Clinical validation of an automatic classification algorithm applied on cleavage stage embryos: analysis for blastulation, euploidy, implantation, and live-birth potential.

STUDY QUESTION: Is a commercially available embryo assessment algorithm for early embryo evaluation based on the automatic annotation of morphokinetic timings a useful tool for embryo selection in IVF cycles? SUMMARY ANSWER: The classification provided by the algorithm was shown to be significantly predictive, especially when combined with conventional morphological evaluation, for development to blastocyst, implantation, and live birth, but not for euploidy. WHAT IS KNOWN ALREADY: The gold standard for embryo selection is still morphological evaluation conducted by embryologists. Since the introduction of time-lapse technology to embryo culture, many algorithms for embryo selection have been developed based on embryo morphokinetics, providing complementary information to morphological evaluation. However, manual annotations of developmental events and application of algorithms can be time-consuming and subjective processes. The introduction of automation to morphokinetic annotations is a promising approach that can potentially reduce subjectivity in the embryo selection process and improve the workflow in IVF laboratories. STUDY DESIGN, SIZE, DURATION: This observational, retrospective cohort study was performed in a single IVF clinic between 2018 and 2021 and included 3736 embryos from oocyte donation cycles (423 cycles) and 1291 embryos from autologous cycles with preimplantation genetic testing for aneuploidies (PGT-A, 185 cycles). Embryos were classified on Day 3 with a score from 1 (best) to 5 (worst) by the automatic embryo assessment algorithm. The performance of the embryo classification model for blastocyst development, implantation, live birth, and euploidy prediction was assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: All embryos were monitored by a time-lapse system with an automatic cell-tracking and embryo assessment software during culture. The embryo assessment algorithm was applied on Day 3, resulting in embryo classification from 1 to 5 (from highest to lowest developmental potential) depending on four parameters: P2 (t3-t2), P3 (t4-t3), oocyte age, and number of cells. There were 959 embryos selected for transfer on Day 5 or 6 based on conventional morphological evaluation. The blastocyst development, implantation, live birth, and euploidy rates (for embryos subjected to PGT-A) were compared between the different scores. The correlation of the algorithm scoring with the occurrence of those outcomes was quantified by generalized estimating equations (GEEs). Finally, the performance of the GEE model using the embryo assessment algorithm as the predictor was compared to that using conventional morphological evaluation, as well as to a model using a combination of both classification systems. MAIN RESULTS AND THE ROLE OF CHANCE: The blastocyst rate was higher with lower the scores generated by the embryo assessment algorithm. A GEE model confirmed the positive association between lower embryo score and higher odds of blastulation (odds ratio (OR) (1 vs 5 score) = 15.849; P < 0.001). This association was consistent in both oocyte donation and autologous embryos subjected to PGT-A. The automatic embryo classification results were also statistically associated with implantation and live birth. The OR of Score 1 vs 5 was 2.920 (95% CI 1.440-5.925; P = 0.003; E = 2.81) for implantation and 3.317 (95% CI 1.615-6.814; P = 0.001; E = 3.04) for live birth. However, this association was not found in embryos subjected to PGT-A. The highest performance was achieved when combining the automatic embryo scoring and traditional morphological classification (AUC for implantation potential = 0.629; AUC for live-birth potential = 0.636). Again, no association was found between the embryo classification and euploidy status in embryos subjected to PGT-A (OR (1 vs 5) = 0.755 (95% CI 0.255-0.981); P = 0.489; E = 1.57). LIMITATIONS, REASONS FOR CAUTION: The retrospective nature of this study may be a reason for caution, although the large sample size reinforced the ability of the model for embryo selection. WIDER IMPLICATIONS OF THE FINDINGS: Time-lapse technology with automated embryo assessment can be used together with conventional morphological evaluation to increase the accuracy of embryo selection process and improve the success rates of assisted reproduction cycles. To our knowledge, this is the largest embryo dataset analysed with this embryo assessment algorithm. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by Agencia Valenciana de Innovació and European Social Fund (ACIF/2019/264 and CIBEFP/2021/13). In the last 5 years, M.M. received speaker fees from Vitrolife, Merck, Ferring, Gideon Richter, Angelini, and Theramex, and B.A.-R. received speaker fees from Merck. The remaining authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

[Application of a protocol for the management of adnexal masses: savings in clinically unnecessary activity and costs].

