Tumor Necrosis Factor-Alpha and Pregnancy: Focus on Biologics. An Updated and Comprehensive Review.

Tumor necrosis factor-α (TNF-α) is a central regulator of inflammation, and TNF-α antagonists may be effective in treating inflammatory disorders in which TNF-α plays a major pathogenic role. TNF-α has also been associated with inflammatory mechanisms related to implantation, placentation, and pregnancy outcome. TNF-α is secreted by immune cells and works by binding to TNFR1 and TNFR2 cell receptors. TNF-α is also related to JAK/STAT pathways, which opens up hypothetical new targets for modifying. The accurate balance between Th1 cytokines, mainly TNF-α, Th17, and Th2, particularly IL-10 is essential to achieve good obstetric outcomes. TNF-α targeted therapy could be rational in treating women with obstetric complication related to overproduction of TNF-α, such as recurrent pregnancy loss, early and severe pre-eclampsia, and recurrent implantation failure syndrome, all "idiopathic" or related to aPL positivity. Along the same lines, Th1 cytokines, mainly TNF- α, play a leading pathogenic role in rheumatic and systemic autoimmune diseases occurring in women and, to a lesser extent, in men of reproductive age. These disorders have to be clinically silent before pregnancy can be recommended, which is usually only possible to achieve after intensive anti-inflammatory and immunosuppressive treatment, TNF-α blockers included. Physicians should be aware of the theoretic potential but low embryo-fetal toxicity risk of these drugs during pregnancy. From an updated review in May 2016, we can conclude that TNF-α blockers are useful in certain "refractory" cases of inflammatory disorders related to poor obstetric outcomes and infertility. Furthermore, TNF-α blockers can be safely used during the implantation period and pregnancy. Breastfeeding is also permitted with all TNF-α inhibitors. Since data on the actual mechanism of action of JAK-STAT in inflammatory obstetric disorders including embryo implantation are scarce, for the time being, therapeutic interventions in this setting should be discouraged. Finally, adverse effects on sperm quality, or causing embryo-fetal anomalies, in men treated with TNF inhibitors have not been described.

Estandarización en la técnica de pretratamiento y tinción para la realización de la morfología espermática humana automatizada tipo asma (assisted sperm morphometry analysis) / Standardization of pretreatment and staining technique to perform automatic human sperm morphology with asma (assisted sperm morphometry analyses)

Objetivo. Estandarización de la técnica de pretratamiento y tinción para la automatización de la morfología espermática con metodología tipo computer-assisted sperm morphometry analysis, con el analizador de sémenes SCA 5.4 (Sperm Class Analyzer, Microptic). Material y métodos. La automatización de la morfología se ha realizado con el analizador de semen SCA 5.4 (Microptic S.L., Barcelona, España). El método de tinción ha sido una modificación del equipo Hemacolor (Merck). Se procesan entre 69 y 125 sémenes, los cuales han sido muestras escogidas aleatoriamente de nuestro laboratorio. Resultados. El pretratamiento de la muestra de semen escogido, debido a los resultados obtenidos, fue una centrifugación suave durante 5 min a 300g, se descarta el plasma seminal y se homogeneiza suavemente el sedimento con 0,2ml del propio plasma seminal. Este pretratamiento ya se comprobó que no artefactaba los espermatozoides. La tinción que se ha escogido es el kit Hemacolor (Merck), pero con modificaciones. Se tampona el fijador con buffer fosfato pH 7,2 al 10%, se reduce los tiempos aconsejados por el fabricante a fijación durante 5seg, tinción con eosina 30 seg y tinción con azur 2 seg. Finalmente se lava 30 seg con tampón fosfato pH 7,2, como indica el fabricante. Tras dicho pretratamiento y tinción estandarizadas se hallan los coeficientes de variación del pretratamiento y valores de referencia para nuestra metodología. Conclusiones. La automatización de la morfología espermática reduce los coeficientes de variación de la determinación, aumentando su fiabilidad técnica y eliminando la subjetividad que conlleva un análisis manual. Esta estandarización constituye el primer paso para el estudio del valor diagnóstico de la morfología avanzada en la infertilidad y en enfermedades urológicas (AU)

Objective. Standardization of pretreatment and staining technique to realize morphological semen evaluation with computer-assisted sperm morphometry analysis system, with semen system analyzer SCA 5.4 (Sperm Class Analyzer, Microptic). Material and methods. Morphological analysis was performed with semen system analyzer SCA 5.4 (Microptic SL, Barcelona, Spain). The staining method was a modification of Hemacolor kit (Merck). Between 60 and 125 semen samples were chosen randomly from our laboratory. Results. Pretreatment of semen samples was a centrifugation for 5minutes at 300g, seminal plasma was rejected and the pellet was homogenized with .2mL of the seminal plasma itself. Not change in sperm morphology have been found with this pretreatment. Staining was performed with Hemacolor kit (Merck) but with some modifications. Fixer has been buffered with phosphate buffer pH 7.2 at 10%, time recommended by the manufacturer has been reduced. Fixation for 5 seconds, 30 seconds with Eosin staining and 2 seconds with staining Azur. Finally it was washed for 30 seconds with pH 7.2 phosphate buffer, as indicated by the manufacturer. After the pretreatment and staining we have got reference values for our methodology. Conclusions. An automation methodology to perform sperm morphology reduces coefficient variations of determination thereby we can increase its technical reliability and remove the subjectivity of the manual analysis. This standardization can be the first step to study the diagnostic value of advanced morphology in infertility and urological diseases (AU)

