Analyzing Cell-free Genomic DNA in Spent Culture Media: Noninvasive Insight into the Blastocysts.

A commonly accepted standard protocol for noninvasive techniques for the genetic evaluation of an embryo remains elusive due to inconclusiveness regarding the volume of spent media to be acquired and the possibility of acquiring the same for subsequent analysis. Single embryo culture is imperative for standardizing noninvasive preimplantation testing using cell-free DNA (cf-DNA) released by individual developing embryos. This study aims to compare the development dynamics of single-drop embryonic culture against with group embryonic culture to establish a standardized protocol for noninvasive Preimplantation Genetic Testing (PGT) in bovine. A total of 239 cumulus-oocyte complexes were aspirated and subjected to in vitro maturation and fertilization. Among these, 120 embryos of day 3 were transferred to single-drop culture until the blastocyst stage. The single-drop culture drops were prepared using microdrops of 30 µL. At the blastocyst stage, spent media from all single-drop embryos were utilized for extracting cell-free genomic DNA to standardize the protocol. The blastocyst rate indicates no significant difference between the two culture methods, suggesting that single-drop culture is suitable for the process. Additionally, the extracted spent media yielded sufficient quantities of cf-DNA, supporting its potential use for PGT ( p < 0.05). These findings support the hypothesis that single-drop embryo culture is a viable method for cf-DNA extraction and confirm the potential of using DNA fragments from spent media as a reliable source for noninvasive PGT.

A prospective study of the proteome of equine pre-implantation embryo.

The present study was conducted to investigate the global proteome of 8-day-old equine blastocysts. Follicular dynamics of eight adult mares were monitored by ultrasonography and inseminated 24 h after the detection of a preovulatory follicle. Four expanded blastocysts were recovered, pooled, and subjected to protein extraction and mass spectrometry. Protein identification was conducted based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, and PepExplorer). Enrichment analysis was performed using g:Profiler, Panther, and String platforms. After the elimination of identification redundancies among search tools (at three levels, based on identifiers, peptides, and cross-database mapping), 1977 proteins were reliably identified in the samples of equine embryos. Proteomic analysis unveiled robust metabolic activity in the 8-day equine embryo, highlighted by an abundance of proteins engaged in key metabolic pathways like the TCA cycle, ATP biosynthesis, and glycolysis. The prevalence of chaperones among highly abundant proteins suggests that regulation of protein folding, and degradation is a key process during embryo development. These findings pave the way for developing new strategies to improve equine embryo media and optimize in vitro fertilization techniques.

Vitrification preservation of good-quality blastocysts for more than 5 years reduces implantation and live birth rates.