BACKGROUND: Evaluate whether the implementation of an adnexal masses protocol, based on the GI-RADS system, allows a correct management of these masses, avoiding unnecessary clinical activity produced by overdiagnosis and overtreatment, as well as cost savings. METHODS: Retrospective cohort study (July 2015 - June 2017) including women treated at the Gynaecology clinic of the Hospital Universitario Rey Juan Carlos (Móstoles, Madrid), with detection of an adnexal mass in high resolution echography. Adnexal masses were classified by the GI-RADS system, and together with the echographic image and menopausal status, surgery or follow-up was decided. RESULTS: A total of 154 women were studied, 24?% with images suggesting malignancy (G4 and G5). Surgery was performed on 33.1?% of adnexal masses; 33.3?% of them were ovarian carcinoma, mainly (88.2?%) in postmenopausal women with echographic images suggesting malignancy. Three point two percent of patients rejected the recommended surgery. During follow-up 21.4?% of the masses disappeared, 61 patients were only monitored due to a stable mass and two (1.3?%) due to surgical risk. Eventually, 96 (62.3?%) surgeries were avoided, achieving a 57,683 Euro saving. CONCLUSIONS: The application of a protocol based on the GI-RADS classification system avoided unnecessary surgeries, as well as the consequences and economical cost produced by them. Thus, this protocol is a useful and practical tool for the monitoring and treatment of adnexal masses.

A new system of sperm cryopreservation: evaluation of survival, motility, DNA oxidation, and mitochondrial activity.

BACKGROUND: Sperm vitrification (V) is a method for cryopreservation, without the use of conventional cryoprotectants, by plunging the sperm suspension directly into liquid nitrogen (LN25). OBJECTIVE: This study aimed to compare the new system of V with conventional freezing (CF) protocol using fresh spermatozoa as reference (C). MATERIAL AND METHODS: Prospective cohort study. A total of 47 sperm samples from men attending the infertility clinic at Instituto Valenciano de Infertilidad Valencia. The sperm V solution was 0.3 M trehalose-sucrose and plunged directly in liquid nitrogen in microdroplets of 5-10 lL, using a new system collector of V. Sperm viability indicators such as sperm motility, vitality rates, mitochondrial function, and sperm DNA oxidation were assessed before and after cryopreservation. Sperm motility and vitality analysis were performed according to published guidelines of the World Health Organization (WHO, 2010). Mitochondrial function was evaluated using JC-1 (fluorescent cationic dye, 5,50,6,60-tetrachloro-1-10,3,30-tetraethyl-benzamidazolocarbocyanin iodide). Sperm DNA oxidation was determined using a fluorescent assay (Oxy-DNA test) for the detection of 8-oxoguanine. The evaluation was carried out before and after cryopreservation using flow cytometry. Statistical analysis was performed using ANOVA and chi-square test, and p < 0.05 was considered statistically significant. RESULT(S): Sperm parameters, including progressive motility, total motility, and viability, observed after cryopreservation were as follows: C = 74.9% [1] 12.3, CF = 27.2% [1] 8.4, V = 42.3% [1] 9.3, p < 0.001; C = 90.1 [1] 6.8, CF = 42.0 [1] 12.9, V = 61.4 [1] 11.8, p < 0.001; C = 90.0% [1] 7.4, CF = 42.5% [1] 14.6, V = 70.9% [1] 6.5, p < 0.001, respectively. Regarding Oxy-DNA and mitochondrial activity, they were significantly affected in both groups (V and CF) when compared to the control group. DISCUSSION: The sperm V and CF have negative impact on sperm parameters as well as DNA integrity and mitochondrial activity. However, sperm V presented improved sperm motility recovery, similar levels of DNA oxidation, and, moreover, a slightly increase in mitochondrial activity when compared to the conventional method. CONCLUSION(S): V as an optimal protocol for sperm cryopreservation.

A comparison of morphokinetic markers predicting blastocyst formation and implantation potential from two large clinical data sets.

