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• The placenta is the first organ to form in mammals and is required for establishment of a maternal–fetal vascular interface capable of supplying the bioenergetic needs of the developing conceptus. • Multiple placental cell types engage in highly varied functions, from attachment, invasion, and vascular remodeling to cell fusion, hormone production, and nutrient transport. • Multiple mechanisms allow transport of waste and nutrients across the placenta, including diffusion, transporter protein-mediated (facilitated diffusion and active transporters), and receptor-mediated mechanisms. • The placenta is not an inert transport interface. It consumes 40%–60% of the oxygen and glucose delivered to the uterus at term. Thus conditions that alter placental metabolism can indirectly affect nutrient transport to the fetus. • Maternal and fetal health alter placental function, which in turn influences fetal adaptations and contributes to in utero fetal programming. • Abnormal placentation and placental infections can lead to preeclampsia (PE), fetal growth retardation, or preterm birth, which can have lifelong bearing on health. • In the United States, iatrogenic delivery is responsible for almost half the births that occur between 28 and 37 weeks of gestation, primarily caused by placental pathologies such as PE or intrauterine growth restriction. • Efforts to standardize placental examination after delivery are in progress so that connections between specific placental problems and poor outcomes can be better defined. In parallel, new advanced imaging techniques and biomarkers for placental function are being developed.

Alu elements are retrotransposons related to epigenetic modifications. To date, the role of epigenetics in hypertrophic scars from burn remains unknown. Here, our aim was to examine the pathophysiology of hypertrophic scars from an epigenetic perspective. For that, we performed a cross-sectional analytical study using tissue and blood samples from burned and healthy patients (n = 23 each) to detect Alu methylation levels and patterns. The results of the combined bisulfite restriction analysis technique were categorized into four groups based on the methylation status at the CpG dinucleotides from the 5' to the 3' ends of the Alu sequence: hypermethylated (mCmC), hypomethylated (uCuC), and partially methylated (uCmC and mCuC). Alu methylation levels were significantly lower in hypertrophic scar tissues than in normal skin (29.37 ± 2.49% vs. 35.56 ± 3.18%, p = 0.0002). In contrast, the levels were significantly higher in white blood cells from blood samples of burned patients than in those of control blood samples (26.92 ± 4.04% vs. 24.58 ± 3.34%, p = 0.0278). Alu total methylation (mC) and the uCmC pattern were significantly lower, whereas uCuC was significantly higher, in hypertrophic scar tissues than in normal skin (p < 0.0001). Receiver operating characteristic analysis indicated that the uCmC and uCuC patterns are useful as hypertrophic scar DNA methylation markers after burn, with 91.30% sensitivity and 96.23% specificity and 100% sensitivity and 94.23% specificity, respectively. Our findings suggest that epigenetic modifications play a major role in hypertrophic scar pathogenesis, and may be the starting point for developing a novel technique for burn scar treatment.

Background: Female fertility refers to the capacity to produce oocytes and achieve fertilization and pregnancy, and it is impaired by age, disease, environment and social pressure. However, no effective therapy that restores female reproductive ability has been established. Mesenchymal Stromal Cells (MSCs) exhibit multilineage differentiation potential and have attracted considerable attention as a tool for restoring female fertility. Methods: This study used human umbilical cord-MSCs (Huc-MSCs) to restore fertility in aging female mice and mice with chemotherapy-induced damage through the rescue of ovarian function and reconstruction of the fallopian tubes and uterus. In our study, two mouse models were generated: aging mice (35 weeks of age) and mice with chemotherapy-induced damage. Results: The effect of MSCs on the ovaries, fallopian tubes and uterus was evaluated by analyzing gonadal hormone levels and by performing morphological and statistical analyses. The levels of estradiol (E2) and follicle-stimulating hormone (FSH) exhibited significant recovery after Huc-MSC transplantation in both aging mice and chemotherapy-treated mice. Huc-MSC treatment also increased the number of primordial, developing and preovulatory follicles in the ovaries of mice. Moreover, MSCs were shown to rescue the morphology of the fallopian tubes and uterus through mechanisms such as cilia regeneration in the fallopian tubes and reformation of glands and endometrial tissue in the uterus. Conclusion: Huc-MSCs may represent an effective treatment for restoring female fertility through recovery from chemotherapy-induced damage and rescue of female reproductive organs from the effects of aging.

Objective: The experiment was conducted to evaluate the effects of maternal undernutrition during late pregnancy on the expressions of genes involved in growth and development in ovine fetal perirenal brown adipose tissue (BAT).Methods: Eighteen ewes with singleton fetuses were allocated to three groups at day 90 of pregnancy: restricted group 1 (RG1, 0.33 MJ metabolisable energy [ME]/kg body weight [BW]0.75/d, n = 6), restricted group 2 (RG2, 0.18 MJ ME/kg BW0.75/d, n = 6), and a control group (CG, ad libitum, 0.67 MJ ME/kg BW0.75/d, n = 6). The fetuses were removed at day 140 of pregnancy. All data were analyzed by using the analysis of variance procedure.Results: The perirenal fat weight (p = 0.0077) and perirenal fat growth rate (p = 0.0074) were reduced in RG2 compared to CG. In fetal perirenal BAT, the protein level of uncoupling protein 1 (UCP1) (p = 0.0001) was lower in RG1 and RG2 compared with CG and UCP1 mRNA expression (p = 0.0265) was decreased in RG2. The protein level of myogenic factor 5 (Myf5) was also decreased in RG2 (p = 0.0001). In addition, mRNA expressions of CyclinA (p = 0.0109), CyclinB (p = 0.0019), CyclinD (p = 0.0015), cyclin-dependent kinase 1 (CDK1) (p = 0.0001), E2F transcription factor 1 (E2F1) (p = 0.0323), E2F4 (p = 0.0101), and E2F5 (p = 0.0018) were lower in RG1 and RG2. There were decreased protein expression of peroxisome proliferator-activated receptor-γ (PPARγ) (p = 0.0043) and mRNA expression of CCAAT/ enhancer-binding protein-α (C/EBPα) (p = 0.0307) in RG2 and decreased PPARγ mRNA expression (p = 0.0008) and C/EBPα protein expression (p = 0.0015) in both RG2 and RG1. Furthermore, mRNA expression of bone morphogenetic protein 4 (BMP4) (p = 0.0083) and BMP7 (p = 0.0330) decreased in RG2 and peroxisome proliferator-activated receptor co-activator-1α (PGC-1α) reduced in RG2 and RG1.Conclusion: Our observations support that repression of regulatory factors promoting differentiation and development results in the inhibition of BAT maturation in fetal perirenal fat during late pregnancy with maternal undernutrition.