STUDY QUESTION: Does vitrification cryopreservation of embryos for more than 5 years affect the pregnancy outcomes after frozen embryo transfer (FET)? SUMMARY ANSWER: Vitrification cryopreservation of good-quality blastocysts for more than 5 years is associated with a decrease in the implantation rate (IR) and live birth rate (LBR). WHAT IS KNOWN ALREADY: Previous studies have predominantly focused on embryos cryopreserved for relatively short durations (less than 5 years), yet the impact of extended cryopreservation duration on pregnancy outcomes remains a controversial issue. There is a relative scarcity of data regarding the efficacy and safety of storing embryos for 5 years or longer. STUDY DESIGN, SIZE, DURATION: This retrospective study involved 36 665 eligible vitrified-thawed embryo transfer cycles from 1 January 2016 to 31 December 2022, at a single fertility center in China. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were divided into three groups according to embryo storage time: Group 1 consisted of 31 565 cycles, with storage time of 0-2 years; Group 2 consisted of 4458 cycles, with a storage time of 2-5 years; and Group 3 included 642 cycles, with storage time exceeding 5 years. The main outcome measures were IR and LBR. Secondary outcome variables included rates of biochemical pregnancy, multiple pregnancy, ectopic pregnancy, and miscarriage, as well as neonatal outcomes. Reproductive outcomes were analyzed as binary variables. Multivariate logistic regression analysis was used to explore the effect of preservation time on pregnancy outcomes after correcting for confounding factors. In addition, we also assessed neonatal outcomes, such as large for gestational age (LGA) and small for gestational age (SGA). MAIN RESULTS AND THE ROLE OF CHANCE: IRs in the three groups (0-2, 2-5, and >5 years) were 37.37%, 39.03%, and 35.78%, respectively (P = 0.017), and LBRs in the three groups were 37.29%, 39.09%, and 34.91%, respectively (P = 0.028). After adjustment for potential confounding factors, compared with the 0-2 years storage group, prolonged embryo vitrification preservation time (2-5 years or >5 years) did not affect secondary outcomes such as rates of biochemical pregnancy, multiple pregnancy, ectopic pregnancy, and miscarriage (P > 0.05). But cryopreservation of embryos for more than 5 years reduced the IR (adjusted odds ratio (aOR) 0.82, 95% CI 0.69-0.97, P = 0.020) and LBR (aOR 0.76, 95% CI 0.64-0.91, P = 0.002). Multivariate stratified analysis also showed that prolonging the cryopreservation time of blastocysts (>5 years) reduced the IR (aOR 0.78, 95% CI 0.62-0.98, P = 0.033) and LBR (aOR 0.68, 95% CI 0.53-0.87, P = 0.002). However, no effect on cleavage embryos was observed (P > 0.05). We further conducted stratified analyses based on the number and quality of frozen blastocysts transferred, and the results showed that the FET results after transfers of good-quality blastocysts in the >5 years storage group were negatively affected. However, the storage time of non-good-quality blastocysts was not significantly associated with pregnancy outcomes. Regarding the neonatal outcomes (of singletons), embryo vitrification preservation time had no effect on preterm birth rates, fetal birth weight, or neonatal sex ratios. However, as the storage time increased, rates of SGA (5.60%, 4.10%, and 1.18%) decreased, while rates of LGA (5.22%, 6.75%, and 9.47%) increased (P < 0.05). After adjusting for confounding factors, the increase in LGA and the decrease in SGA were significantly correlated with the duration of storage time. LIMITATIONS, REASONS FOR CAUTION: This was a retrospective study using data from a single fertility center, even though the data had been adjusted, our findings still need to be validated in further studies. WIDER IMPLICATIONS OF THE FINDINGS: With the full implementation of the two-child policy in China, there may be more patients whose embryos have been frozen for a longer time in the future. Patients should be aware that the IR and LBR of blastocysts are negatively affected when the cryopreservation time is longer than 5 years. Couples may therefore consider shortening the time until FET treatment. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Nature Science Foundation of China (No. 82101672), Science and Technology Projects in Guangzhou (No. 2024A03J0180), General Guidance Program for Western Medicine of Guangzhou Municipal Health Commission (No. 20231A011096), and the Medical Key Discipline of Guangzhou (2021-2023). None of the authors have any conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Production of bovine embryos by piezo-ICSI using capacitated spermatozoa selected by fluorescence-activated cell sorting (FACS-piezo-ICSI).

Intracytoplasmic sperm injection (ICSI) remains inefficient in cattle. One reason could lie in the injection of oocytes with sperm that have not undergone molecular changes associated with in vivo capacitation and fertilizing ability. This study aimed to enhance the efficiency of bovine intracytoplasmic sperm injection (piezo-ICSI) by employing fluorescent-activated cell sorting (FACS) to select the sperm population before injection based on capacitation markers. First, we evaluated the effects of incubating thawed sperm for 2 hours with different capacitating inductors: heparin, methyl-beta-cyclodextrin (MßCD), and dibutyryl cyclic AMP (dbcAMP), alone or in combinations in a basal capacitating (C) medium (Sp-TALP). Sperm capacitation and quality markers were evaluated by flow cytometry, revealing heparin as the most effective inducer of sperm capacitation changes. It, therefore, this treatment was chosen as the sperm pretreatment for FACS-piezo-ICSI. Two cell populations showing high capacitating levels (Heparin-HCL) and low capacitating levels (Heparin-LCL) of the markers associated with sperm capacitation i(Ca2+) levels and acrosome integrity were selected by FACS and used for sperm injection. Pronuclear formation was significantly higher when ICSI was performed with Heparin-HCL sperm than with Heparin-LCL and the control group (Heparin unsorted) groups (50 %, 10 %, and 20 %, respectively). Furthermore, injecting Heparin-HCL sperm resulted in a higher blastocyst rate (22.5 %) than Heparin-LCL (10 %) and the control group (15.2 %). In conclusion, heparin treatment effectively induced changes associated with sperm capacitation. The combination of Heparin-HCL treatment and FACS enabled precise selection of capacitated sperm before ICSI, enhancing the efficiency of this technology in the bovine species.