PURPOSE: To demonstrate whether the standard morphokinetic markers used for embryo selection have a similar relationship to blastocyst formation and implantation in two large clinical data sets. METHODS: This is a retrospective cohort analysis striving to answer two distinct questions utilizing data sets from two large IVF clinics. Blastocysts (BL) and implanted blastocysts (I) in both clinics, IVI-Valencia (BL = 11,414, I = 479) and WMC (BL = 15,902; I = 337), were cultured in a time-lapse system (EmbryoScope, Vitrolife, Sweden). The study was designed to assess the relationship between early morphokinetic hallmarks and BL development, with a secondary analysis of implantation rates following single-embryo day 3 and day 5 transfers. RESULTS: We performed a detailed graphical analysis for t3, t5, duration of the second cell cycle (cc2) (t3-t2), and the ratio (t5-t3)/(t5-t2). The t5 timing was not affected between the clinics. However, Weill Cornell Medicine's (WCM) proportions were significantly affected by having BL vs. not. A significant decrease of blastocysts with longer t5 in WCM data, while t5 was more informative in the IVI data set for the implantation rate. CONCLUSIONS: Morphokinetic intervals for early cleavages were distributed differently between the clinics. Incorporation of embryo-selection algorithms depends on the individual clinic's selected developmental hallmarks, all of which must be validated before incorporation into clinical practice.

New insights into the performance of multigene panel testing: Two novel nonsense variants in BRIP1 and TP53 in a young woman with breast cancer.

Li-Fraumeni syndrome is an autosomal-dominant disorder caused by germline mutations in the tumour suppressor gene TP53. Here we report the case of a family whose index case was a woman diagnosed with bilateral breast cancer at the age of 18 and who had a non-informative result after BRCA1 and BRCA2 testing. After extending the study through multigene panel testing, two clinically relevant variants in the TP53 and BRIP1 genes, respectively, were found. Afterwards, the patient developed a glioblastoma. Both tumours were consistent with Li-Fraumeni syndrome. Thanks to the possibility of studying different genes related with hereditary breast and ovarian cancer, it was possible to find out the gene variant that caused the early onset cancers in the patient. Furthermore, genetic counselling was provided to the index case and her family.

High sperm DNA fragmentation delays human embryo kinetics when oocytes from young and healthy donors are microinjected.

BACKGROUND: Time-lapse monitoring (TLM) technology has been implemented in the clinical setting for the culture and selection of human embryos. Many studies have assessed the association between sperm DNA fragmentation (sDNAf) and clinical outcomes after ART, but little is known about the influence of sDNA on embryo morphokinetics. OBJECTIVES: The objective of this retrospective study, which includes 971 embryos from 135 consecutive ICSI cycles (56 cases with own oocytes, 79 with oocytes from young and healthy donors), was to assess if sDNAf has an impact on embryo morphokinetics. MATERIALS AND METHODS: Samples used to perform ICSI were analyzed by the flow cytometry TUNEL assay, and embryo development was assessed through an EmbyoScope® system. The association between sDNAf and the timings of cell cleavage was analyzed by categorizing the first variable into quartiles: ≤6.50%; 6.51-10.70%; 10.71-20.15%; >20.15%. RESULTS: In cases where sDNAf was above 20.15% (the upper quartile), embryos derived from donated oocytes (n = 644) showed significantly slower divisions. Such association was not observed in embryos obtained from the patients' own oocytes (n = 327). The embryo cleavage pattern (either normal, direct from 1 to 3 blastomeres, direct from 1 to 4 blastomeres, incomplete, reversed or asynchronous) was independent of the sDNAf level. Blastocyst arrival rate was 63.0% and the rate of good quality embryos (transferred and frozen embryos divided by the number of zygotes) was 45.49%. Neither parameter was related to the levels of sDNAf. DISCUSSION: According to our results, the association between high sDNAf and donated oocytes led to delayed cell division. To our knowledge, this is the first study suggesting that sDNAf can delay human embryo cleavage timings when oocytes from donors are inseminated. CONCLUSIONS: This finding may indicate that, in the presence of increased DNA damage, time is needed before the first embryonic cell division for the activation of the optimal DNA repairing machinery in higher quality oocytes.