Fil: Saban, Melina. Hospital Británico. Unidad de Endocrinología, Metabolismo, Nutrición y Diabetes; Argentina Fil: Costilla, Melisa. Pontificia Universidad Católica Argentina. Instituto de Investigaciones Biomédicas; Argentina Fil: Klecha, Alicia Juana. Pontificia Universidad Católica Argentina. Instituto de Investigaciones Biomédicas; Argentina Fil: Di Cugno, Mariana. Hospital Británico. Unidad de Endocrinología, Metabolismo, Nutrición y Diabetes; Argentina Fil: Curriá, Marina Inés. Hospital Británico. Unidad de Endocrinología, Metabolismo, Nutrición y Diabetes; Argentina Fil: Cremaschi, Graciela A. Pontificia Universidad Católica Argentina. Instituto de Investigaciones Biomédicas; Argentina Fil: Barreiro Arcos, María Laura. Pontificia Universidad Católica Argentina. Instituto de Investigaciones Biomédicas; Argentina Abstract: Background: Graves’ disease is an autoimmune disorder characterised by excessive production of thyroid hormones, which induces increased cellular metabolism in most tissues and increased production of reactive oxygen species (ROS). The aim of this work was to analyse the effect of ROS on cell viability and the expression of catalase (CAT), glutathione peroxidase-1 (GPx1), superoxide dismutase (SOD-1) and DNA methyltransferase-1 (DNMT-1) in peripheral blood mononuclear cells (PBMC) from patients with newly diagnosed Graves’ disease or treated with methimazole. Patients and methods: For this study, women patients with newly diagnosed Graves’ disease (n = 18), treated with methimazole (n = 6) and healthy subjects (n = 15) were recruited. ROS were evaluated by flow cytometry, and the viability/apoptosis of PBMC was analysed by flow cytometry and fluorescence microscopy. Genomic expression of CAT, GPx-1, SOD-1 and DNMT-1 was quantified by real-time PCR. Results: We found high levels of ROS and increased expression of CAT, GPx-1, SOD-1 and DNMT-1 in PBMC from patients with newly diagnosed Graves’ disease. Methimazole treatment reversed these parameters. Cell viability was similar in all study groups. Conclusions: ROS induces the expression of CAT, GPx-1, and SOD-1. The activity of these enzymes may contribute to the protection of PBMC from the harmful effect of free radicals on cell viability. Increased expression of DNMT-1 may be associated with aberrant methylation patterns in immunoregulatory genes contributing to autoimmunity in Graves’ disease. Resumen: Antecedentes: La enfermedad de Graves es un trastorno autoinmune caracterizado por una producción excesiva de hormonas tiroideas, que induce un aumento del metabolismo celular en la mayoría de los tejidos y una mayor producción de especies reactivas de oxígeno (ROS). El objetivo de este trabajo fue analizar el efecto de las ROS sobre la viabilidad celular y la expresión de catalasa (CAT), glutatión peroxidasa-1 (GPx-1), superóxido dismutasa (SOD-1) y ADN metiltransferasa-1 (DNMT-1) en células mononucleares de sangre periférica (PBMC) de pacientes con enfermedad de Graves recién diagnosticada o tratados con metimazol. Pacientes y métodos: Se seleccionó a mujeres con enfermedad de Graves recién diagnosticada (n = 18), tratadas con metimazol (n = 6) y a sujetos sanos (n = 15). La producción de ROS fue evaluada por citometría de flujo. La viabilidad y apoptosis de las PBMC fue analizada por citometría de flujo y microscopía de fluorescencia. La expresión genómica de CAT, GPx-1, SOD-1 y DNMT-1 fue cuantificada por PCR en tiempo real. Resultados: Encontramos altos niveles de ROS y una mayor expresión de CAT, GPx-1, SOD-1 y DNMT-1 en PBMC de pacientes con enfermedad de Graves recién diagnosticada. El tratamiento con metimazol revirtió estos parámetros. La viabilidad celular fue similar en todos los grupos de estudio. Conclusiones: Las ROS inducen la expresión de CAT, GPx-1 y SOD-1. La actividad de estas enzimas podría contribuir a la protección de las PBMC del efecto nocivo de los radicales libres sobre la viabilidad celular. El aumento de la expresión de DNMT-1 podría estar asociado con patrones de metilación aberrantes en genes inmunorreguladores que contribuyen a la autoinmunidad en la enfermedad de Graves.

Background Platelets play an important role in the pathogenesis of inflammatory and proliferative vascular changes. The aim of this study was to investigate whether human platelets are able to induce transplant arteriosclerosis in a humanized C57/Bl6-Rag2-/-γc-/- mouse xenograft model. Methods Nonactivated and in vitro activated human platelets were analyzed and phenotyped for surface markers by flow cytometry. Side branches of human mammary arteries were implanted into the infrarenal aorta of recipients, followed by daily application of human platelets and histological analyzed on day 30 after transplantation. Results Human platelets collected by apheresis had low levels of platelet activation markers. However, after in vitro activation, expression was markedly increased. 60 min after injection in recipient mice, nonactivated human platelets become significantly activated. Increased adhesion of platelets to the vascular endothelium was detected by in vivo fluorescence microscopy. After intravenous injection of nonactivated or activated platelets, human xenografts showed pronounced intimal proliferation. Immunohistological analysis showed that the group treated with activated human platelets exhibited significantly increased intragraft protein expression of ICAM-1 and PDGF receptor β and SMC migration into the neointima. Conclusions These data demonstrate that an isolated daily application of both in vivo and in vitro activated human platelets results in the development of transplant arteriosclerosis in a humanized mouse transplantation model.Visual Supplemental Abstract; http://links.lww.com/TP/C278.

Introduction The transplantation of mesenchymal stem cells (MSCs) in patients with premature ovarian failure (POF) could lead to clinical improvement. The transplantation to the ovaries among other transplantation methods have been reported in various animal models, however, there is little evidence regarding the optimal method, including the clinical safety and the efficiency for the treatment of age associated ovarian hypofunction. Objectives To establish the most effective transplantation route of MSCs, explore the resistance to therapy, its safety and role in the natural aging process of the ovaries. Methods Highly purified MSCs were injected intraperitoneally, directly into the ovaries or tail-intravenously in mice animal model. The ovarian function, quantity and quality of oocytes, cell viability/apoptosis, were evaluated, applying chemiluminescence analysis (CLIA), western blotting, immunofluorescence staining, transmission electron microscope (TEM), TdT mediated dUTP Nick End Labeling (TUNEL) assay and other techniques. The organ tumorigenicity was also evaluated by long-term observation and histopathological examination. The efficiency of MSCs was further verified in non-human primates by the most effective transplantation route. Results The 32nd week was ultimately determined as the time point of MSCs transplantation. Our results showed that the intra-ovarian injection was the best transplantation method with a more conspicuous effect. With deeper investigations, we found that the transplanted MSCs showed an effective influence on the follicular number, promoted follicle maturation and inhibited cell apoptosis, which was further verified in non-human primates. In addition, the long-term observation and the histopathological examinations ruled out neoplasms or obvious prosoplasia after MSCs transplantation. Conclusion MSCs transplantation by intra-ovarian injection could within a month exert the most conspicuous anti-age-associated ovarian hypofunction effects, which may improve the quantity and quality of oocytes by changing the mitochondrial structure, regulating mitochondrial function and attenuating cell apoptosis to increase the storage of the follicle pool without a remarkable potential of tumorigenicity.

What is the reproductive potential of embryos that achieve blastulation on day 7 followed by preimplantation genetic testing for aneuploidies (PGT-A) for infertility patients with slow embryo development?This was a retrospective cohort study in a private IVF clinic of consecutive female infertility patients (n = 2966) aged 24-48 (36.3 ± 3.8) years who underwent frozen embryo transfer (FET) of a single euploid blastocyst.The women underwent single euploid FET of an embryo that achieved blastulation on day 5 (n = 1880), day 6 (n = 986) or day 7 (n = 100). Day 7 embryos resulted in lower implantation and live birth rates compared with both day 5 and day 6 embryos (P 0.001). The day 5, day 6 and day 7 groups had 68.5%, 55.2% and 36.0% live birth rates, respectively. The day 7 group was older than the day 5 group (P 0.001); comparing age-matched cohorts, the day 7 group still had lower implantation and live birth rates (P 0.0001 and P 0.001, respectively). Embryo grade was unrelated to live birth rates. Day 7 embryos of expansion grade 5 or 6 or trophectoderm grade A were more likely to be euploid compared with expansion grade 3 or trophectoderm grade B.Euploid day 7 embryos represented reduced implantation potential, even when controlling for maternal age. Of all day 7 embryos that underwent PGT-A, euploidy was associated with expansion grade 5 or 6 and trophectoderm grade A. These results can help providers manage patient expectations in cases where infertile women have slow embryo development.

OBJECTIVE To evaluate the effect of intrauterine administration of activated peripheral blood mononuclear cells (PBMC) on intrauterine insemination (IUI) success rates. METHODS This prospective double-blind randomized parallel clinical trial included 213 patients undergoing IUI at the Fertilys clinic. PBMC were isolated on the day of ovulation (day 0; D0) and stimulated with phytohemagglutinin (PHA) and human chorionic gonadotropin (hCG) for 48 hours (day 2; D2). Patients in the PBMC group (n = 108) underwent in utero administration of 1.106 cells on D2, while patients in the control group (n = 105) were administered sperm-washing medium. Distribution of CD4 T lymphocyte populations (n = 61) was assessed on D0 and D2. Pregnancy and live birth rates were also evaluated. RESULTS Demographic and clinical characteristics, pregnancy rates, and live birth rates were not significantly different between the PBMC and control groups. Significantly higher levels of T helper (Th) 2, Th22, and T regulatory cells (P < 0.0001) and lower levels of Th17 cells were observed in hCG-activated PBMC at D2 than at D0. CONCLUSION Intrauterine administration of PBMC was not beneficial in IUI patients. New clinical approaches to better identify patients requiring endometrium immunomodulation needs to be addressed.

