano en pacientes y donantes (Capítulo III). Después de probar varios métodos para la recolección de fluidos uterinos y oviductales humanos, se encontró que el FO humano (FOH) podría recolectarse ex vivo mediante el método descrito previamente por Carrasco et al., 2008 en modelos animales. Sin embargo, no se encontró ningún método o dispositivo en el mercado que permitiera la recolección de FU humano (FUH) in vivo. Por esta razón, se desarrolló un dispositivo novedoso para la recolección de fluidos humanos que ha sido objeto de una solicitud de patente en el presente. El establecimiento de un acuerdo de cooperación con la red de Biobancos en Murcia ha permitido crear la primera colección de biobancos de fluidos reproductivos humanos (Capítulo IV). En este capítulo, se describen el flujo de trabajo y los métodos de manipulación, registro, trazabilidad, transporte y almacenamiento de los fluidos, así como los números y tipos de muestras disponibles en Biobanc-Mur para uso futuro. En el Capítulo V, se seleccionaron un conjunto de parámetros físico-químicos, actualmente utilizados para las certificaciones incluidas en los controles de calidad (CC) requeridos para la venta de medios de cultivo humanos, y se optimizaron los métodos para evaluarlos. En el capítulo VI, se realizó un análisis proteómico de alta resolución de los fluidos del tracto reproductivo femenino durante la fase secretora del ciclo menstrual, lo que constituye una contribución novedosa al conocimiento del proteoma oviductal y uterino. El conjunto de muestras evaluado, de solo 3 individuos, fue muy valioso en comparación con otros estudios porque fue posible analizar los fluidos reproductivos y el plasma de las mismas mujeres, evitando el sesgo debido a la variabilidad individual. Los estudios anteriores se han centrado en FUH, pero nuestro estudio demuestra que FOH también es un líquido rico en proteínas esenciales que podrían ser un objetivo para mejorar las tasas de fecundación y el desarrollo temprano del embrión si se utilizan en los medios de cultivo. Se necesitan más estudios con diseños similares y con procedimientos operativos estándar establecidos para corroborar nuestros resultados / hipótesis y encontrar marcadores consistentes en las secreciones del tracto reproductor femenino. Por otro lado, establecer correlaciones entre la demografía y el estilo de vida de los donantes con sus resultados de FIV es de gran utilidad para los usuarios. Una mejor comprensión de la dinámica folicular y la respuesta ovárica a la estimulación con gonadotropina de los donantes podría optimizar el reclutamiento del donante y los tratamientos de FIV en el futuro. Por lo tanto, en el capítulo VII, se estudiaron las características demográficas y de estilo de vida en relación con la respuesta ovárica a la hiperestimulación entre las donantes de ovocitos y se encontraron evidencias de que algunos factores clave y potencialmente modificables pueden afectar los rendimientos de ovocitos en mujeres jóvenes sanas. Específicamente, encontramos que las mujeres con un índice de masa corporal (IMC) en el extremo superior del IMC normal, así como las mujeres con un IMC en el rango de sobrepeso, tenían un mayor rendimiento de ovocitos MII. Además, las mujeres que tomaron siestas que duraron más de 30 minutos tuvieron un mayor rendimiento de ovocitos, mientras que las mujeres que hicieron ejercicio durante al menos 5 horas cada semana tuvieron un rendimiento de ovocitos más bajo que las mujeres que no tomaron siestas y las mujeres que no hicieron ejercicio, respectivamente. En resumen, se lograron varios hitos importantes dentro de esta tesis: i) el desarrollo de un dispositivo para recolectar el fluido uterino humano in vivo; ii) La creación del primer biobanco de fluidos reproductivos humanos. La conciencia de la población sobre este biobanco y su impacto en las parejas infértiles puede contribuir para obtener más donaciones de donantes sanos; iii) el establecimiento de los rangos de seguridad para los parámetros utilizados para certificar la calidad de los fluidos reproductivos humanos almacenados en el biobanco; y iv) el análisis epidemiológico y proteómico de los fluidos reproductivos humanos, cuyas conclusiones resultantes permitirán mejorar el conocimiento sobre los componentes de los fluidos reproductivos y elaborar un método para utilizarlos como suplementos para los medios de cultivo de embriones. En conjunto, la estrategia desarrollada en esta tesis podría contribuir a prevenir alteraciones epigenéticas y mejorar la salud de la descendencia derivada de las TRA mediante sistemas de cultivo más naturales durante el desarrollo de embriones preimplantacionales in vitro.The current literature, reviewed in the chapter I, provide increasing evidence about the potential benefits of female reproductive fluids as supplements