Cáncer de páncreas familiar asociado a mutación en el gen BRCA2 / Familial pancreatic cancer asociated to germline mutation of BRCA2

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Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development.

STUDY QUESTION: What is the origin and composition of cell-free DNA in human embryo spent culture media? SUMMARY ANSWER: Cell-free DNA from human embryo spent culture media represents a mix of maternal and embryonic DNA, and the mixture can be more complex for mosaic embryos. WHAT IS KNOWN ALREADY: In 2016, ~300 000 human embryos were chromosomally and/or genetically analyzed using preimplantation genetic testing for aneuploidies (PGT-A) or monogenic disorders (PGT-M) before transfer into the uterus. While progress in genetic techniques has enabled analysis of the full karyotype in a single cell with high sensitivity and specificity, these approaches still require an embryo biopsy. Thus, non-invasive techniques are sought as an alternative. STUDY DESIGN, SIZE, DURATION: This study was based on a total of 113 human embryos undergoing trophectoderm biopsy as part of PGT-A analysis. For each embryo, the spent culture media used between Day 3 and Day 5 of development were collected for cell-free DNA analysis. In addition to the 113 spent culture media samples, 28 media drops without embryo contact were cultured in parallel under the same conditions to use as controls. In total, 141 media samples were collected and divided into two groups: one for direct DNA quantification (53 spent culture media and 17 controls), the other for whole-genome amplification (60 spent culture media and 11 controls) and subsequent quantification. Some samples with amplified DNA (N = 56) were used for aneuploidy testing by next-generation sequencing; of those, 35 samples underwent single-nucleotide polymorphism (SNP) sequencing to detect maternal contamination. Finally, from the 35 spent culture media analyzed by SNP sequencing, 12 whole blastocysts were analyzed by fluorescence in situ hybridization (FISH) to determine the level of mosaicism in each embryo, as a possible origin for discordance between sample types. PARTICIPANTS/MATERIALS, SETTING, METHODS: Trophectoderm biopsies and culture media samples (20 µl) underwent whole-genome amplification, then libraries were generated and sequenced for an aneuploidy study. For SNP sequencing, triads including trophectoderm DNA, cell-free DNA, and follicular fluid DNA were analyzed. In total, 124 SNPs were included with 90 SNPs distributed among all autosomes and 34 SNPs located on chromosome Y. Finally, 12 whole blastocysts were fixed and individual cells were analyzed by FISH using telomeric/centromeric probes for the affected chromosomes. MAIN RESULTS AND THE ROLE OF CHANCE: We found a higher quantity of cell-free DNA in spent culture media co-cultured with embryos versus control media samples (P ≤ 0.001). The presence of cell-free DNA in the spent culture media enabled a chromosomal diagnosis, although results differed from those of trophectoderm biopsy analysis in most cases (67%). Discordant results were mainly attributable to a high percentage of maternal DNA in the spent culture media, with a median percentage of embryonic DNA estimated at 8%. Finally, from the discordant cases, 91.7% of whole blastocysts analyzed by FISH were mosaic and 75% of the analyzed chromosomes were concordant with the trophectoderm DNA diagnosis instead of the cell-free DNA result. LIMITATIONS, REASONS FOR CAUTION: This study was limited by the sample size and the number of cells analyzed by FISH. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to combine chromosomal analysis of cell-free DNA, SNP sequencing to identify maternal contamination, and whole-blastocyst analysis for detecting mosaicism. Our results provide a better understanding of the origin of cell-free DNA in spent culture media, offering an important step toward developing future non-invasive karyotyping that must rely on the specific identification of DNA released from human embryos. STUDY FUNDING/ COMPETING INTEREST: This work was funded by Igenomix S.L. There are no competing interests.

Epidemiology and Risk Factors for Cancer After Lung Transplantation.