Vitrification of human blastocysts for couples undergoing assisted reproduction: an updated review.

Over the past 40 years there has been a worldwide critical change in the field of assisted reproduction technology (ART), leading to the increased application of single blastocyst transfer, which is extremely important to avoid the risks of multiple pregnancy and associated complications for both mother and babies. Indeed, advancements in ART over the last few decades have been obtained thanks to several improvements, including ovarian stimulation, embryo culture conditions and, of course, progress in cryopreservation methods, especially with the application of vitrification. The ability to cryopreserve human embryos has improved significantly with vitrification compared to the initially adopted slow-freezing procedures. Since the introduction of vitrification, it has become the gold standard method to effectively cryopreserve human blastocysts. However, some new protocols are now being explored, such as the short warming procedure and even shorter exposure to the equilibration solution before vitrification, which seem to provide optimal results. Therefore, the main aim of the current narrative review, will be to illustrate the benefit of vitrification as an effective method to cryopreserve the human blastocyst and to illustrate new protocols and variations which in future may increase the performance of vitrification protocols.

Effect of cryopreservation on Odc1 and RhoA genes expression in diapausing mouse blastocysts.

The possibility of embryo cryopreservation is important for applying the genome resource banking (GRB) concept to those mammalian species that exhibit embryonal diapause in their early development. Odc1 encodes ODC1, which is a key enzyme in polyamine synthesis. RhoA is an essential part of Rho/ROCK system. Both Odc1 and RhoA play an important role in preimplantation embryo development. Studying these systems in mammalian species with obligate or experimentally designed embryonic diapause may provide insight into the molecular machinery underlying embryo dormancy and re-activation. The effect of cryopreservation procedures on the expression of the Odc1 and RhoA in diapausing embryos has not been properly studied yet. The purpose of this work is to address the possibility of cryopreservation diapausing embryos and to estimate the expression of the Odc1 and RhoA genes in diapausing and non-diapausing embryos before and after freeze-thaw procedures using ovariectomized progesterone treated mice as a model. Both diapausing and non-diapausing in vivo-derived embryos continued their development in vitro after freezing-thawing as evidenced by blastocoel re-expansion. Although cryopreservation dramatically decreased the expression of the Odc1 and RhoA genes in non-diapausing embryos, no such effects have been observed in diapausing embryos where these genes were already at the low level before freeze-thaw procedures. Future studies may attempt to facilitate the re-activation of diapausing embryos, for example frozen-thawed ones, specifically targeting Odc1 or Rho/ROCK system.

Individually Cultured Bovine Zygotes Successfully Develop to the Blastocyst Stage in an Extremely Confined Environment.

The possibility of detecting the developmental competence of individually cultured embryos through analysis of spent media is a major current trend in an ART setting. However, individual embryo culture is detrimental compared with high-density group culture due to the reduced concentration of putative embryotropins. The main aim of this study was to identify an individual culture system that is not detrimental over high-density group culture in the bovine model. Blastocyst rates and competence were investigated in a conventional (GC) group, semi-confined group (MG), and individual culture (MS) in a commercial microwell device. Main findings showed that: (1) individual embryos can be continuously cultured for 7 days in ~70 nL microwells (MS) without detrimental effects compared with the GC and MG; (2) MS and MG blastocysts had a reduced number of TUNEL-positive cells compared to GC blastocysts; (3) though blastocyst mean cell numbers, mitochondrial activity, and lipid content were not different among the three culture conditions, MS blastocysts had a higher frequency of small-sized lipid droplets and a reduced mean droplet diameter compared with GC and MG blastocysts. Overall, findings open the way to optimize the development and competence of single embryos in an ART setting.

