Phospholipase C Zeta in Human Spermatozoa: A Systematic Review on Current Development and Clinical Application

During fertilization, the fusion of the spermatozoa with the oocytes causes the release of calcium from the oocyte endoplasmatic reticulum. This, in turn, triggers a series of calcium ion (Ca2+) oscillations, a process known as oocyte activation. The sperm-specific factor responsible for oocyte activation is phospholipase C zeta (PLCζ). Men undergoing intracytoplasmic sperm injection (ICSI) with their spermatozoa lacking PLCζ are incapable of generating Ca2+ oscillation, leading to fertilization failure. The immunofluorescence assay is the most used technique to assess the expression and localization of PLCζ and to diagnose patients with reduced/absent ability to activate the oocytes. In these patients, the use of assisted oocyte activation (AOA) technique can help to yield successful ICSI results and shorten the time of pregnancy. However, the production of a stable PLCζ recombinant protein represents a new powerful therapeutic approach to treating individuals with this condition. We aim to conduct a systematic review focusing on the expression, level, and localization of PLCζ, discussing the novel genetic mutation associated with its impairment. In addition, we highlight the benefits of AOA, looking at new and less invasive methods to diagnose and treat cases with PLCζ dysfunction.This research was funded by the Cátedra Human Fertility (A-GE-Cátedra Human Fertility 35.90.6Q..00.01) and Departamento de Biotecnología of the Universidad de Alicante (VIGROB-186)

Levels of dioxin-like PCBs in low-volume serum samples of male patients attending fertility clinics

An accurate and easy method for the extraction, cleanup, and HRGC-HRMS analysis of dioxin-like PCBs (DL-PCBs) in low-volume serum samples (1 mL) was developed. Serum samples were extracted several times using n-hexane and purified by acid washing. Recovery rates of labeled congeners ranged from 70 to 110 % and the limits of detection were below 1 pg/g on lipid basis. Although human studies are limited and contradictory, several studies have shown that DL-PCBs can have adverse effects on the male reproductive system. In this way, the present method was applied to 21 serum samples of male patients attending fertility clinics. The total levels obtained for the patients ranged from 6.90 to 84.1 pg WHO-TEQ/g lipid, with a mean value of 20.3 pg WHO-TEQ/g lipid. The predominant PCBs (the sum of PCB 118, 156, and 105) contributed 67 % to the mean concentration of total DL-PCBs in the samples analyzed.Support for this work was provided by the CTQ2013-41006-R project from the Ministry of Economy and Competitiveness (Spain), the PROMETEOII/2014/007 project from the Valencian Community Government (Spain), and the Chair of Human Fertility of the University of Alicante (Spain)

Optimization of the Analytical Method for Dioxin-Like PCBs in Low Volume Samples of Human Serum of Patients Facing Fertility Problems

Several studies indicate that human semen quality and fertility have declined over the last decades. According to the estimation of World Health Organization about 8% of couples at the global level experience some forms of infertility problem during their reproductive lives. This percentage means that 50 to 80 million people have problems with fertility. Several lifestyle-related (obesity, smoking) and environmental (exposure to traffic exhaust fumes, dioxins, combustion products) factors appear to negatively affect human fertility, emphasizing the importance of environmental/lifestyle impacts throughout the life course.Support for this work was provided by: Ministry of Education and Science (Spain) (CTQ2008-05520 project); Valencian Community Government (Spain) (PROMETEO/2009/043/FEDER and ACOMP2011/224 projects); Chair Human Fertility of University of Alicante

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Ensayo comparativo entre congelación lenta y vitrificación espermática

