Testing an artificial intelligence algorithm to predict fetal heartbeat of vitrified-warmed blastocysts from a single image: predictive ability in different settings.

STUDY QUESTION: Could an artificial intelligence (AI) algorithm predict fetal heartbeat from images of vitrified-warmed embryos? SUMMARY ANSWER: Applying AI to vitrified-warmed blastocysts may help predict which ones will result in implantation failure early enough to thaw another. WHAT IS KNOWN ALREADY: The application of AI in the field of embryology has already proven effective in assessing the quality of fresh embryos. Therefore, it could also be useful to predict the outcome of frozen embryo transfers, some of which do not recover their pre-vitrification volume, collapse, or degenerate after warming without prior evidence. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study included 1109 embryos from 792 patients. Of these, 568 were vitrified blastocysts cultured in time-lapse systems in the period between warming and transfer, from February 2022 to July 2023. The other 541 were fresh-transferred blastocysts serving as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Four types of time-lapse images were collected: last frame of development of 541 fresh-transferred blastocysts (FTi), last frame of 467 blastocysts to be vitrified (PVi), first frame post-warming of 568 vitrified embryos (PW1i), and last frame post-warming of 568 vitrified embryos (PW2i). After providing the images to the AI algorithm, the returned scores were compared with the conventional morphology and fetal heartbeat outcomes of the transferred embryos (n = 1098). The contribution of the AI score to fetal heartbeat was analyzed by multivariate logistic regression in different patient populations, and the predictive ability of the models was measured by calculating the area under the receiver-operating characteristic curve (ROC-AUC). MAIN RESULTS AND THE ROLE OF CHANCE: Fetal heartbeat rate was related to AI score from FTi (P < 0.001), PW1i (P < 0.05), and PW2i (P < 0.001) images. The contribution of AI score to fetal heartbeat was significant in the oocyte donation program for PW2i (odds ratio (OR)=1.13; 95% CI [1.04-1.23]; P < 0.01), and in cycles with autologous oocytes for PW1i (OR = 1.18; 95% CI [1.01-1.38]; P < 0.05) and PW2i (OR = 1.15; 95% CI [1.02-1.30]; P < 0.05), but was not significantly associated with fetal heartbeat in genetically analyzed embryos. AI scores from the four groups of images varied according to morphological category (P < 0.001). The PW2i score differed in collapsed, non-re-expanded, or non-viable embryos compared to normal/viable embryos (P < 0.001). The predictability of the AI score was optimal at a post-warming incubation time of 3.3-4 h (AUC = 0.673). LIMITATIONS, REASONS FOR CAUTION: The algorithm was designed to assess fresh embryos prior to vitrification, but not thawed ones, so this study should be considered an external trial. WIDER IMPLICATIONS OF THE FINDINGS: The application of predictive software in the management of frozen embryo transfers may be a useful tool for embryologists, reducing the cancellation rates of cycles in which the blastocyst does not recover from vitrification. Specifically, the algorithm tested in this research could be used to evaluate thawed embryos both in clinics with time-lapse systems and in those with conventional incubators only, as just a single photo is required. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the Regional Ministry of Innovation, Universities, Science and Digital Society of the Valencian Community (CIACIF/2021/019) and by Instituto de Salud Carlos III (PI21/00283), and co-funded by European Union (ERDF, 'A way to make Europe'). M.M. received personal fees in the last 5 years as honoraria for lectures from Merck, Vitrolife, MSD, Ferring, AIVF, Theramex, Gedeon Richter, Genea Biomedx, and Life Whisperer. There are no other competing interests. TRIAL REGISTRATION NUMBER: N/A.

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.

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.

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.

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.

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.

Similar morphokinetic patterns in embryos derived from obese and normoweight infertile women: a time-lapse study.

STUDY QUESTION: Does female obesity affect the dynamic parameters of embryo quality assessed by time-lapse analysis? SUMMARY ANSWER: Female obesity does not affect the dynamic embryo quality as determined by image acquisition and time-lapse analysis. WHAT IS KNOWN ALREADY: Female obesity impairs natural and assisted reproduction but there is no agreement on the specific contribution of gametes, embryos or endometrial receptivity. In this preliminary study the dynamic parameters of embryo quality are assessed for the first time by time-lapse analysis. STUDY DESIGN, SIZE, DURATION: Two-year cohort retrospective study comparing embryos from three groups of patients according to the presence of infertility and/or obesity. PARTICIPANTS AND SETTING: Participants attended a University-affiliated private clinic where ICSI was performed. Using an IVF incubator with a built-in camera designed to automatically acquire images at defined time points, we monitored individual embryos from 89 patients: 71 embryos from 13 obese infertile women, 242 embryos from 45 normoweight infertile women and 111 embryos from 31 normoweight fertile oocyte donors. The chronological pattern of cell divisions (timings of cell cleavages) and other morphologic features (time-dependent cell size and nucleation) was recorded. MAIN RESULTS AND THE ROLE OF CHANCE: Embryos from obese and normoweight infertile women showed similar cleavage patterns, but occurring more slowly, to those from fertile donors. These differences were statistically significant for t2 (time of cleavage to two-blastomere embryo) (P = 0.016), t3 (P = 0.014), t4 (P = 0.003) and t5 (P = 0.040). LIMITATIONS, REASONS FOR CAUTION: These are preliminary data from a retrospective analysis with a limited sample size. GENERALIZABILITY TO OTHER POPULATIONS: Not recommended until further studies using time-lapse analysis of a larger sample have been performed. STUDY FUNDING/COMPETING INTEREST(S): None.