Cancer is the third most common cause of death among lung transplant (LT) recipients who survive for more than 1 year. The purpose of this study was to analyze the incidence and risk factors for cancer after LT in a Spanish cohort. The epidemiology and risk factors for cancer were retrospectively analyzed in LT recipients from 2 cities in Spain, Madrid and Barcelona. Of the 1353 LT patients initially included in the study, 125 (9.2%) developed cancer after a mean of 3.7 years. This frequency was 5-fold higher than in the general population. The most prevalent tumors were skin cancer (32%), lymphoproliferative disease (18%), and lung cancer (16.5%). In 4 patients, lung cancer was diagnosed on the day of the operation. The risk of cancer increased with age >55 year (hazard ratio [HR] 2.89 [1.64-5.09]; P < .001), in men (HR 2.8 [1.4-5.6]; P = .004), and in heavy smokers (>20 pack-years) (HR 2.94 [1.64-5.27]; P < .001). Other factors such as sun exposure were not found to be risk factors. In conclusion, prevalence of cancer is high in LT recipients in a Mediterranean country. Skin tumors, lymphoproliferative disease, and lung cancer are the most prevalent cancers. Age, male sex, and smoking were the main risk factors for cancer in this population.

An homozygous mutation in KCNK3 is associated with an aggressive form of hereditary pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare devastating disease characterized by a high genetic heterogeneity with several related genes recently described, including BMPR2,TBX4 and KCNK3. The association between KCNK3 and PAH has been recently identified, but the prognosis and phenotype associated with these mutations have been poorly described. We studied a series of 136 idiopathic and hereditary PAH Spanish patients for BMPR2, TBX4 and KCNK3 mutations. We report the results of KCNK3 in which we were able to describe two new mutations (p.Gly106Arg and p.Leu214Arg) in three patients. The first one was found in a patient belonging to a consanguineous Romani family, who carried a homozygous mutation in KCNK3 and developed a severe and early form of the disease. To the best of our knowledge, this is the first time that a homozygous mutation in KCNK3 is reported in a PAH patient. The second one was found in a patient who presented at the young adult age a severe form of the disease. The present report supports the contribution of KCNK3 mutations to the genetic etiology of PAH and strongly suggests that mutations in KCNK3 follow incomplete dominance with worsening of the clinical features in homozygous patients.

How much have we learned from time-lapse in clinical IVF?

Can the time-lapse system (TLS) identify the best embryo for transfer? Although there are several studies that support this hypothesis, more research is required to improve the quality of the current evidence and also to assess live birth rate, miscarriage, stillbirth or clinical pregnancy in order to choose between a TLS or conventional incubation. In addition, although some authors report on effectiveness and safety in the use of TLS monitoring of embryo development in vitro, other authors that have not found relevant differences between the two systems for the culture and subsequence embryo selection. On the other hand, TLS has emerged as a novel technology and has been introduced into clinical practice in many laboratories to perform embryo morphology evaluation and study developmental kinetics in ART. However, most studies only assess blastocyst formation or implantation rate as the primary end-point and additional data are required, for example, about live birth, monozygotic twinning rates and health problems. Furthermore, the features of populations studies are varied; for example, female and male age, seminal characteristics and female factor. The embryo culture conditions and culture medium used also vary. For this review, a search of PubMed was conducted to retrieve relevant studies regarding use of TLS in embryo incubation and selection, and compare them with standard embryo culture and evaluation.

What is that? HIV-negative plasmablastic lymphoma with intramuscular masses.

Plasmablastic lymphoma is a relatively new clinical entity described as a distinct subtype of diffuse large B-cell lymphoma, although in the last decade several case reports and series have been published. This case is presented because of its rarity, as this pathology is rare in immunocompetent patients and intramuscular masses are present. We report the case of a 63-year-old male with no significant clinical background. He was referred to the emergency department of our hospital with a 10-day history of pain on the left side of the chest that was described as burning and spreading to the right side. On physical examination, he had no fever or recent weight loss. The abdomen was soft and non-distended, and no peritoneal signs were present but he had three palpable masses located in the soft tissues of the breast, right gluteal region and left leg. Histological examination of the biopsy specimens disclosed the diagnosis of plasmablastic lymphoma. To our knowledge, this will be the second case report referring to intramuscular masses in the English language literature.