The impact of a second embryo biopsy for preimplantation genetic testing for monogenic diseases (PGT-M) with inconclusive results on pregnancy potential: results from a matched case-control study.

PURPOSE: To evaluate whether a second biopsy, following a first diagnostic failure on blastocysts tested for preimplantation genetic testing for monogenic diseases (PGT-M), allows to obtain genetic diagnosis and to what extent this procedure can influence clinical pregnancy and live birth rates compared to the PGT-M process with a successful genetic diagnosis from the first biopsy. METHODS: Embryos from women who underwent PGT-M in an infertility centre and who had been transferred after two biopsies for genetic analysis (n = 27) were matched in a 1:1 ratio accordingly to women's age (± 1 year) and fertility status (fertile vs infertile), as well as with the study period, with embryos who were transferred after receiving a conclusive PGT result straight after the first biopsy (n = 27). The main evaluated outcome was clinical pregnancy rate following embryo transfers in which healthy embryos were transferred after only one biopsy and those in which an embryo was transferred after being re-biopsied. Live birth rate was the secondary outcome. RESULTS: Clinical pregnancy rate was 52% (95% CI: 34-69) following the transfer of a single-biopsy blastocyst and 30% (95% CI: 16-48) following the transfer of a re-biopsied blastocyst. The likelihood to have a healthy baby was 33% (95% CI: 19-52) following the transfer of a blastocyst biopsied once and 22% (95% CI: 11-41) following the transfer of a re-biopsied blastocyst. CONCLUSIONS: The re-biopsy intervention seems to considerably reduce the pregnancy potential of a blastocyst. However, a greater sample size is necessary to clarify this issue definitively.

Primary sex ratio in euploid embryos of consanguine couples after IVF/ICSI.

PURPOSE: To assess the primary sex ratio (males-to-females at time of conception) in blastocysts from consanguine couples undergoing IVF/ICSI treatments and its correlation with chromosomal constitution. METHOD: A total of 5135 blastocysts were analyzed by preimplantation-genetic testing for aneuploidy (PGT-A) with next-generation sequencing (NGS) from November 2016 to December 2020. From those, a total of 1138 blastocysts were from consanguine couples (CS) and 3997 from non-consanguine couples (NCS). Only blastocysts presenting normal sex chromosome constitution with or without autosomal aneuploidies were included. Primary sex ratio (PSR) of biopsied blastocysts was compared between CS and NCS couples. RESULTS: Expanded blastocysts derived from CS had 47.7% XY versus 52.3% XX constitutions, presenting a PSR of 0.91. In NCS, 48.9% of expanded blastocysts were XY and 51.2% XX, with a less pronounced PSR of 0.95. When stratifying embryos by ploidy, euploid embryos from CS had the lowest PSR (0.87) with 46.6% XY versus 53.4% XX blastocysts (OR 0.89, 95% CI 0.70-1.14; NS), but it did not achieve statistical significance. The lower PSR seemed rather related to euploid embryos from first-degree cousins (PSR = 0.80 versus 0.98 in second-degree cousins, NS). Euploid embryos from NCS presented a PSR of 0.96, with 49.1% XY versus 50.9% XX blastocysts (OR 0.98, 95% CI 0.79-1.22; NS). Significant differences in prevalence of euploidy of specific chromosomes were encountered between CS and NCS. CONCLUSIONS: The primary sex ratio was generally similar in expanded blastocysts from consanguine and non-consanguine couples, with a slight decrease in primary sex ratio of euploid blastocysts from consanguine couples.