Heart transplantation is the gold standard of treatments for end-stage heart failure, but its use is limited by extreme shortage of donor organs. The time "window" between procurement and transplantation sets the stage for myocardial ischemia/reperfusion injury, which constrains the maximal storage time and lowers use of donor organs. Given mesenchymal stem cell (MSC)-derived paracrine protection, we aimed to evaluate the efficacy of MSC-conditioned medium (CM) and extracellular vesicles (EVs) when added to ex vivo preservation solution on ameliorating ischemia/reperfusion-induced myocardial damage in donor hearts.Mouse donor hearts were stored at 0°C-4°C of1-hour cold ischemia (1hr-I), 6hr-I + vehicle, 6hr-I + MSC-CM, 6hr-I + MSC-EVs, and 6hr-I + MSC-CM from MSCs treated with exosome release inhibitor. The hearts were then heterotopically implanted into recipient mice. At 24 hours postsurgery, myocardial function was evaluated. Heart tissue was collected for analysis of histology, apoptotic cell death, microRNA (miR)-199a-3p expression, and myocardial cytokine production.Six-hour cold ischemia significantly impaired myocardial function, increased cell death, and reduced miR-199a-3p in implanted hearts versus1hr-I. MSC-CM or MSC-EVs in preservation solution reversed the detrimental effects of prolong cold ischemia on donor hearts. Exosome-depleted MSC-CM partially abolished MSC secretome-mediated cardioprotection in implanted hearts. MiR-199a-3p was highly enriched in MSC-EVs. MSC-CM and MSC-EVs increased cold ischemia-downregulated miR-199a-3p in donor hearts, whereas exosome-depletion neutralized this effect.MSC-CM and MSC-EVs confer improved myocardial preservation in donor hearts during prolonged cold static storage and MSC-EVs can be used for intercellular transport of miRNAs in heart transplantation.

Background: Diabetic retinopathy (DR) is a severe complication of diabetes; however, the pathogenesis of DR has not been completely clarified, which is mostly dependent on the molecular pathology. To investigate key serum-derived miRNAs associated with DR. Methods: miRNA expression profile arrays of human umbilical vein endothelial cells (HUVECs) treated with glucose were downloaded from the Gene Expression Omnibus (GEO) database (GSE74296). Weighted gene co-expression network analysis (WGCNA) was performed to obtain hub miRNAs, which were verified in HUVECs treated with 40 mM and 5 mM glucose, respectively. Meanwhile, serum samples of patients with DR and healthy controls were collected, and EVs were extracted from the patients’ serum by ultracentrifugation. Hub miRNAs associated with endothelial dysfunction were verified in healthy individuals before and after treatment of patients with DR, by qRT-PCR. Results: These miRNAs were categorized into six modules, among which miR-26b-5p had a strong association with other modules. This miRNA was also one of the hyperglycemia-induced miRNAs related to endothelial dysfunction. miR-26b-5p was upregulated in HUVECs treated with 40 mM glucose and in the serum of patients with DR before and after treatment Furthermore, miR-26b-5p was slightly up-regulated in serum-derived EVs but not in serum without EVs in DM patients. Conclusion: Our results suggest that EVs derived from miR-26b-5p are up-regulated in the serum of patients with DR.

Cartilage does not naturally heal, and cartilage lesions from trauma and wear-and-tear can lead to eventual osteoarthritis. To address long-term repair, tissue engineering of functional biologic implants to treat cartilage lesions is desirable, but the development of such implants is hindered by several limitations, including (1) donor tissue scarcity due to the presence of diseased tissues in joints, (2) dedifferentiation of chondrocytes during expansion, and (3) differences in functional output of cells dependent on donor age. Toward overcoming these challenges, (1) costal cartilage has been explored as a donor tissue, and (2) methods have been developed to rejuvenate the chondrogenic phenotype of passaged chondrocytes for generating self-assembled neocartilage. However, it remains unclear how the rejuvenation processes are influenced by donor age and, thus, how to develop strategies that specifically target age-related differences. Using histological, biochemical, proteomic, and mechanical assays, this study sought to determine the differences among neocartilage generated from neonatal, juvenile, and adult donors using the Yucatan minipig, a clinically relevant large animal model. Based on the literature, a relatively young adult population of animals was chosen due to a reduction in functional output of human articular chondrocytes after 40 years of age. After isolation, costal chondrocytes were expanded, rejuvenated, and self-assembled, and the neocartilages were assessed. The aggregate modulus values of neonatal constructs were at least 1.65-fold of those from the juvenile or adult constructs. Poisson's ratio also significantly differed among all groups, with neonatal constructs exhibiting values 49% higher than adult constructs. Surprisingly, other functional properties such as tensile modulus and glycosaminoglycan content did not significantly differ among groups. Total collagen content was slightly elevated in the adult constructs compared to neonatal and juvenile constructs. A more nuanced view using bottom-up mass spectrometry showed that Col2a1 protein was not significantly different among groups, but protein content of several other collagen subtypes (i.e., Col1a1, Col9a1, Col11a2, and Col12a1) was modulated by donor age. For example, Col12a1 protein content in adult constructs was found to be 102.9% higher than neonatal-derived constructs. Despite these differences, this study shows that different aged donors can be used to generate neocartilages of similar functional properties. IMPACT STATEMENT: Tissue-engineered neocartilage can be generated with functional properties that mimic native cartilage tissue. However, cell sourcing challenges hinder clinical translation of tissue-engineered cartilage. Chondrocytes can be expanded and rejuvenated for the generation of functional self-assembled cartilage, making an allogeneic approach feasible. However, it is currently unclear if donor age impacts functional properties. In this study, using the Yucatan minipig as a clinically relevant large animal model, we demonstrate that functional properties of self-assembled neocartilage are relatively consistent regardless of donor age, suggesting that a wider range of donor ages may be used for cartilage tissue engineering than previously expected.

Implantation timing is key for a successful pregnancy. Short delay in embryo implantation caused by targeted gene ablation produced a cascading problem in the later stages of the pregnancy. Although several delayed implantation models have been established in wild mice, almost none of them is suitable for investigating the delay on the late events of pregnancy. Here, we report a new delayed implantation model established by the intraperitoneally administration of letrozole at 5 mg/kg body weight on the day 3 of pregnancy. In these mice, initiation of implantation was induced at will by the injection of estradiol (E2). When the estradiol (3 ng) was injected on day 4 of pregnancy (i.e., without delay), the embryo implantation restarted, and the pregnancy continued normally. However, high dose of estrogen (25 ng) caused compromised implantation. We also found that only 67% of the female mice could be pregnant normally and finally gave birth when the injection of estradiol (3 ng) was on day 5 of pregnancy (i.e., one day delay). Most of the failed pregnancies had impaired decidualization, decreased plasma progesterone levels and compromised angiogenesis. Progesterone supplementation could rescue decidualization failure in the mice. Collectively, we established a new model of delayed implantation by letrozole, which can be easily used to study the effect and mechanisms of delay of embryo implantation on the progression of late pregnancy events.

Objective: Male genital tract infections have been associated with infertility, and Escherichia coli has drawn increasing attention as an important bacterium in this context. This investigation aimed to characterize and compare the distributions of O-antigen serogroups of E. coli in the semen samples of fertile and infertile men. Methods: In this case-control study, semen samples were collected from 618 fertile and 1,535 infertile men. The E. coli-positive samples were evaluated in terms of concentration, morphology, viability, and motility parameters according to the World Health Organization 2010 guidelines. Finally, different serogroups of E. coli were identified by multiplex polymerase chain reaction targeting the O-antigen variations of the bacterium.Results: The prevalence of E. coli among fertile men was significantly higher than among infertile men (p

Objective To develop an efficient clinical-grade freezing protocol towards experimental clinical cryopreservation of testicular tissues in prepubertal boys suffering from cancer. Design Experimental cryopreservation of testicular tissue. Setting Hadassah Hebrew University Medical Center, Jerusalem, Israel. Patients Adult patients undergoing orchiectomy for various tumors, and prepubertal boys scheduled for gonadotoxic treatment. Intervention: None Main outcome measure(s) Histopathological analysis of tissue architecture, structural integrity, and cellular morphology, was performed for control and frozen-thawed cryopreserved tissues. The number of seminiferous tubules per testicular section was calculated. The survival of spermatogonial stem cells (SSCs) and Sertoli cells of control and frozen-thawed cryopreserved tissues was analyzed by immunofluoresence staining. Results Uncontrolled Slow-Freezing (USF), Controlled Slow-Freezing (CSF), and vitrification similarly preserved the integrity of the adult testicular tissues, and the survival of SSCs and Sertoli cells. CSF of prepubertal testicular tissues effectively preserved their architecture, the number of tubules, SSCs and Sertoli cells. In addition, we observed SSC loss following chemotherapy in prepubertal boys, reemphasizing the importance of fertility preservation prior to gonadotoxic treatment. Conclusion Future fertility restoration for male survivors of pediatric cancers depends on the development of an optimal prepubertal testicular tissue cryopreservation method. Our findings demonstrate the effectiveness of CSF for cryopreservation of human prepubertal testicular tissues, and may contribute to more effective banking of these tissues, and potential fertility restoration.