La congelación de espermatozoides es una técnica que supuso un importante paso para el desarrollo de la reproducción asistida. Esta es una técnica habitual y fundamental en las clínicas de reproducción que ha permitido lograr mejores resultados en las técnicas de reproducción asistida. A pesar de sus ventajas, durante la congelación, el espermatozoide sufre un gran número de alteraciones que dan lugar a la pérdida de función en el espermatozoide debido principalmente a: las bajas temperaturas a las que son sometidos, la cristalización de agua intracelular y los cambios osmóticos como consecuencia de la entrada y salida de crioprotector. Debido a los daños que se han observado, se está comenzando a desarrollar otro tipo de congelación ultrarápida llamada vitrificación, en la que los efectos que produce la entrada y salida de crioprotectores y los cambios de osmolaridad se ven disminuidos. Esta vitrificación es una técnica que consiste en el uso de crioprotectores diferentes y en un descenso de temperaturas brusco. Los primeros ensayos que se llevaron a cabo no resultaron exitosos debido a la poca tolerancia que presentan los espermatozoides a las altas tasas de crioprotector de las que se partieron. Ensayos posteriores en los que se reducía la concentración de crioprotector, se eliminaba el crioprotector permeable y la vitrificación se hacía mediante la aplicación directa de la solución espermática dentro de nitrógeno permitieron obtener mejores resultados. Es por ello que nos propusimos hacer un estudio en tres fases: una primera fase donde se realizara una valoración general de la congelación lenta y buscar un conjunto de biomarcadores útiles para la evaluación del criodaño. Posteriormente una segunda fase, en la que se realizó un estudio comparativo entre la congelación lenta y la vitrificación espermática, testando los biomarcadores elegidos en fase primera. Y, por último, en la tercera fase, un ensayo comparativo dentro de un ciclo de reproducción asistida donde la mitad del ciclo fuera fecundado con espermatozoides congelados y la otra mitad con espermatozoides vitrificados. Los resultados generales obtenidos mostraron que tras la congelación de muestras nomozoospérmicas y de baja calidad hay un conjunto de biomarcadores que dependen del estado inicial de la muestra y otros que dependen de la técnica de congelación. Que además se recomienda incluir en el estudio básico de la muestra seminal la fragmentación del ADN, el daño en el citoesqueleto y en el acrosoma, ya que son indicadores del criodaño. Que la nueva técnica de vitrificación permite criopreservar mejor las muestras seminales que la técnica de congelación lenta. Y que es seguro aplicar la técnica de vitrificación dentro de los ciclos de TRA, ya que se obtienen fecundaciones, desarrollos y niños nacidos normales.Cátedra Human Fertility, Ivf Spain Foundatio

First birth of a healthy infant following intra-cytoplasmic sperm injection using a new permeable cryoprotectant-free sperm vitrification protocol

Purpose: The purpose of this study is to present the first birth of healthy infant born following ICSI using the new permeable cryoprotectant-free sperm vitrification protocol Easy-Sperm®. Principal results: A 39 years old woman and his 40 years old partner underwent egg donation treatment at IVF-Spain Alicante (Spain). Half of the mature oocytes obtained from a young and healthy donor were fertilized by ICSI, using slow-frozen spermatozoa and the other half with vitrified spermatozoa. A total of 5 blastocysts were obtained on day 5 (3 resulting from vitrified spermatozoa and 2 from frozen sperm). The best embryo, with AA quality (derived from one of the oocytes fertilized with vitrified sperm) was transferred. The woman conceived and, following a normal pregnancy, delivered a healthy boy. Conclusions: To the best of our knowledge, this is the first case report of a successful pregnancy and delivery of a healthy infant from ICSI with permeable vitrified spermatozoa in an oocyte donation program with transfer on blastocyst stage.This study was funded by private funding (IVF-Spain Foundation and Human Fertility Chair)

Effectiveness of human spermatozoa biomarkers as indicators of structural damage during cryopreservation

Human spermatozoa cryopreservation techniques are used to maintain and protect male fertility in cases such as infertility and malignancy treatments. However, during cryopreservation, the spermatozoa's metabolic rate is reduced and they undergo dramatic functional and structural changes owing to exposure to cryoprotectants and freezing-thawing procedures. While the effects of cryopreservation on cells are documented, to date the induced cryodamage on structural and/or functional sperm biomarkers is not well established at multivariate scale. To address this question, we performed basic sperm analysis, sperm DNA fragmentation assessment, spontaneous acrosome reaction measurement, and cytoskeleton evaluation after thawing samples from subjects with normal and low-quality semen. A cryodamage rate was used to determine the effects of the freeze-thaw process on spermatozoa. In addition, a Principal Component Analysis (PCA) was used for data reduction and to evaluate sperm-specific patterns during the cryopreservation process. We found that the vitality, progressive motility and sperm count from low-quality samples after cryopreservation show higher damage rates (≥40%) than in normal sperm samples. However, cytoskeleton, DNA, tail and mid-piece and acrosome display the highest cryodamage rates (∼50–99%) and are equally susceptible to cryopreservation-induced damage in both low- and normal-quality semen samples. Overall, the evaluation of these parameters provides meaningful information about different aspects of sperm functionality after cryopreservation.We would like to appreciate financial support provided by the Cátedra Human Fertility (Universidad de Alicante, Alicante, Spain) grant number 60931013

Mejoramiento, mantenimiento y/o rehabilitación vías terciarias en cinco departamentos cafeteros