Situación actual del estado nutricional del yodo en gestantes de la región de Murcia, España / Current situation of nutritional state of iodide in pregnant women in Murcia región, Sapin / Situação atual do estado nutricional do iodo em gestantes da região de Múrcia, Espanha

El objetivo de este trabajo consistió en identificar los niveles de yodo en le primer trimestre del embarazo en mujeres atendidas en los centros de salud , pertenecientes al área sanitaria I de Murcia (España) , asi como la realacion de dichos niveles con la ingesta y los suplementos de yodo. por otra parte se realizo el estudio de la funcion tiroidea en el mismo grupo. Se realizó un estudio descriptivo funcional. El grupo de estudio consistió en 37 mujeres embarazadas, que habían acudido a la primera visita en su centro de salud correspondiente desde los meses de marzo hasta agosto de 2011. las variables de estudio fueron: niveles de yodo en orina medidos en una muestra aislada de orina, frecuencia de la ingesta de yodo relacionada con la alimentación y suplementos (datos obtenidos mediante una entrevista personal y un cuestionario estructurado realizado entre participantes del estudio), niveles de hormona tiroestimulante TSH y T4 libre ambas obtenidas a partir de muestras de sangre. El 86% del grupo en estudio presentaba deficiencias de yodo. Hubo diferencias estadísticamente significativas en cuanto a la excresión urinaria de yodo ,en la ingesta de sal iodada y de pescado. Sin embargo cualquier otra variable no presento diferencias significativas. Se hallaron tres embarazadas con hipotiroidismo subclínica y una presentó hipertiroidismo sub clínico. Además,estos resultados mostraron que los niveles de TSH pueden ser mas bajos en embarazadas que en la población general. En base a los datos de ioduria obtenidos, los suplementos de yodo en este grupo se consideraron insuficientes. Además se concluye que sería recomendable incluir la determinación sistemática de T 4 libre y TSH en el primer trimestre del embarazo, para corregir una posible disfunción tiroidea tan pronta como sea posible y así evitar daños al feto...

Situación actual del estado nutricional del yodo en gestantes de la región de Murcia, España / Current situation of nutritional state of iodide in pregnant women in Murcia region, Spain / SituaþÒo atual do estado nutricional do iodo em gestantes da regiÒo de Múrcia, Espanha

El objetivo de este trabajo consistió en identificar los niveles de yodo en el primer trimestre del embarazo en mujeres atendidas en centros de salud, pertenecientes al área sanitaria I de Murcia (España), así como la relación de dichos niveles con la ingesta y los suplementos de yodo. Por otra parte se realizó el estudio de la función tiroidea en el mismo grupo. Se realizó un estudio descriptivo funcional. El grupo de estudio consistió en 37 mujeres embarazadas, que habían acudido a la primera visita en su centro de salud correspondiente desde los meses de marzo hasta agosto de 2011. Las variables de estudio fueron: niveles de yodo en orina medidos en una muestra aislada de orina, frecuencia de la ingesta de yodo relacionada con la alimentación y suplementos (datos obtenidos mediante una entrevista personal y un cuestionario estructurado realizado entre las participantes del estudio), niveles de hormona tiroestimulante TSH y T4 libre ambas obtenidas a partir de una muestra de sangre. El 86% del grupo en estudio presentaba deficiencia de yodo. Hubo diferencias estadísticamente significativas en cuanto a la excreción urinaria de yodo, en la ingesta de sal iodada y de pescado. Sin embargo, cualquier otra variable no presentó diferencias significativas. Se hallaron tres embarazadas con hipotiroidismo subclínico y una presentó hipertiroidismo subclínico. Además, estos resultados mostraron que los niveles de TSH pueden ser más bajos en embarazadas que en la población general. En base a los datos de ioduria obtenidos, los suplementos de yodo en este grupo se consideraron insuficientes. Además, se concluye que sería recomendable incluir la determinación sistemática de T4 libre y TSH en el primer trimestre del embarazo, para corregir una posible disfunción tiroidea tan pronto como sea posible y así evitar daños al feto.(AU)