Recently, we reported the differential expression of kisspeptinergic system in the bubaline hypothalamus and corpus luteum. Here, we document the expression of kisspeptin (Kp) and its receptor (Kiss1r) in the ovarian follicles of the buffalo with respect to the functional status. Follicles of ≥10 to ≤13 mm diameter (n = 45) were retrospectively categorized into active (n = 18), intermediate (n = 16) and atretic (n = 11) follicles based on the concentrations of intrafollicular progesterone and estradiol. The P4:E2 ratio was significantly lower in the active follicle (0.43 ± 0.08) than that of the intermediate (3.46 ± 0.53) and atretic (28.4 ± 10.6) follicles (P

International audience; Objective: To explore the regulatory role of soluble CD146 (sCD146) and its interaction with galectin-1 (Gal1) in placenta-mediated complications of pregnancy.Design: Prospective pilot and experimental studies.Setting: University-affiliated hospital and academic research laboratory.Patient(s): One hundred fifteen women divided into three groups: 30 healthy, nonpregnant women, 50 women with normal pregnancies, and 35 with placenta-mediated pregnancy complications.Intervention(s): Wound-healing experiments were conducted to study trophoblast migration.Main outcome measure(s): Quantification of sCD146 and Gal1 by enzyme-linked immunosorbent assay. Analysis of trophoblast migration by wound closure.Result(s): Concomitant detection of sCD146 and Gal1 showed lower sCD146 and higher Gal1 concentrations in women with normal pregnancies compared with nonpregnant women. In addition, follow-up of these women revealed a decrease in sCD146 associated with an increase in Gal1 throughout pregnancy. In contrast, in women with preeclampsia, we found significantly higher sCD146 concentrations compared with women with normal pregnancies and no modification of Gal1. We emphasize the opposing effects of sCD146 and Gal, since, unlike Gal1, sCD146 inhibits trophoblast migration. Moreover, the migratory effect of Gal1 was abrogated with the use of an anti-CD146 blocking antibody or the use of small interfering RNA to silence VEGFR2 expression. This suggests that trophoblast migration is mediated though the interaction of Gal1 with CD146, further activating the VEGFR2 signaling pathway. Significantly, sCD146 blocked the migratory effects of Gal1 on trophoblasts and inhibited its secretion, suggesting that sCD146 acts as a ligand trap.Conclusion(s): Soluble CD146 could be proposed as a biomarker in preeclampsia and a potential therapeutic target. Clinical trial registration number: NCT 01736826.Trial registration: ClinicalTrials.gov NCT01736826.

Objective: The present study evaluated the efficiency of embryo development and pregnancy of somatic cell nuclear transfer (SCNT) embryos using different source-matured oocytes in Camelus dromedarius.Methods: Camelus dromedarius embryos were produced by SCNT using in vivo- and in vitro- matured oocytes. In vitro embryo developmental capacity of reconstructed embryos was evaluated. To confirm the efficiency of pregnancy and live birth rates, a total of 72 blastocysts using in vitro- matured oocytes transferred into 45 surrogates and 95 blastocysts using in vivo- matured oocytes were transferred into 62 surrogates by transvaginal method.Results: The collected oocytes derived from ovum pick up showed higher maturation potential into metaphase II oocytes than oocytes from the slaughterhouse. The competence of cleavage, and blastocyst were also significantly higher in in vivo- matured oocytes than in vitro- matured oocytes. After embryo transfer, 11 pregnant and 10 live births were confirmed in in vivo- matured oocytes group, and 2 pregnant and 1 live birth were confirmed in in vitro- matured oocytes group. Furthermore, blastocysts produced by in vivo-matured oocytes resulted in significantly higher early pregnancy and live birth rates than in vitromatured oocytes.Conclusion: In this study, SCNT embryos using in vivo- and in vitro-matured camel oocytes were successfully developed, and pregnancy was established in recipient camels. We also confirmed that in vivo-matured oocytes improved the development of embryos and the pregnancy capacity using the blastocyst embryo transfer method.

The American journal of pathology 192(2), 239-253 (2022). doi:10.1016/j.ajpath.2021.10.020, Published by Elsevier, New York [u.a.]

Preeclampsia evolves in 2 stages: a placental problem that generates signals to the mother to cause a range of responses that comprise the second stage (preeclampsia syndrome). The first stage of early-onset preeclampsia is poor placentation, which we here call malplacentation. The spiral arteries are incompletely remodeled, leading to later placental malperfusion, relatively early in the second half of pregnancy. The long duration of the first stage (several months) is unsurprisingly associated with fetal growth restriction. The first stage of late-onset preeclampsia, approximately 80% of total cases, is shorter (several weeks) and part of a process that is common to all pregnancies. Placental function declines as it outgrows uterine capacity, with increasing chorionic villous packing, compression of the intervillous space, and fetal hypoxia, and causes late-onset clinical presentations such as "unexplained" stillbirths, late-onset fetal growth restriction, or preeclampsia. The second stages of early- and late-onset preeclampsia share syncytiotrophoblast stress as the most relevant feature that causes the maternal syndrome. Syncytiotrophoblast stress signals in the maternal circulation are probably the most specific biomarkers for preeclampsia. In addition, soluble fms-like tyrosine kinase-1 (mainly produced by syncytiotrophoblast) is the best-known biomarker and is routinely used in clinical practice in many locations. How the stress signals change over time in normal pregnancies indicates that syncytiotrophoblast stress begins on average at 30 to 32 weeks' gestation and progresses to term. At term, syncytiotrophoblast shows increasing markers of stress, including apoptosis, pyroptosis, autophagy, syncytial knots, and necrosis. We label this phenotype the "twilight placenta" and argue that it accounts for the clinical problems of postmature pregnancies. Senescence as a stress response differs in multinuclear syncytiotrophoblast from that of mononuclear cells. Syncytiotrophoblast irreversibly acquires part of the senescence phenotype (cell cycle arrest) when it is formed by cell fusion. The 2 pathways converge on the common pathologic endpoint, syncytiotrophoblast stress, and contribute to preeclampsia subtypes. We highlight that the well-known heterogeneity of the preeclampsia syndrome arises from different pathways to this common endpoint, influenced by maternal genetics, epigenetics, lifestyle, and environmental factors with different fetal and maternal responses to the ensuing insults. This complexity mandates a reassessment of our approach to predicting and preventing preeclampsia, and we summarize research priorities to maximize what we can learn about these important issues.

Testicular sperm extraction (TESE) is an invasive surgery for achieving the spermatozoa in cases with azoospermia. In these patients, the number of retrieved spermatozoa is limited and the optimal cryo-storage is very critical for their fertility preservation. Therefore, single sperm vitrification has been introduced for preservation of low number of spermatozoa. The goal was to assess the efficacy of sperm freezing medium (SFM) and sucrose medium as cryoprotectants for single sperm vitrification in cases with severe oligozoospermia and azoospermia. A total of 20 ejaculates from severe oligozoospermia and 20 testicular samples from azoospermia were processed. Twenty-five sperm cells were collected using ICSI injection pipette and transferred to a cryoprotectant droplet placed on the Cryotech, then vitrified by plunging in liquid nitrogen. Sperm motility, viability, fine-morphology, mitochondrial activity and DNA fragmentation index (DFI) were assessed before and after vitrification. Sperm motility, viability and the percentage of cells with mitochondrial activity were significantly decreased after vitrification in both severe oligozoospermic and testicular samples in either cryoprotectants. However, the rates of post-warm sperm motility and the cells with mitochondrial activity increased significantly in sucrose medium in both severe oligozoospermic and testicular samples compared to SFM. In testicular samples, the DFI of spermatozoa vitrified in SFM was significantly higher than those vitrified with sucrose medium. Sperm motility, viability, mitochondrial activity, and DNA integrity were better preserved in sucrose medium than SFM after single cell vitrification. The presented method may be a useful candidate for successful freezing of individual sperm cells in clinical setting.