Is morphokinetic analysis the answer?

Efforts aimed at improving pregnancy rates have focused on the search for additional markers of viability to supplement current criteria for embryo selection. Time-lapse technology represents a powerful tool in assisted reproduction for evaluating embryos dynamically. Whilst standard methods of embryo assessment are based on subjective morphology evaluation at discrete time points, thereby limiting the information produced for embryo selection, time-lapse recording introduces several additional morphokinetic parameters for embryo evaluation. This additional information can improve implantation rates and reproductive outcomes. This review surveys available knowledge on time-lapse imaging to provide an overview of the advantages and applications of this technology.

Non-diphtheriae Corynebacterium species: an emerging respiratory pathogen.

The purpose of the study was to describe the microbiological and clinical features of ten cases of lower respiratory tract infection due to Corynebacterium striatum, Corynebacterium propinquum and Corynebacterium pseudodiphtheriticum. Respiratory samples were recovered from hospitalised patients who were diagnosed of pneumonia and exacerbations of chronic respiratory infections. The samples were Gram-stained and seeded on conventional bacterial growing media. Bacteria were identified by matrix-assisted linear desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility was tested by the disk diffusion method. All patients presented an acute respiratory onset, most of them in the context of an underlying disease and/or immunosuppression. In all patients, the microscopical examination of Gram-stained respiratory samples showed numerous polymorphonuclear cells and Gram-positive bacilli, suggestive of the Corynebacterium morphotype. A pure culture growth of Corynebacterium was obtained in the majority (72 %) of samples. The conclusions are that non-diphtheriae Corynebacterium species are an emerging cause of respiratory infection among patients with chronic respiratory disease and/or immunosuppression, and cannot always be considered as mere colonisers. The microorganism's predominance in Gram-stained purulent respiratory samples together with abundant growth in the culture is the key for the microbiological diagnosis.

Malnutrition in an elderly population without cognitive impairment living in nursing homes in Spain: study of prevalence using the Mini Nutritional Assessment test.

BACKGROUND: The prevalence of malnutrition in institutionalized elderly people is generally high. A good nutritional status is related to a decrease in mortality and costs of morbidity treatments. Therefore, it is essential to know the nutritional status in order to establish action policies. However, there are not enough studies about malnutrition in institutionalized elderly in Spain. OBJECTIVES: The objectives of this survey were to assess the prevalence of malnutrition and risk of malnutrition in elderly people living in nursing homes in the province of Albacete (Spain) using the Mini Nutritional Assessment (MNA®) test, to analyze their distribution according to age, gender and different nursing home features, and to identify the MNA items that best predict the MNA total score. METHODS: A cross-sectional study was conducted with data collected from 895 elders living in 34 nursing homes all over the province of Albacete, including facilities located in the main city, towns and villages. Prevalence of malnutrition and risk of malnutrition were assessed using the MNA full form test. Groups of malnutrition were compared by using mean MNA scores. Stepwise linear regression analyses were used to identify the items in the MNA which best predicted the MNA total scores. RESULTS: According to the MNA, the prevalence of malnutrition among elderly people staying in Albacete province nursing homes was 2.8%, and the prevalence of being at risk of malnutrition was 37.3%. MNA total score was significantly higher in men (24.3 ± 2.9) than in women (23.7 ± 3.3) and significantly higher in residents living in nursing homes located in towns and villages (24.2 ± 2.8) than in those who were living in institutions placed in the main city (23.7 ± 3.4). The MNA score was found to decrease with increasing age, except for individuals aged 90 years or older. CONCLUSIONS: The prevalence of malnutrition and risk of malnutrition was 40.1%. Female gender and living in institutions located in the main city were identified as malnutrition risk factors. In general, an increase in malnutrition with age was detected, except for people in the last years of their lives. The MNA questions that best predicted the nutritional status were the ones relating to the anthropometrical and self-assessments.