Existe una oportunidad de mejorar el sistema vial rural y la calidad de vida de los habitantes de las zonas rurales con el mejoramiento de vías terciarias mediante el uso y la construcción de placa huellas que permitan contrarrestar las dificultades que actualmente se presentan en algunas zonas de influencia cafetera de los departamentos de Cauca, Valle del Cauca, Quindío, Risaralda y Caldas. Una vez identificados los grupos que por la falta de buenas condiciones en las vías; tienen dificultad con el transporte de sus productos agrícolas, los excesivos costos del transporte y que adicionalmente han sido víctimas del conflicto armado; se gestionó recursos ante el OCAD PAZ logrando a través del INVIAS la adjudicación de 5 contratos de obra en alianza con la Federación Nacional de Cafeteros; y los comités de los departamentos involucrados en la ejecución de los contratos, estructuren y desarrollen un proyecto integral brindando una oportunidad de desarrollo para los municipios como Buenaventura, Dagua, Dovio, Balboa, Buenos Aires, Cajibio, Corinto, El Tambo, Florencia, Jambalo, López de Micay, Mercedes, Piamonte, Santa Rosa, Santander de Quilichao, Caldono, entre otros.There is an opportunity to improve the rural road system and the quality of life of the inhabitants of rural areas with the improvement of the territorial roads through the use and construction of the plate footprints that must counteract the difficulties that arise in the areas of Coffee influence of the departments of Cauca, Valle del Cauca, Quindío, Risaralda and Caldas. Once the groups that due to the lack of good conditions on the tracks; they have difficulties with the transport of their agricultural products, the excessive transportation costs and that have been additionally victims of the armed conflict; resources were managed before OCAD PAZ, through INVIAS, the awarding of 5 work contracts in partnership with the National Federation of Coffee Growers; and the committees of the departments involved in executing the contracts, structuring and developing an integral project, providing a development opportunity for the municipalities such as Buenaventura, Dagua, Dovio, Balboa, Buenos Aires, Cajibio, Corinto, El Tambo, Florencia, Jambalo , Lopez de Micay, Mercedes, Piedmont, Santa Rosa, Santander de Quilichao, Caldono, among others

New permeable cryoprotectant-free vitrification method for native human sperm

STUDY QUESTION: Is permeable cryoprotectant-free vitrification of native sperm samples a good alternative to conventional slow freezing? SUMMARY ANSWER: The permeable cryoprotectant-free sperm vitrification protocol tested in this study renders considerably better recovery rates of good quality sperm compared to slow freezing. WHAT IS KNOWN ALREADY: Slow freezing is currently the most commonly used technique for sperm cryopreservation, though this method has been repeatedly shown to have negative effects on both structural and functional sperm features. New alternative methods such as vitrification have been established as a successful alternative in other reproductive cell types, but vitrification of spermatozoa is still a rather unexplored methodology, with limited studies showing its efficacy in male gametes. STUDY DESIGN SIZE, DURATION: This study included 18 normozoospermic sperm samples from patients seeking ART treatment between 2014 and 2015. The effects of a new vitrification protocol on functional and structural sperm quality parameters in comparison to fresh and slow-frozen samples were assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: All samples were divided into three aliquots: fresh (F), slow freezing–thawing (S) and vitrification-warming (V). Sperm concentration, motility, morphology, vitality, DNA fragmentation, cytoskeleton integrity and spontaneous acrosome reaction were assessed and compared between the groups. MAIN RESULTS AND THE ROLE OF CHANCE: Results showed improved preservation of sperm features after vitrification compared to conventional freezing. Permeable cryoprotectant-free vitrification presented a significantly higher percentage of live spermatozoa, than slow freezing, better preservation of acrosomes was achieved in vitrified samples and DNA fragmentation was reduced approximately one-third on average compared to slow freezing. Regarding tubulin assay, three different labelling patterns were observed. The frequency of these labelling patterns was similar in F and V groups but this was not the case of the S group. The multivariate analysis of all sperm quality parameters studied revealed that the V group presented features that are closer to the F group than the S group, indicating that samples are better preserved through vitrification than slow freezing. LIMITATIONS REASONS FOR CAUTION: This validation has been undertaken only on normozoospermic sperm samples. It would be necessary to compare these results in pathological samples and also to evaluate the influence of the application of this methodology on clinical outcomes. WIDER IMPLICATIONS OF THE FINDINGS: The sperm vitrification protocol here described warrants better maintenance of sperm quality parameters than traditional freezing methods and may be a good alternative to preserve sperm samples from patients seeking IVF treatment. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by IVF-Spain Foundation. The authors have no conflicts of interest to declare.This study was funded by IVF-Spain Foundation