Objective To analyze the effect of a fertilin-derived peptide (cFEE) on in vitro maturation of human oocytes. Design Randomized study. Setting Fertility center in an academic hospital. Patients Not applicable. Intervention Human immature germinal vesicle-stage oocytes (n=1629), donated for research according to French bioethics laws were randomly allocated to groups treated with 1 μM, 100 μM cFEE or to a control group. They were incubated at 37° C in 6% CO2 and 5% O2 and their maturation was assessed using time-lapse microscopy over 24 hours. In vitro-maturated metaphase II oocytes were analyzed for chromosomal content using microarray-comparative genomic hybridization (CGH-array) and their transcriptomes were analyzed using Affymetrix Clariom D microarrays. Main outcome measures The percentage of oocytes undergoing maturation in vitro was observed. Aneuploidy and euploidy were assessed for all chromosomes, and differential gene expression was analyzed in oocytes treated with cFEE compared to the control to obtain insights into its mechanism of action. Results cFEE significantly increased the percentage of oocytes that matured in vitro and improved euploidy in meiosis II oocytes by upregulation of FMN1 and FLNA genes, both of which encode proteins involved in spindle structure. Conclusion cFEE improves human oocyte maturation in vitro and reduces aneuploidy. It might prove useful for treating oocytes prior to fertilization in Assisted Reproduction Technology and for in vitro maturation in fertility preservation programs to improve oocyte quality and the chances for infertile couples to conceive.

OBJECTIVE To investigate the chondroprotective effects of autologous platelet-rich plasma (PRP), ampicillin-sulbactam (AmpS), or PRP combined with AmpS (PRP+AmpS) in an in vitro chondrocyte explant model of bovine Staphylococcus aureus–induced septic arthritis. SAMPLE Autologous PRP and cartilage explants obtained from 6 healthy, adult, nonlactating Jersey-crossbred cows. ProcedureS Autologous PRP was prepared prior to euthanasia using an optimized double centrifugation protocol. Cartilage explants collected from grossly normal stifle joints were incubated in synovial fluid (SF) alone, S aureus–inoculated SF (SA), or SA supplemented with PRP (25% culture medium volume), AmpS (2 mg/mL), or both PRP (25% culture medium volume) and AmpS (2 mg/mL; PRP+AmpS) for 24 hours. The metabolic activity, percentage of dead cells, and glycosaminoglycan content of cartilage explants were measured with a resazurin-based assay, live-dead cell staining, and dimethylmethylene blue assay, respectively. Treatment effects were assessed relative to the findings for cartilage explants incubated in SF alone. RESULTS Application of PRP, AmpS, and PRP+AmpS treatments significantly reduced S aureus–induced chondrocyte death (ie, increased metabolic activity and cell viability staining) in cartilage explants, compared with untreated controls. There were no significant differences in chondrocyte death among explants treated with PRP, AmpS, or PRP+AmpS. CLINICAL RELEVANCE In this in vitro explant model of S aureus–induced septic arthritis, PRP, AmpS, and PRP+AmpS treatments mitigated chondrocyte death. Additional work to confirm the efficacy of PRP with bacteria commonly associated with clinical septic arthritis in cattle as well as in vivo evaluation is warranted.

Cryopreservation of single seminiferous tubule is significant for fertility preservation, especially for male patients with cryptorchidism, Y-chromosome deletion and orchitis. However, there are few studies on cryopreservation of single seminiferous tubule. This study proposes several improved strategies for cryopreservation of single seminiferous tubule in mice. First, single seminiferous tubule was cryopreserved with modified slow freezing and vitrification methods. The results showed that the apoptosis negative rates of spermatogenic cells in single seminiferous tubule with modified slow freezing method were significantly higher than vitrification with plastic slide. Then, plastic slide and two metal vitrification carriers with high thermal conductivity, copper mesh and aluminum foil, were used to vitrify single seminiferous tubule. The metal carriers could improve the outcome of vitrification than plastic slide. The apoptosis negative rates of spermatogenic cells in the aluminum foil group was significantly higher than that in the copper mesh group. Finally, single seminiferous tubule was perfused with CPAs by micro-injection technique and vitrified. The results of cryo-microscopy experiments showed that the ice crystals formed inside the injected seminiferous tubule was reduced during the cooling process, the apoptosis negative rate of spermatocytes, spermatids and Sertoli cells were significantly higher than that of the non-injection group, indicating that the injection technique can effectively improve the effect of vitrification. This study has potential clinical value for in-vitro culture of spermatogonial stem cells and autologous testicular tissue grafting in patients with azoospermia.

Spontaneous loss is seen 12-15 % in all pregnancies. More than %50 of those patients do not have a defined etiology, so they are named unexplained Recurrent Pregnancy Loss (URPL). Studies in recent years have made us think that Pregnancy Losses may be related to insufficient decidualization and undetected prothrombotic problems. Tyro3 receptor kinase (RTK) is a member of TAM receptors and has been reported to have an important role in migration, Epithelial-mesenchymal transition (EMT), and platelet aggregation. There is not sufficient knowledge about expression levels of Tyro3 RTK in human first-trimester URPL decidua (URPLD). By immunohistochemistry, immunoblotting, and qPCR, we investigated the expression of Tyro3 RTK in the decidua of human first-trimester termination healthy pregnancy (TPD)(n:6) and URPLD(n:6), and human endometrium (CE)(n:6). Our results suggest that Tyro3 RTK expression significantly decreases with pregnancy and then returns to levels of CE for URPLD. Considering the role of Tyro3 RTK in platelet activation and thrombus formation, it suggests that this decrease observed in TPD may prevent the formation of prothrombosis which can prevent placental flow, and thus, hemostasis could be regulated in pregnancy through Tyro3 RTK. Although Tyro3 RTK decrease in TPD, locally increase on TPD, and also decrease on URPLD of Tyro3 RTK on DSCs makes us think, which Tyro3 RTK has a regulating role for EMT formation on DSCs and may participate in implantation and survival processing of the embryo through DSCs. If those predictions are supported by further functional analyses, one of the causes of URPLD can be enlightened.

Chlamydia trachomatis (CT) infection is the most prevalent sexually transmitted bacterial disease worldwide. However, unlike that in female infertility, the role of CT infection in male infertility remains controversial. The objective of this retrospective study was to explore the impacts of CT infection in the genital tract on sperm quality, sperm acrosin activity, antisperm antibody levels, and inflammation in a large cohort of infertile males in China. A total of 7154 semen samples were collected from infertile male subjects, 416 of whom were CT positive (CT+ group) and 6738 of whom were CT negative (CT- group), in our hospital between January 2016 and December 2018. Routine semen parameters (semen volume, pH, sperm concentration, viability, motility, morphology, etc.), granulocyte elastase levels, antisperm antibody levels, and sperm acrosin activity were compared between the CT+ and CT- groups. Our results showed that CT infection was significantly correlated with an abnormally low semen volume, as well as an increased white blood cell count and granulocyte elastase level (all P < 0.05) in the semen of infertile males; other routine semen parameters were not negatively impacted. The antisperm antibody level and sperm acrosin activity were not affected by CT infection. These findings suggested that CT infection might contribute to inflammation and hypospermia but does not impair sperm viability, motility morphology, and acrosin activity or generate antisperm antibodies in the infertile males of China.