for embryo culture media in human species. From those evidences, we hypothesize that it is possible to collect, characterize and validate the use of these fluids in the human species, and to store them in a biobank fulfilling all the sanitary conditions and legal requirements for their future use in biomedical research or in clinical trials. In order to assess the viability of this hypothesis, four main objectives were defined in this thesis described in the chapter II. The first one was the development of a procedure for the collection of human oviductal (HOF) and UF in patients and donors (Chapter III). After testing several methods for the collection of human uterine and oviductal fluids, it was found that human OF (HUF) could be collected ex vivo by the method previously described by Carrasco et al 2008 in animal models. However, it was not found any method or device in the market allowing the collection of human UF (HUF) in vivo. For this reason, a novel device for human fluids collection was developed, which has been submitted for patenting at the present. The establishment of a cooperation agreement with the Biobank network in Murcia has allowed to create the first biobank collection of human reproductive fluids, (Chapter IV). In this chapter, the workflow and the methods for manipulation, registration, traceability, transportation and storage of the fluids is described as well as the numbers and types of samples available at Biobanc-Mur for future use. In Chapter V, a set of physico-chemical parameters, currently used for the certifications included in the quality controls (QC) required for the selling of human culture media, were selected, and the methods to assess them were optimized. In chapter VI, a high-throughput proteomic analysis of female reproductive tract fluids during the secretory phase of the menstrual cycle was performed, which constitutes a novel contribution to the knowledge of the oviductal and uterine proteome. The evaluated set of samples, of only 3 individuals, was highly valuable compared to other studies because it was possible to test the reproductive fluids and plasma of the same women, avoiding bias due to individual variability. Previous studies have focused on HUF, but our study demonstrates that HOF is also a rich fluid with essential proteins that could be a target to improve fertilization rates and early embryo development, if used in the culture media. More studies with similar designs and with established standard operating procedures are needed to corroborate our results/hypothesis and find consistent markers in secretions of female reproductive tract. On the other hand, establishing correlations between demographic and lifestyle of donors with their IVF outcomes has great utility for providers. A better understanding of follicular dynamics and ovarian response to gonadotropin stimulation of the donors could optimize the donor’s recruitment and IVF treatments going forward. Hence, in chapter VII, demographic and lifestyle characteristics in relation to ovarian response to hyperstimulation among oocyte donors were studied We evaluated a variety of demographic and lifestyle factors in relation to peak E2, total oocyte yield and MII oocyte yield among oocyte donors in Spain, and found evidence that some key and potentially modifiable factors may impact oocyte yields among otherwise young healthy women. Specifically, we found that women with a BMI on the high end of normal BMI, as well as women with a BMI in the overweight range, had a higher yield of MII oocytes. In addition, women who took naps lasting more than 30 minutes had a higher oocyte yield, whereas women who exercised for at least 5 hours every week had a lower oocyte yield than women who did not take naps and women who did not exercise, respectively. In summary, several important milestones were achieved within this thesis: i) the development of a device to collect human uterine fluid in vivo; ii) the creation of the first biobank of human reproductive fluids. The awareness of the population for this biobank and its impact on infertile couples may contribute for more donations from healthy donors; iii) the establishment of the safety ranges for the parameters used to certificate the quality of the human reproductive fluids stored in the biobank; and iv) the epidemiologic and proteomics analysis of human reproductive fluids, which resulting conclusions will allow to improve the knowledge about the reproductive fluids constituents, and to elaborate a method to use them as supplements for the embryo culture media. As a whole, the strategy developed in this thesis could contribute to prevent epigenetic alterations and to improve the health of ART-derived offspring by means of more natural culture systems during the in vitro preimplantational embryo development