Preimplantation genetic testing for aneuploidy: predictive embryonic factors.

PURPOSE: In a preimplantation genetic testing for aneuploidy (PGT-A) cycle, does the blastocyst quality before biopsy, or the day of biopsy, or the embryo hatching status have an impact on either euploidy or the rate of embryo survival after freezing? METHODS: This was a retrospective study including 6130 biopsied blastocysts coming from 1849 PGT-A cycles performed in our center (2016-2022). Embryos were categorized according to the inner cell mass and trophectoderm quality, using Gardner's scoring (excellent: AA; good: AB, BA, BB; poor: AC, CA, BC, CB, CC); the day of biopsy (5 or 6); and their hatching status (fully hatched blastocysts [FHB] or non-fully hatched blastocysts [nFHB]). The independent relationship between each group and both euploidy and survival rate was assessed. RESULTS: Excellent-quality embryos were more euploid than both good- and poor-quality embryos (52.69%, 39.69%, and 26.21%; p < 0.001), and day 5-biopsied embryos were more euploid than day 6-biopsied embryos (39.98% and 34.80%; p < 0.001). Survival rates of excellent-quality (92.26%) and good-quality (92.47%) embryos were higher than survival rates in the poor-quality group (84.61%) (p = 0.011 and p = 0.002). Day 5-biopsied embryos survived better than day 6-biopsied embryos (93.71% vs. 83.69%; p < 0.001) and FHB had poorer survival than nFHB (78.61% vs. 93.52%; p < 0.001). CONCLUSIONS: Excellent-quality and day 5-biopsied embryos are more prone to be euploid than good and poor or day 6-biopsied embryos, respectively. Poor-quality, day 6-biopsied embryos, and FHB have significantly lower survival after biopsy and vitrification.

Exploring the impacts of senescence on implantation and early embryonic development using totipotent cell-derived blastoids.

INTRODUCTION: Advanced maternal age is associated with reduced implantation and pregnancy rates, yet the underlying mechanisms remain poorly understood, and research models are limited. OBJECTIVES: Here, we aim to elucidate the impacts of senescence on implantation ability by employing blastoids to construct a novel research model. METHODS: We used a novel three-dimensional system with totipotent blastomere-like cells (TBLCs) to construct TBL-blastoids and established senescence-related embryo models derived from oxidative stress-induced TBLCs. RESULTS: Morphological and transcriptomic analyses revealed that TBL-blastoids exhibited characteristic blastocyst morphology, cell lineages, and a higher consistency in developmental rate. TBL-blastoids demonstrated the ability to develop into postimplantation structures in vitro and successfully implanted into mouse uteri, inducing decidualization and forming embryonic tissues. Importantly, senescence impaired the implantation potential of TBL-blastoids, effectively mimicking the impaired implantation ability and reduced pregnancy rates associated with advanced age. Furthermore, analysis of differentially expressed genes (DEGs) in human homologous deciduae revealed enrichment in multiple fertility-related diseases and other complications of pregnancy. The genes implicated in these diseases and the common DEGs identified in the lineage-like cells of the two types of TBL-blastoids and deciduae may represent potential targets for addressing impaired implantation potential. CONCLUSION: These results unveiled that TBL blastoids are an improved model for investigating implantation and early postimplantation, offering valuable insights into pregnancy-related disorders in women with advanced age and potential targets for therapeutic interventions.

Artificial insemination of all ejaculated sperm fractions accelerates embryo development and increases the uterine vascularity in the pig.