Linfocitos T reguladores, tolerancia maternofetal y aborto recurrente: nuevas oportunidades terapéuticas / Regulatory T cells, maternal-foetal immune tolerance and recurrent miscarriage: new therapeutic challenging opportunities

Because maternal alloreactive lymphocytes are not depleted during pregnancy, local and/or systemic mechanisms have to play a key role in altering the maternal immune response. Peripheral T regulatory cells (pTregs) at the maternal-foetal interface are necessary in situ to prevent early abortion, but only those pTregs that have been previously exposed to paternal alloantigens. It has been showed that pregnancy selectively stimulates the accumulation of maternal Foxp3+ CD4+CD25+ (Foxp3Tregs) cells with foetal specificity. Interestingly, after delivery, foetal-specific pTregs persist at elevated levels, maintain tolerance to pre-existing foetal antigen, and rapidly re-accumulate during subsequent pregnancy. pTreg up-regulation could be hypothesized as a possible future therapeutic strategy in humans (AU)

Considerando que los linfocitos T alorreactivos no son completamente eliminados durante la gestación, parece necesario que haya otros mecanismos locales y/o generales que colaboren en la modificación de la respuesta inmunitaria materna. Los linfocitos T reguladores periféricos (Tregs-p) de la interfaz maternofetal previamente expuestos a antígenos paternos son necesarios in situ para prevenir el aborto precoz. Se ha demostrado que durante la gestación se produce una acumulación de Tregs-p Foxp3+ CD4+ CD25+ con especificidad para ciertos antígenos fetales. Tras la gestación, estos Tregs-p con especificidad para antígenos fetales persisten a títulos elevados y mantienen la tolerancia materna, incrementando su número y funcionalidad en la siguiente gestación. El incremento de los Tregs-p podría ser una nueva y esperanzadora estrategia terapéutica en humanos (AU)

Regulatory T cells, maternal-foetal immune tolerance and recurrent miscarriage: new therapeutic challenging opportunities.

Because maternal alloreactive lymphocytes are not depleted during pregnancy, local and/or systemic mechanisms have to play a key role in altering the maternal immune response. Peripheral T regulatory cells (pTregs) at the maternal-foetal interface are necessary in situ to prevent early abortion, but only those pTregs that have been previously exposed to paternal alloantigens. It has been showed that pregnancy selectively stimulates the accumulation of maternal Foxp3(+)CD4(+)CD25(+) (Foxp3Tregs) cells with foetal specificity. Interestingly, after delivery, foetal-specific pTregs persist at elevated levels, maintain tolerance to pre-existing foetal antigen, and rapidly re-accumulate during subsequent pregnancy. pTreg up-regulation could be hypothesized as a possible future therapeutic strategy in humans.

A versatile strategy for preimplantation genetic diagnosis of haemophilia A based on F8-gene sequencing.

Preimplantation genetic diagnosis (PGD) of hemophilia A (HA) and other X-linked diseases through sex selection implies that male embryos will be systematically discarded, even though 50% are unaffected. The objective of the present work was to develop a PGD protocol for direct mutation identification that could be applied to first polar bodies (1PBs) in several HA clinical cases. Single buccal cells from controls and patients, and 1PBs were subjected to primer extension preamplification (PEP) PCR followed by amplification of F8 gene coding and intronic flanking regions, and direct sequencing. Moreover, multiplex fluorescent amplification of four short tandem repeats was adapted to a single cell preamplification in order to rule out contamination and allele drop-out, and for confirmatory indirect diagnosis. A couple at risk of HA transmission, with a familial mutation characterized as a 41-bp duplication in exon 14 of the F8 gene, was selected for the first clinical study. After optimizing the protocol, the complete F8 gene coding sequence was obtained from single cells to demonstrate the sensitivity of our methodology although in any clinical case only the relevant region, not the whole gene, must be amplified. The woman enrolled in the first clinical case has completed the first in-vitro fertilization cycle, and seven oocytes were analyzed with concordant results by both linkage analysis and direct sequencing method. Only one oocyte, among those diagnosed as mutation free, developed to embryo at day 3. It was transferred but pregnancy was not achieved. This PGD procedure enables non-affected and noncarrier embryo selection in families with any point or small-range mutation in the F8 gene, without the need for further custom-made modifications.