Supplementation of bovine follicular fluid during in vitro maturation increases oocyte cumulus expansion, blastocyst developmental kinetics and blastocyst cell number.

Bovine follicular fluid (bFF) is the natural milieu for oocyte growth and development. However, its value as supplementation to in vitro maturation medium is still questioned due to inconsistent results. In this study we hypothesized that adding 10% of follicular fluid as well as heat treating it to inhibit the complement system, would produce higher quality embryos. To do so, experiments were conducted to compare the effect of bFF and heat-treated bFF (bFFin) on oocyte competence assessed by different parameters such as nuclear and cytoplasmic maturation, IVF efficiency, in vitro embryo development and embryo survivability post-vitrification. No differences on nuclear maturation nor cortical granules migration were observed but differences were found on oocyte’s cumulus cell expansion, with bFF group having the highest increase (79.0±3.7%). bFFin had a negative impact on IVF efficiency (58.6±3.2%), but no differences were found between bFF (62.9±3.2%) and control (72.8±3.0%). Although the cleavage and blastocyst rate were similar between groups, the day 6 embryo development rate was higher in bFFin group, suggesting an accelerated developmental kinetics. Hatched blastocysts from the bFF group showed a higher cell count than the control group (241.3±20.1 and 185.8± 10.0, respectively), and bFFin embryos showed values in between (214.9±14.0). No difference on survivability post-vitrification was found between groups, although the blastocyst stage had a significant impact on the survival rate across all groups. In conclusion, using bFF as supplementation to maturation medium showed a higher benefit when comparing to the standard supplementation by having oocytes with higher cumulus expansion rate, faster development of embryos and higher number of cells per embryo. Inactivation of bFF lowered IVF efficiency but didn’t compromise blastocyst development and quality.Horizon 2020 Marie Sklodowska-Curie Innovative Training Network (REPBIOTECH675526); Ministry of Economy and Competitiveness (Spain) (AGL2015-66341-R MINECO-FEDER) and Fundación Séneca (20040/GERM/16

Which Low-Abundance Proteins are Present in the Human Milieu of Gamete/Embryo Maternal Interaction?

The improvement of the embryo culture media is of high relevance due to its influence on successful implantation rates, pregnancy, neonatal outcomes, and potential effects in adult life. The ideal conditions for embryo development are those naturally occurring in the female reproductive tract, i.e., the oviductal and uterine fluids. To shed light on the differences between chemical and natural media, we performed the first comparative study of the low abundance proteins in plasma, uterine, and oviductal fluid collected, simultaneously, from healthy and fertile women that underwent a salpingectomy. The rationale for this design derives from the fact that high-abundant proteins in these fluids are usually those coming from blood serum and frequently mask the detection of low abundant proteins with a potentially significant role in specific processes related to the embryo&ndash;maternal interaction. The proteomic analysis by 1D-nano LC ESI-MSMS detected several proteins in higher amounts in oviductal fluid when compared to uterine and plasma samples (RL3, GSTA1, EZRI, DPYSL3, GARS, HSP90A). Such oviductal fluid proteins could be a target to improve fertilization rates and early embryo development if used in the culture media. In conclusion, this study presents a high-throughput analysis of female reproductive tract fluids and contributes to the knowledge of oviductal and uterine secretome

Seminovaginal microbiome: it takes two to tango

Infertility, adverse pregnancy outcomes, and genital infections are conditions that can affect any couple in the global population. The reproductive tract microbiome appears to play a crucial role in the physiology of both the female and male reproductive tracts. Despite the presence of thousands of microbes in body fluids shared during unprotected sexual intercourse, they have traditionally been studied separately, with greater emphasis on the female (mostly vaginal) microbiome. Consequently, the concept of “seminovaginal microbiome” emerges to address both microbial niches as a whole that would provide more detailed understanding and potential solutions to the reproductive success. This systematic review discusses the state-of-the-art of the complementary microbiome, encompassing its diversity and composition, and how it is linked to the couples’ health and disease, the success of assisted reproductive techniques and pregnancy, and the occurrence of microbe-associated diseases such as sexually transmitted diseases, prostatitis, bacterial vaginosis, and candidiasis. Additionally, the microbial interplay in homosexual couples and transsexual individuals is discussed