The aim of this work was to identify iodine levels in the first term of pregnancy in women attending health care centers of Sanitary Area I of Murcia (Spain), and the relationship of said levels with food intake and iodine supplements. Apart from that, thyroid gland function was assessed in the same group. A descriptive transversal study was performed. The sample group consisted of 37 pregnant women who had a first appointment at the corresponding midwifery service from March to August 2011. The variables studied were urinary iodine levels through isolated sample collection, frequency of intake of iodine related to food and iodine supplements (data obtained through a face to face interview and a structured questionnaire administered among the participats in the study), and levels of thyroid-stimulating hormone TSH and free T4 obtained from a blood sample. Eighty-six per cent of the study group had iodine deficiency. Statistically significant differences were found in urinary iodine excretion, in intake of iodine salt and of sea food. However, no other variable studied presented any statistically significant difference. Three pregnant women were found with subclinical hypothyroidism and one presented subclinical hyperthyroidism. Furthermore, these results showed that TSH levels can be lower in pregnant women than in the general population. On the basis of the normal urinary iodine excretion obtained, the supply of iodine in the study group is insufficient. Moreover, It would be desirable to include the systematic determination of free T4 and TSH in the first term of pregnancy so as to to correct a likely thyroid dysfunction as soon as possible in order to avoid possible damage to the foetus.(AU)

O objetivo deste trabalho consistiu em identificar os níveis de iodo no primeiro trimestre de gravidez em mulheres atendidas em centros de saúde, pertencentes O área sanitária I de Múrcia (Espanha), bem como na relaþÒo de tais níveis com a ingestÒo e os suplementos de iodo. Por outra parte foi realizado o estudo da funþÒo tireoidiana no mesmo grupo. Foi feito um estudo descritivo funcional. O grupo de estudo consistiu em 37 mulheres grávidas, que tinham assistido O primeira visita no centro de saúde correspondente, desde marþo até agosto de 2011. As variáveis de estudo foram: níveis de iodo em urina, medidos numa amostra isolada de urina, frequÛncia da ingestÒo de iodo relacionada com a alimentaþÒo e suplementos (dados obtidos através de uma entrevista pessoal e um questionário estruturado realizado entre as participantes do estudo), níveis de horm¶nio tiroestimulante TSH e T4 livre, ambas obtidas a partir de uma amostra de sangue. 86% do grupo em estudo apresentava deficiÛncia de iodo. Houve diferenþas estatisticamente significativas quanto O excreþÒo urinária de iodo, na ingestÒo de sal iodado e de peixe. Entretanto, qualquer outra variável nÒo apresentou diferenþas significativas. Foram encontradas trÛs mulheres grávidas com hipotireoidismo subclínico e uma apresentou hipertireoidismo subclínico. Além disso, estes resultados mostraram que os níveis de TSH podem ser mais baixos em mulheres grávidas que na populaþÒo geral. Com base nos dados de iodúria obtidos, os suplementos de iodo neste grupo foram considerados insuficientes. Além do mais, seria recomendável incluir a determinaþÒo sistemática de T4 livre e TSH no primeiro trimestre da gravidez, para corrigir uma possível disfunþÒo tireoidiana assim que for possível e, desse modo, evitar danos no feto.(AU)

A founder EIF2AK4 mutation causes an aggressive form of pulmonary arterial hypertension in Iberian Gypsies.

Pulmonary arterial hypertension (PAH) is a pathological condition characterized by a persistent and progressive elevation of pulmonary vascular resistance with devastating consequences if untreated. In the past recent years, several genes have been related to PAH, however, the molecular defect remains unknown in a significant proportion of patients with familial PAH (∼20%). During the past few years, we have observed that PAH shows a particular behavior in Iberian Gypsies, with more aggressive course and frequently affecting multiple members of the same family. We studied five Gypsy families in whom at least one individual from each family developed a severe form of PAH and in whom no mutation had been identified in the common genes. We applied SNP-array-based homozygosity mapping in three families and obtained, among others, one of which included the gene EIF2AK4, recently reported in patients with PAH from group-1' pulmonary veno-occlusive disease (PVOD) and pulmonary capillary hemangiomatosis (PCH). Subsequently, we sequenced EIF2AK4 and found a homozygous mutation in all five families: c.3344C>T(p.P1115L). The majority of our patients required early lung transplantation. Hence, this mutation appeared with a more severe phenotype than previously reported for other EIF2AK4 mutations. The finding of this novel mutation is important for genetic counseling and calculation of population recurrence risks.