Assisted reproductive techniques (ARTs), such as artificial insemination, in vitro fertilization, and cryopreservation of gametes/zygotes, have been developed to improve breeding and reproduction of livestock, and for the treatment of human infertility. Their widespread use has contributed to improvements in human health and welfare. However, in dogs, only artificial insemination using frozen semen is readily available as an ART to improve breeding and control genetic diversity. A recent priority in sperm cryopreservation is the development of alternatives to egg yolk, which is widely used as a component of the sperm extender. Egg yolk can vary in composition among batches and is prone to contamination by animal pathogens. The latter can be a problem for international exchange of cryopreserved semen. Low-density lipoprotein and skim milk are promising candidates for use as extenders, to ensure fertility after artificial insemination. Although not tested for its effects on fertility following artificial insemination, polyvinyl alcohol may also be a useful alternative to egg yolk as an extender. The development of cryopreservation techniques for canine embryos lags behind that for other mammals, including humans. However, given the success of non-surgical embryo transfer in 2011, studies have sought to refine this approach for practical use. Research on sperm cryopreservation has yielded satisfactory results. However, investigation of other approaches, such as cryopreservation of oocytes and gonadal tissues, remains insufficient. Techniques for the efficient induction of estrus may aid in the development of successful canine ARTs.

Made available in DSpace on 2022-05-01T09:47:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 This study aimed to evaluate the immunomodulatory effect of vitamin D (VD) on the NLRP1 and NLRP3 inflammasomes in placental explants from preeclamptic (PE) and normotensive (NT) pregnant women. Placental explants from eight PE and eight NT pregnant women were cultured with or without hydrogen peroxide (H2O2), VD or H2O2 + VD. Gene and protein expression of NLRP1, NLRP3, HMGB1, caspase-1, IL-1β, TNF-α and IL-18 were determined by qPCR and Western blotting/ELISA. Compared to NT pregnant women, the endogenous gene expression of NLRP1, NLRP3, HMGB1, IL-1β, TNF-α and IL-18 was significantly higher in explants from PE and became decreased after VD treatment. Similarly, VD decreased the protein expression of NLRP1, NLRP3, caspase-1, HMGB1, IL-1β, TNF-α and IL-18 in PE. Placental explants from NT cultured with H2O2 showed increased gene and protein expression of NLRP1, NLRP3, caspase-1, IL-1β, TNF-α and HMGB1, while H2O2 was also able to increase TNF-α and caspase-1 gene expression in PE. Treatment with H2O2 + VD decreased gene/protein expression of NLRP1, NLRP3, caspase-1, HMGB1, IL-1β, TNF-α and IL-18 in PE and NT explants with H2O2. NLRP1 and NLRP3 are upregulated in the PE. VD may play an immunomodulatory role in the placental inflammation and downregulates oxidative stress induced in vitro by H2O2. Botucatu Medical School Sao Paulo State University (Unesp) Institute of Biosciences Sao Paulo State University (Unesp) Botucatu Medical School Sao Paulo State University (Unesp) Institute of Biosciences Sao Paulo State University (Unesp)

In preeclamptic pregnancies, a variety of intrauterine alterations lead to abnormal placentation, release of inflammatory and/or antiangiogenic factors, and subsequent fetal growth restriction with significant potential to cause a primary insult to the developing fetal lung. Thus, modulation of the maternal intrauterine environment may be a key therapeutic avenue to prevent preeclampsia-associated developmental lung injury. A biologic therapy of interest is mesenchymal stromal cell–derived extracellular vesicles (MEx), which we have previously shown to ameliorate preeclamptic physiology through intrauterine immunomodulation. To evaluate the therapeutic potential of MEx to improve developmental lung injury in experimental preeclampsia, using the heme oxygenase-1–null mouse (Hmox1(−)(/−)) model, preeclamptic pregnant dams were administered intravenous antenatal MEx treatment during each week of pregnancy followed by analysis of fetal and postnatal lung tissues, amniotic fluid protein profiles, and lung explant and amniotic fluid cocultures in comparison with control and untreated preeclamptic pregnancies. We first identified that a preeclamptic intrauterine environment had a significant adverse impact on fetal lung development, including alterations in fetal lung developmental gene profiles in addition to postnatal alveolar and bronchial changes. Amniotic fluid proteomic analysis and fetal lung explant and amniotic fluid cocultures further demonstrated that maternally administered MEx altered the expression of multiple inflammatory mediators in the preeclamptic intrauterine compartment, resulting in the normalization of fetal lung branching morphogenesis and developmental gene expression. Our evaluation of fetal and postnatal parameters overall suggests that antenatal MEx treatment may provide a highly valuable preventative therapeutic modality for amelioration of lung development in preeclamptic disease.

Objective To evaluate whether platelet-rich plasma (PRP) optimizes endometrial thickness (EMT) and improves live birth rates (LBR) in women with refractory thin endometrium due to varied aetiology during fresh in vitro fertilization (IVF) and frozen-thawed embryo transfer (FET). Methods A prospective interventional study was conducted at the ART Centre of a tertiary care academic hospital. Twenty-two infertile women with refractory thin endometrium (0.05). PRP led to significant increase in EMT in tuberculosis (TB), diminished ovarian reserve (DOR) and polycystic ovary syndrome (PCOS) (p0.05). Overall, clinical pregnancy and LBR reached up to 20% and 25%, respectively. No adverse reactions were reported. Conclusions PRP enhances EMT significantly during fresh and FET cycles in thin endometrium associated with TB, PCOS and DOR, thus improving the CPR and LBR in these low prognosis patients.

Objective To demonstrate the non-inferiority of Clinical Pregnancy Rates from Progestin-Primed Ovarian Stimulation compared to the GnRH Antagonist Protocol when the freeze-all and blastocyst transfer strategy is applied. Methods A retrospective study included all IVF cycles performed at Pro-Criar Reproductive Medicine Center, Belo Horizonte, Minas Gerais, Brazil, between May 2018 and May 2019 using a GnRH antagonist analogue or oral progestins to block the LH peak in IVF/intra-cytoplasmic sperm injection (ICSI) cycles for infertility treatment. Results The primary outcome of our study was Clinical Pregnancy Rate at the first ET (Blastocyst), which were 58.4% in the progestin group and 54.9% in the antagonist group (p=0.735), a finding consistent with most studies published to date using different progestins. The mean number of retrieved oocytes was 11 in the antagonist group and 9 oocytes in the progestin group (p=0.009). The fertilization rate was 80% for both groups (p=0.935). The rate of blastocyst formation per cycle was 50% in the antagonist group and 55.6% in the progestin group (p=0.106). The stimulation lasted a mean of 10 days in the two groups (p=0.403) and did not vary with patient age in either group. The gonadotropin dose used was higher in the antagonist group (2025 IU) than in the progestin group (1950 IU) (p=0.057). In addition, the blockade was effective: there was only one case of spontaneous ovulation, which corresponded to less than 1% of the cycles. Conclusions Progestin-Primed Ovarian Stimulation is a non-inferior alternative to the GnRH Antagonist Protocol in patients undergoing assisted reproductive techniques. An incidence compatible with the 0.34 to 8% risk described in the literature for failure to control the premature LH surge in antagonist protocol cycles.

To research the local action of endometriosis on follicular inflammatory microenvironment of patients undergoing in vitro fertilization (IVF) cycles. A prospective case control study was organized at Kamal AL-Samarai IVF center (Baghdad/Iraq), January 2021 - May 2021. The study included (80) infertile women undergoing IVF cycles were split to two groups: Patients with endometriosis group (n = 40) and Patients without endometriosis group (n = 40). Follicular fluid (FF) and blood samples were gathered at the period of oocyte pick-up. Levels of MCP-1 mRNA were investigated with the reverse transcription polymerase chain reaction (RT-PCR) technique. Concentrations of cytokines (monocyte chemo attractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10)) were investigated in FF by sandwich enzyme-linked immunosorbent assay (ELISA). The mRNA expression of MCP-1 was significantly (P = 0.00) higher in patients with endometriosis group (1.95 ± 0.69) compared with patients without endometriosis group (0.40 ± 0.15). The studied cytokines concentrations in FF of women with endometriosis group were significantly (P = 0.00) higher than those without endometriosis group: MCP-1 (548.07 ± 199.43 pg/ml vs. 45.98 ± 18.72 pg/ml), TNF-α (912.15 ± 316.12 pg/ml vs. 261.89 ± 90.69 pg/ml), IL-10 (258.94 ± 68.82 pg/ml vs. 52.12 ± 14.46 pg/ml). The immune milieu of FF of patients with endometriosis is modified, which could explain the effect of endometriosis on infertility. Furthermore, elevated MCP-1 expression in endometriosis patients suggesting systemic inflammatory changes.