The semen of boar is characterized by ejaculation in well-differentiated fractions with specific concentration, composition, and volume. The 'sperm-rich fraction' (SRF), the most concentrated seminal fraction, is habitually collected in insemination centers to make artificial insemination (AI) doses. The absence of the other fractions in AI doses could alter the uterine reaction to AI and not trigger essential responses that could maximize fertility. Thus, there is an urge to ascertain the impact of different ejaculate fractions on the uterus after AI to optimize the semen doses. This work analyzed specific parameters related to fertility in pregnant artificially inseminated sows (n = 15) with ac-cumulative fractions of the semen of boars (n = 6): F1, composed of the sperm-rich fraction (SRF); F2, composed of F1 plus the intermediate fraction; F3, composed of F2 plus the post-SRF. Non-inseminated sows (n = 5) were included as control (C). The different types of seminal dose did not affect the number of ovulated follicles (CL; corpora lutea, p > 0.05) but did affect the embryo development (p < 0.05). The proportion of embryos in morula stages was significantly higher in AI-F1 sows (84.4%, p < 0.05). Morulas and blastocysts were balanced in AI-F2 or AI-F3 (p > 0.05). Independently of the type of seminal dose (F1, F2, or F3), we observed by immunohistochemistry that AI significantly increased uterine vascularization, although with some anatomical differences. The cranial region of the uterine horns was significantly more vascularized in AI-F1 or AI-F2 sows (26.7 ± 2.3 and 28.6 ± 2.0%, respectively), and AI-F3 showed significantly less vascularization at that point (17.8 ± 1.6%, p < 0.05). To summarize, the synergistic effect of all ejaculate fractions accelerates embryo development, at least during the preimplantation period, and increases the uterine reaction to AI in certain parts of the uterus.

Fast and furious: pregnancy outcome with one-step rehydration in the warming protocol for human blastocysts.

RESEARCH QUESTION: Do embryos warmed using a one-step rehydration protocol with a more efficient workflow result in comparable pregnancy rates to the standard multi-step rehydration protocol? DESIGN: A retrospective cohort study of 3439 frozen embryo transfers (FET). Clinical outcomes of 833 FETs using a one-step rehydration protocol were reviewed and compared with results from the control group (2606 FETs using standard multi-step rehydration protocol). Primary outcome was ongoing pregnancy rate. Secondary outcomes were survival, positive pregnancy, clinical pregnancy, implantation and miscarriage rates. RESULTS: Survival rates were identical between the two groups (99.5%). Clinical pregnancy rate was 63.0% in the one-step warming protocol, comparable to 59.9% in the multi-step rehydration protocol. A significant increase was observed in the ongoing pregnancy rate with 60.4% in the one-step rehydration versus 55.4% in the multi-step rehydration group (P = 0.011); implantation rate was 63.6% versus 57.0% (P = 0.0005). The miscarriage rate of 4.0% in the one-step rehydration protocol was significantly lower compared with 7.6% in the multi-step rehydration protocol (P = 0.0001). Comparable outcomes persisted even when the analysis was extended to embryos that had and had not undergone preimplantation genetic testing (PGT), as well as day of development of the blastocysts. When controlling for variables of age, PGT, blastocyst development day and embryo expansion, rapid warming significantly increased chances of an ongoing pregnancy (adjusted OR 1.264, 95% CI 1.076 to 1.484). CONCLUSION: A one-step rehydration protocol resulted in identical survival rates and improved ongoing pregnancy rates compared with the multi-step rehydration technique.

Single-Cell mRNA-sncRNA Co-sequencing of Preimplantation Embryos.

The development of single-cell multiomics has provided the ability to systematically investigate cellular diversity and heterogeneity in different biological systems via comprehensive delineations of individual cellular states. Single-cell RNA sequencing in particular has served as a powerful tool to the study of the molecular circuitries underlying preimplantation embryonic development in both the mouse and human. Here we describe a method to elucidate the cellular dynamics of the embryo further by performing both single-cell RNA sequencing (Smart-Seq2) and single-cell small non-coding RNA sequencing (Small-Seq) on the same individual embryonic cell.

PDGFRß Activation Induced the Bovine Embryonic Genome Activation via Enhanced NFYA Nuclear Localization.