The use of morphokinetics as a predictor of  implantation: a multicentric study to define and validate an algorithm for embryo selection.

STUDY QUESTION: Can we use morphokinetic markers to select the embryos most likely to implant and are the results likely to be consistent across different clinics? SUMMARY ANSWER: Yes, morphokinetic markers can be used to select the embryos most likely to implant and the results were similar in different IVF clinics that share methods and organization to some extent. WHAT IS KNOWN ALREADY: With the introduction of time-lapse technology several authors have proposed the use of kinetic markers to improve embryo selection. The majority of these markers can be detected as early as Day 2 of development. Morphology remains the gold standard but kinetic markers have been proven as excellent tools to complement our decisions. Nevertheless, the majority of time-lapse studies are based on small data sets deriving from one single clinic. STUDY DESIGN, SIZE, DURATION: Retrospective multicentric study of 1664 cycles of which 799 were used to develop an algorithm (Phase 1 of the study) and 865 to test its predictive power (Phase 2 of the study). PARTICIPANTS/MATERIALS, SETTING, METHODS: University-affiliated infertility centres patients undergoing first or second ICSI cycle using their own or donated oocytes. Embryo development was analysed with a time-lapse imaging system. Variables studied included the timing to two cells (t2), three cells (t3), four cells (t4) and five cells (t5) as well as the length of the second cell cycle (cc2 = t3 - t2) and the synchrony in the division from two to four cells (s2 = t4 - t3). Implantation (IR) and clinical pregnancy (CPR) rates were also analysed. MAIN RESULTS AND THE ROLE OF CHANCE: During Phase 1 of the study we identified three variables most closely related to implantation: t3 (34-40 h), followed by cc2 (9-12 h) and t5 (45-55 h). Based on these results we elaborated an algorithm that classified embryos from A to D according to implantation potential. During Phase 2 of the study the algorithm was validated in a different group of patients that included 865 cycles and 1620 embryos transferred. In this phase of the study, embryos were categorized based on the algorithm and significant differences in IR were observed between the different categories ('A' 32%, 'B' 28%, 'C' 26%, 'D' 20% and 'E' 17%, P < 0.001). In addition we identified three quality criteria: direct cleavage from one to three cells, uneven blastomere size in second cell cycle and multinucleation in third cell cycle. LIMITATIONS, REASONS FOR CAUTION: The retrospective nature of the study limits its potential value, although the use of one database to generate the algorithm (embryos from this database were not selected by any morphokinetic criteria) and one database to validate it reinforces our conclusions. WIDER IMPLICATIONS OF THE FINDINGS: The elaboration of an algorithm based on a larger database derived from different (albeit related) clinics raises the possibility that such algorithms could be applied in different clinical settings.

Biomarkers of pulmonary rejection.

Immunologic complications after lung transplantation (LT) include acute cellular rejection (ACR), antibody-mediated rejection (AMR), and most forms of chronic allograft dysfunction (CAD). ACR is an inflammatory process in which the reaction is mediated by the T-cell population. Most episodes of ACR fully recover with treatment, but repeated bouts are considered to be a risk factor for CAD. Biomarker cytokines interleukin (IL)-10, IL-15, IL-6, CCL5, CCR2 and IFNγ may play significant roles in this complication. Formerly bronchiolitis obliterans syndrome (BOS) or chronic rejection or most forms of CAD were considered to be immunologic complications not amenable therapeutic measures. CAD, the main limitation for long-term survival in LT, is characterized histologically by airway epithelial cell apoptosis and luminal fibrosis in the respiratory bronchioles causing airflow obstruction and, in some cases, lung parenchymal affectations causing restrictive lung disease. Several biomarkers have been studied in CAD, IL-6, IL-8, IL-17, IL-23, IL-13, IFN γ, and TGF ß cytokines, pH, bile acid, and tripsine of gastroesophageal reflux and toll-like receptors of innate immunity. Herein we have reviewed the literature of biomarkers involved in lung rejection.