Semen analysis has long been used to evaluate male fertility. Recently, several sperm function tests have been developed. Of those, the sperm DNA fragmentation index (DFI), which describes the status of the sperm DNA, is thought to be a suitable parameter for evaluating male fertility. However, there have been no large-scale studies on the sperm DFI of Japanese men. Therefore, we investigated the feasibility of using an in-house flow cytometry-based sperm DFI analysis based on the sperm DNA fragmentation test of sperm chromatin structure assay (SCSA) to assess male fertility in Japan. This study enrolled 743 infertile and 20 fertile Japanese men. To evaluate reproducibility, inter- and intraobserver precision was analyzed. A receiver operating characteristic curve analysis was used to set a cutoff value for the sperm DFI to identify men who could father children by timed intercourse or intrauterine insemination. The variability of the sperm DFI among fertile volunteers was determined. The relationship between semen parameters and the sperm DFI was assessed by Spearman's rho test. A precision analysis revealed good reproducibility of the sperm DFI. The cutoff value of sperm DNA fragmentation in infertile men was 24.0%. Semen volume had no relationship with the sperm DFI. Sperm concentration, sperm motility, total motile sperm count, and percentage of normal-shaped sperm were significantly and negatively correlated with the sperm DFI. The median sperm DFI was smaller in fertile volunteers (7.7%) than that in infertile men (19.4%). Sperm DNA fragmentation analysis can be used to assess sperm functions that cannot be evaluated by ordinary semen analysis.

In cattle there are two types of the corpus luteum (CL): homogeneous (CLhom) and cavitary (CLcav). Although they are considered equal in their hormonal activity, the function of the CLcav is questioned by many veterinarians. In consequence, females with the CLcav are considered less valuable for assisted reproductive techniques such as embryo transfer (ET), where recipients with the CLhom are preferred. The aim of our study was to compare the two types of CLs regarding morphological endpoints, serum progesterone (P4) concentrations and final pregnancy rate (PR) in recipient heifers after ET. The morphological type of the CL and the final PR after ET of 432 Holstein Friesian heifers were analyzed. Oestrus was synchronized with two i.m. inj. of 0.5 μg cloprostenol 14 days apart. The ET took place on the day 7 of the estrous cycle, only animals with visible oestrum were chosen for the procedure. Clinical and transrectal US examinations of ovaries were performed on the day of ET. The presence of the CLhom or CLcav was determined, and the CL diameter and cross-sectional area were measured. If present, measurements of the cavities were also taken. Only embryos recovered immediately prior to the ET at the morula or blastocyst stage were transferred to the randomly chosen recipient that underwent initial selection regardless of the CL morphology. Additionally, from randomly selected heifers (N = 53, CLhom = 33; CLcav = 20) blood samples for serum P4 concentration analysis were collected. Pregnancy was determined by transrectal palpation 2 months after ET. The medium-sized CLcav was larger in diameter (P The presence of a CLcav in the recipient heifers did not negatively affect the potential of the CL to maintain pregnancy. On the contrary, the CLcav may give the embryo better chances of surviving the time of pregnancy recognition and in consequence, may have a positive effect on PR in heifers.

RESEARCH QUESTION Is ovarian response associated with individualized follitropin delta dosing regimen comparable across different ethnic populations? DESIGN Post-hoc analysis of ovarian response in 800 IVF/intracytoplasmic sperm injection (ICSI) patients (170 Japanese women and 630 White women) undergoing stimulation with individualized follitropin delta dosing based on serum anti-Mullerian hormone concentration and body weight in two randomized controlled trials conducted in Japan (NCT03228680) and in Europe, North America and South America (NCT01956110). RESULTS On average, Japanese women weighed 10 kg less, which affected the total follitropin delta dose, compared with White women (83.5 ± 28.9 versus 90.2 ± 25.2 µg). At the end of stimulation, serum FSH concentrations were not significantly different between Japanese and White women (median 14.3 versus 14.0 IU/l), whereas serum oestradiol concentrations were significantly higher in Japanese women (median 6517 versus 5298 pmol/l, P < 0.0001). Japanese and White women had a similar number of oocytes retrieved with no significant differences among all women who started stimulation (9.3 ± 5.4 versus 9.5 ± 5.7), potential low responders (7.2 ± 3.7 versus 7.6 ± 4.6) or potential high responders (10.8 ± 5.9 versus 11.0 ± 6.0). At each level of ovarian response, serum oestradiol concentrations were significantly higher in Japanese women (P = 0.024). The incidence of early ovarian hyperstimulation syndrome was significantly higher in Japanese women compared with White women; overall (10.0% versus 2.2%, P = 0.0124) and at similar serum oestradiol concentrations (P = 0.0137). CONCLUSIONS The individualized follitropin delta dosing provides similar serum FSH concentrations and similar oocyte yield in Japanese and White IVF/ICSI patients, but the oestradiol response is higher in Japanese women.

Aneuploidy is the leading genetic cause of miscarriage and infertility in women and occurs frequently in oocytes. Spindle formation and positioning are two critical events that must be regulated tightly to avoid erroneous chromosome segregation. Following nuclear envelope breakdown (NEBD), the spindle is assembled centrally before migrating towards the cortex to allow the first asymmetric division. The biological significance of the primary central positioning of the spindle is unknown. Because the spindle forms where NEBD occurs, whether positioned at the center or at the cortex of the cell, full-grown GV (prophase I) oocytes were collected from CF-1 mice (6-8 weeks old) and sorted according to the position of the GV into three groups: central, intermediate, and peripheral. Approximately 50% of the cells exhibited a central GV position, while 25% of cells showed a peripheral GV position. These proportions were similar to those obtained by histological evaluation of ovarian oocytes in 6-8 week mice, but not to those of mice aged less than 5 weeks, which tended to have a majority of peripherally positioned GVs. When peripheral GV oocytes were matured in vitro, GVs (88%) migrated towards the center and NEBD (and spindle assembly) occurred either at the center of the cell or during migration. The percentages of NEBD and polar body (PB) extrusion did not vary significantly among groups. Importantly, peripheral GV oocytes had a significant increase of abnormal K-MT attachments at metaphase I (Met I) and showed a significant increase of aneuploidy at metaphase II (Met II) when compared to central GV oocytes. Interestingly, peripheral GV oocytes that were able to achieve full GV relocation to the center were half as likely to experience aneuploidy when compared to peripheral GV oocytes unable to achieve relocation. These results indicate that preferential central spindle formation is an insurance mechanism to protect against incorrect K-MT attachments and aneuploidy.

Objective We sought to investigate the biological effects of pre-reperfusion treatments of the liver after warm and cold ischemic injuries in a porcine donation after circulatory death (DCD) model. Summary of background data DCD represents a severe form of liver ischemia and reperfusion injury that has a profound impact on graft function after liver transplantation. Methods Twenty donor pig livers underwent 60 minutes of in situ warm ischemia after circulatory arrest and 120 minutes of cold static preservation prior to simulated transplantation using an ex vivo perfusion machine. Four reperfusion treatments were compared: Control-Normothermic (N), Control-Subnormothermic (S), regulated hepatic reperfusion (RHR)-N, and RHR-S (n = 5 each). The biochemical, metabolic, and transcriptomic profiles, as well as mitochondrial function were analyzed. Results Compared to the other groups, RHR-S treated group showed significantly lower post-reperfusion aspartate aminotransferase levels in the reperfusion effluent and histologic findings of hepatocyte viability and lesser degree of congestion and necrosis. RHR-S resulted in a significantly higher mitochondrial respiratory control index and calcium retention capacity. Transcriptomic profile analysis showed that treatment with RHR-S activated cell survival and viability, cellular homeostasis as well as other biological functions involved in tissue repair such as cytoskeleton or cytoplasm organization, cell migration, transcription, and microtubule dynamics. Furthermore, RHR-S inhibited organismal death, morbidity and mortality, necrosis, and apoptosis. Conclusion Subnormothermic RHR mitigates IRI and preserves hepatic mitochondrial function after warm and cold hepatic ischemia. This organ resuscitative therapy may also trigger the activation of protective genes against IRI. Subnormothermic RHR has potential applicability to clinical liver transplantation.