Embryonic genome activation (EGA) is a critical step during embryonic development. Several transcription factors have been identified that play major roles in initiating EGA; however, this gradual and complex mechanism still needs to be explored. In this study, we investigated the role of nuclear transcription factor Y subunit A (NFYA) in bovine EGA and bovine embryonic development and its relationship with the platelet-derived growth factor receptor-ß (PDGFRß) by using a potent selective activator (PDGF-BB) and inhibitor (CP-673451) of PDGF receptors. Activation and inhibition of PDGFRß using PDGF-BB and CP-673451 revealed that NFYA expression is significantly (p < 0.05) affected by the PDGFRß. In addition, PDGFRß mRNA expression was significantly increased (p < 0.05) in the activator group and significantly decreased (p < 0.05) in the inhibitor group when compared with PDGFRα. Downregulation of NFYA following PDGFRß inhibition was associated with the expression of critical EGA-related genes, bovine embryo development rate, and implantation potential. Moreover, ROS and mitochondrial apoptosis levels and expression of pluripotency-related markers necessary for inner cell mass development were also significantly (p < 0.05) affected by the downregulation of NFYA while interrupting trophoblast cell (CDX2) differentiation. In conclusion, the PDGFRß-NFYA axis is critical for bovine embryonic genome activation and embryonic development.

Reproductive outcomes with delayed blastocyst development: the clinical value of day 7 euploid blastocysts in frozen embryo transfer cycles.

Embryos of optimal development reach blastocyst stage 116 ± 2 h after insemination. Usable D7 blastocysts represent nearly 5% of embryos in IVF with acceptable pregnancy and live birth rates, however data are still limited. Therefore, this study aimed to analyze the ongoing pregnancy rate (OPR) of D7 blastocysts in single euploid frozen embryo transfer (FET) cycles. An observational study was performed including 1527 FET cycles with blastocysts biopsied on D5 (N = 855), D6 (N = 636) and D7 (N = 36). Blastocysts were classified as good (AA/AB/BA), fair (BB) or poor (AC/BC/CC/CA/CB) (Gardner scoring). FETs were performed in natural cycles (NC) or hormone replacement therapy (HRT) cycles. Patient's age differed significantly between D5, D6 and D7 blastocysts FET cycles (33.2 ± 5.6, 34.4 ± 5.3 and 35.9 ± 5.2, P < 0.001). OPRs were higher when D5 euploid blastocysts were transferred compared with D6 and D7 (56.0% vs. 45.3% and 11.1%, P < 0.001). Poor quality blastocysts were predominant in D7 blastocyst FET cycles (good quality: 35.4%, 27.2%, 5.6%; fair quality: 52.1%, 38.5%, 11.1%; poor quality: 12.5%, 34.3%, 83.3%, P < 0.001 for D5, D6 and D7 blastocysts; respectively). OPR was significantly reduced by D7 blastocyst FETs (OR = 0.23 [0.08;0.62], P = 0.004), patient's BMI (OR = 0.96 [0.94;0.98], P < 0.001), HRT cycles (OR = 0.70 [0.56;0.88], P = 0.002) and poor quality blastocysts (OR = 0.33 [0.24;0.45], P < 0.001). OPR is significantly reduced with D7 compared with D5/D6 euploid blastocysts in FET cycles. The older the patient, the more likely they are to have an FET cycle with blastocysts biopsied on D7, therefore culturing embryos until D7 can be a strategy to increase OPR outcomes in patients ≥38 years.

Effect of L. crispatus M247 Administration on Pregnancy Outcomes in Women Undergoing IVF: A Controlled, Retrospective, Observational, and Open-Label Study.

The aim of our study was to retrospectively evaluate whether the oral administration of L. crispatus (M247) could increase pregnancy and live birth rates in women undergoing assisted reproductive technology procedures. Enrolled women (N = 160) were divided into two groups: treated (N = 80) or untreated (N = 80) with the probiotic strain. The odds ratio (OR) for a treated woman to have a clinical pregnancy (CP) was 1.56. In women aged 30-40 years, M247 increased the probability of a CP in correlation with the progressive rise in BMI, reaching 47% (35% in controls) with a BMI of 35 (OR: 2.00). The CAID statistics showed that in a woman of the blastocyst subgroup, below 43 years, with a BMI over 18.6, treatment with M247 increased the chance of a CP from 28.4% to 44.5% (OR: 2.08; p < 0.05). Considering live births, the rate of the probiotic group was 12.5% versus 7.5% (OR: 1.76). Considering only the blastocyst subgroup, the treatment increased the number of live births by 200% (OR: 3.64; p = 0.05). As confirmed also by statistical indices NNT, NNH, and LHH, the use of M247 demonstrated a risk-benefit ratio to the full advantage of the benefits.