Computerized evaluation of mean residence times in multicompartmental linear system and pharmacokinetics.

Deriving mean residence times (MRTs) is an important task both in pharmacokinetics and in multicompartmental linear systems. Taking as starting point the analysis of MRTs in open or closed (Garcia-Meseguer et al., Bull Math Biol 2003, 65, 279) multicompartmental linear systems, we implement a versatile software, using the Visual Basic 6.0 language for MS-Windows, that is easy to use and with a user-friendly format for the input of data and the output of results. For any multicompartmental linear system of up to 512 compartments, whether closed or open, with traps or without traps and with zero input in one or more of the compartments, this software allows the user to obtain the symbolic expressions, in the most simplified form, and/or the numerical values of the MRTs in any of its compartments, in the entire system or in a part of the system. As far as we known from the literature, such a software has not been implemented before. The advantage of the present software is that it reduces on the work time needed and minimizes the human errors that are frequent in compartmental systems even those that are relatively staightforward. The software bioCelTer, along with instructions, can be downloaded from http://oretano.iele-ab.uclm.es/~fgarcia/bioCelTer/.

Y chromosome microdeletions, sperm DNA fragmentation and sperm oxidative stress as causes of recurrent spontaneous abortion of unknown etiology.

BACKGROUND: The aim of the present study was to evaluate the implication of male factor, in terms of sperm DNA oxidation and fragmentation, and Y chromosome microdeletions in recurrent spontaneous abortion (RSA) of unknown origin in a strictly selected cohort. METHODS: A prospective cohort study was carried out in a private university-affiliated setting. Three groups, each comprised of 30 males, were compared. The first was formed by healthy and fertile sperm donors (SD) with normal sperm parameters (control group), the second by men presenting severe oligozoospermia (SO) without RSA history, and the third by men from couples who had experienced idiopathic RSA. Frequency of Y chromosome microdeletions and mean sperm DNA fragmentation and oxidation were determined. RESULTS: Y chromosome microdeletions were not detected in any of the males enrolled in the study. Moreover, sperm DNA oxidation measurements were not demonstrated to be relevant to RSA. Interestingly, sperm DNA fragmentation was higher in the SO group than in the RSA and the SD groups, and also higher in the RSA group compared with the SD group, but lacked an adequate predictive power to be employed as a discriminative test of RSA condition. CONCLUSIONS: Sperm DNA features and Y chromosome microdeletions do not seem to be related to RSA of unknown origin. Other molecular features of sperm should be studied to determine their possible influence on RSA. Clinicaltrials.gov reference: NCT00447395.

[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.

GnRH agonist versus recombinant HCG in an oocyte donation programme: a randomized, prospective, controlled, assessor-blind study.

The use of gonadotrophin-releasing hormone (GnRH) agonists for triggering ovulation remains controversial. The primary objective of this study was to evaluate the incidence of ovarian hyperstimulation syndrome (OHSS) following GnRH agonist versus recombinant human chorionic gonadotrophin (HCG) as methods for triggering ovulation. A second aim was to compare the clinical outcome and embryo quality according to the two procedures. The cycle characteristics of 100 oocyte donors undergoing ovarian stimulation and IVF outcomes of their 100 oocyte recipients were analysed. Donors were prospectively randomized into two groups on the last day of ovarian stimulation: Group I received a single bolus of 0.2 mg of triptorelin and Group II received 250 microg of recombinant HCG. No differences were observed in the number of oocytes retrieved or in the proportion of metaphase II oocytes between the groups. The OHSS rate was higher in donors that received recombinant HCG ( P = 0.003). Moreover, there was no significant difference between IVF parameters and outcome in the two groups. In conclusion, a GnRH agonist effectively triggers the final oocyte maturation in oocyte donors without negatively affecting implantation, pregnancy or miscarriage rates. Moreover, this regime effectively eliminates the risk of OHSS in this group of women.

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.

Mean lifetime and first-passage time of the enzyme species involved in an enzyme reaction. Application to unstable enzyme systems.

Taking as starting point the complete analysis of mean residence times in linear compartmental systems performed by Garcia-Meseguer et al. (Bull. Math. Biol. 65:279-308, 2003) as well as the fact that enzyme systems, in which the interconversions between the different enzyme species involved are of first or pseudofirst order, act as linear compartmental systems, we hereby carry out a complete analysis of the mean lifetime that the enzyme molecules spend as part of the enzyme species, forms, or groups involved in an enzyme reaction mechanism. The formulas to evaluate these times are given as a function of the individual rate constants and the initial concentrations of the involved species at the onset of the reaction. We apply the results to unstable enzyme systems and support the results by using a concrete example of such systems. The practicality of obtaining the mean times and their possible application in a kinetic data analysis is discussed.

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.