Outcome of Different Endometrial Preparation Protocols Prior to Frozen-Thawed Embryo Transfer on Pregnancy Outcomes in Women with Repeated Implantation Failure.

Aim: To compare the pregnancy outcomes of frozen-thawed embryo transfer (FET) cycles among women with repeated implantation failure (RIF) treated with various endometrial preparation protocols. Methods: A total of 605 women with RIF were retrospectively recruited between January 2017 and December 2020 from Northern Theater General Hospital. Patients were divided into natural cycles, hormone replacement therapy (HRT) cycles, depot gonadotropin-releasing hormone (GnRH) agonist-HRT, and endometrial scratching (ES) plus depot GnRH agonist-HRT. The primary endpoint was clinical pregnancy rate, while secondary endpoints included live birth rate and pain assessment. Results: Of the 605 recruited patients, 63 were undergoing natural cycles, 281 were treated with HRT cycles, 141 treated with depot GnRH agonist-HRT, and 120 treated with ES combined with depot GnRH agonist-HRT. There were significant differences among protocols on clinical pregnancy rate (P=0.029), while no significant difference was observed among protocols on live birth rates (P=0.108). Multivariate analyses suggested that HRT (odds ratio [OR]: 0.50; 95% confidence interval [CI]: 0.28-0.89; P=0.019) and depot GnRH agonist-HRT (OR: 0.49; 95% CI: 0.27-0.91; P=0.021) cycles were associated with a lower clinical pregnancy rate as compared with natural cycles, while no significant difference between ES combined with depot GnRH agonist-HRT and natural cycles for clinical pregnancy rates (OR: 0.72; 95% CI: 0.38-1.36; P=0.313). Moreover, the HRT (OR: 0.70; 95% CI: 0.39-1.28; P=0.239), depot GnRH agonist-HRT (OR: 0.67; 95% CI: 0.35-1.29; P=0.229), and ES combined with depot GnRH agonist-HRT (OR: 1.11; 95% CI: 0.58-2.14; P=0.754) cycles had no significant effects on live birth rate as compared with natural cycles. A total of 87.50% patients treated with ES combined with depot GnRH agonist-HRT reported pain during the procedure. Conclusion: ES and depot GnRH agonists could be considered for RIF women with high-quality blastocysts, 14 days after verified transplantation failure.

Monitoring potentially pathogenic protists in sewage sludge using Metataxonomics.

Intestinal parasites continue to pose a significant threat to human health worldwide, particularly among children. Contaminated water and soil serve as major transmission vehicles for these parasites and intestinal protists are among the most prevalent parasites in both developed and developing nations. Traditionally, parasites have been studied using human or animal fecal samples, while studying them in environmental samples has been challenging due to technical limitations. However, advancements in Next-Generation Sequencing (NGS) and bioinformatic approaches now enable the detection of parasite DNA in environmental samples. In this study, we applied a metataxonomic and phylogenetic strategy to detect and classify DNA of protists present in sewage sludge from two major cities in Colombia: Medellin and Cali. We successfully detected several human pathogenic parasites including Giardia intestinalis, Entamoeba histolytica, and Blastocystis sp., among other protists, in all sludge samples examined. We also investigated the entry and exit of parasite DNA from the San Fernando wastewater treatment plant (WWTP). We observed a higher number of parasite DNA sequences in the plant's influent wastewater, but we also detected the discharge of DNA from pathogenic parasites in both effluent waters and biosolids.