BACKGROUND Impaired paternal genome expression may cause poor embryonic development after in vitro fertilization (IVF). OBJECTIVE To evaluate the expression of male infertility on embryo morphokinetics using a time-lapse incubator and its impact on IVF cycles. MATERIALS AND METHODS This retrospective cohort study followed patients from January 2017 to August 2019. Patients were divided according to the cause of infertility to male factor (study group) and unexplained infertility (control group) and further subdivided according to the severity of male infertility. RESULTS A cohort of 462 patients who underwent IVF cycles, with a total of 3,252 embryos was evaluated. Intracytoplasmic sperm injection (ICSI) was conducted more often in the study group compared to the control group (94% vs. 47%, p

OBJECTIVE To find and explore the relationship among the length of the embryo's main anchoring villus and the week of gestation, embryonic crown-rump length (CRL), and maternal blood human chorionic gonadotropin (hCG) during early pregnancy using ultrasound. METHODS A total of 108 women with singleton pregnancies underwent ultrasound examination of the fetus during the early months of their pregnancy (5+5 -13+6 weeks of pregnancy). The main anchoring villus was detected using HD-Flow and SlowflowHD mode ultrasound. The detection rate and length of the main anchoring villus were recorded. The correlations among main anchoring villus with gestational weeks, CRL, and blood hCG were obtained. RESULTS The main anchoring villus was detected in all women (108/108). It could be found throughout the early gestational weeks from as early as 4+6 weeks. The length of the main anchoring villus increased with gestational age, and was positively correlated with CRL and the logarithm of hCG (P

Cotyledons (n= 167) from 30 Hungarian and 5 Slovakian dairy cattle herds were analysed forCoxiella burnetiiby real-time PCR targeting theIS1111gene. Eighty (88.9%) out of the 90 cotyledons from retained placentas and 31 (40.3%) out of the 77 cotyledons from normally separated placentas tested positive. Seventeen out of the 80 positive samples (21.3%) originating from retained placentas were found to be highly loaded withC. burnetiiwith a cycle threshold (Ct) value lower than or equal to 27.08, ranging between 11.92 and 27.08. The rest of the positive samples from retained fetal membranes and from normally separated placentas were moderately loaded withC. burnetiiDNA. Five out of the ten samples showing the strongest positivity (Ct 11.92–18.28) from retained placentas were genotyped by multispacer sequence typing based on ten loci, which revealed sequence type (ST) 61, a type that had not been detected in Hungary and Slovakia previously. Retained placenta was more likely in cows withC. burnetiiPCR-positive cotyledons (odds ratio: 12.61,P= 0.0023). The highC. burnetiiDNA load found in retained fetal membranes may be a potential risk factor for human infection and may also be associated with the retention of fetal membranes.

Recurrent implantation failure is a major problem in assisted reproductive technology (ART). Although ART systems have evolved rapidly over the decades, it is still difficult to diagnose uterine conditions suitable for embryo transfer (ET) without the use of invasive endometrial procedures. Previous studies in mice showed that leukemia inhibitory factor (LIF) is a well-known endometrial biomarker for uterine implantation capacity, also known as uterine receptivity. This study focused on LIF in the mouse and human cervix as a possible biomarker of implantation capacity. We found that high expression of LIF in the cervical epithelium is strongly correlated with that of the uterine epithelium during the peri-implantation period in mice. Likewise, human cervical epithelia also exhibit elevated levels of LIF in the peri-implantation period. In addition, cervical LIF is downregulated in mice with defective implantation caused by pharmacological treatments. These results indicated that cervical LIF is a possible biomarker that detected uterine receptivity without invasive endometrial damage.

Preeclampsia is a severe pregnancy complication characterized by hypertension and may cause maternal morbidity and mortality. A better understanding of the essential genes involved in preeclampsia pathophysiology is urgently needed. This study investigated the function and molecular mechanisms of pumilio RNA binding family member 1 (PUM1) in extravillous trophoblast cells (EVTs). The interaction between protein and mRNA was verified by RNA pull-down assays, RNA immunoprecipitation assays, and luciferase reporter assays. The mRNA and protein levels of the genes involved were determined by RT-qPCR and western blot assays, respectively. Our results demonstrated that PUM1 could bind to the 3′-untranslated region of low-density lipoprotein receptor-related protein 6 (LRP6) mRNA, resulting in reduced expression of LRP6 mRNA and protein. Repression of PUM1 resulted in enhanced colony formation, cell proliferation, migration, and invasion of EVTs. The PUM1-depletion-mediated promotion effects on EVTs could be abrogated by LRP6 knockdown. PUM1 regulates the growth and mobility of EVTs by modulating LRP6 expression. Developing strategies to balance PUM1 and LRP6 levels may be beneficial for the management of preeclampsia patients.

Summary Chromosome imbalance (aneuploidy) is the major cause of pregnancy loss and congenital disorders in humans. Analyses of small biopsies from human embryos suggest that aneuploidy commonly originates during early divisions, resulting in mosaicism. However, the developmental potential of mosaic embryos remains unclear. We followed the distribution of aneuploid chromosomes across 73 unselected preimplantation embryos and 365 biopsies, sampled from four multifocal trophectoderm (TE) samples and the inner cell mass (ICM). When mosaicism impacted fewer than 50% of cells in one TE biopsy (low-medium mosaicism), only 1% of aneuploidies affected other portions of the embryo. A double-blinded prospective non-selection trial (NCT03673592) showed equivalent live-birth rates and miscarriage rates across 484 euploid, 282 low-grade mosaic, and 131 medium-grade mosaic embryos. No instances of mosaicism or uniparental disomy were detected in the ensuing pregnancies or newborns, and obstetrical and neonatal outcomes were similar between the study groups. Thus, low-medium mosaicism in the trophectoderm mostly arises after TE and ICM differentiation, and such embryos have equivalent developmental potential as fully euploid ones.

Introduction Currently, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be induced to differentiate at the cellular level but not to form mature tissues or organs suitable for transplantation. ESCs/iPSCs form immature teratomas after injection into immunodeficient mice. In humans, immature teratomas often transform into fully differentiated mature teratomas after administration of anticancer agents. Methods We first investigated the ability of cisplatin to induce changes in mouse ESCs/iPSCs in vitro. Next, we designed experiments to analyze ESC/iPSC-derived immature teratoma tissue in vivo after treatment of cisplatin. Groups of six mice carrying ESC- or iPSC-derived teratomas were given either low or high dose intraperitoneal injection of cisplatin, while the control group received saline for 4 weeks. Results Treatment of ESC/iPSC cultures with cisplatin for 3 days caused a dose-related decrease in cell numbers without inducing any morphological changes to the cells. ESC/iPSC-derived teratomas showed lower growth rates with a significantly higher mature components ratio in a concentration dependent manner after cisplatin treatment (P, Highlights • Transformation of immature to mature teratoma after chemotherapy was verified. • Mice bearing ESC/iPSC-derived immature teratomas were used. • Mice were treated with intraperitoneal injection of cisplatin for 4 weeks. • Newly differentiated structures were found only in the tumors of treated mice. • Cisplatin can induce differentiation in ESC/iPSC-derived immature teratomas.

The purpose of the present work was to study effectiveness for early pregnancy diagnosis in cattle of the new enzyme immunoassay (EIA) sandwich kit commercially available based on the measurement of pregnancy-associated glycoproteins (PAGs). 120 Holstein-Friesian cattle of mixed age and parity were comprised from different dairy herds. The pregnant females (n = 68) were diagnosed by ultrasonography at day 35-40 after artificial insemination and confirmed by transrectal exploration at 2-3 months after AI. The non-pregnant females (n = 52) were housed in the absence of males during the experimental period. Blood samples were collected from coccygeal vessels of females into EDTA tubes. The serum was obtained by centrifugation and the serum was stored at - 20 C until assay. The PAG concentrations in pregnant and non-pregnant females were determined in serum by EIA kit. The reproducibility inter- and intra-assay of the PAG-EIA is satisfactory (2.78 and 13.19 %, respectively). The accuracy ( 94.8 %) and the test of parallelism were largely acceptable. No cross-reaction was observed with the different hormones tested at different dilutions. PAG-EIA system gave 100 % sensitivity and negative predictive values. Whereas, specificity and positive predictive value were 91.93 and 71.15 %, respectively. The accuracy of pregnancy diagnosis by PAG-EIA was 87.5 %. In conclusion, the present study shows clearly that the EIA kit can be used to measure PAG in serum cows for the detection of gestation in Algeria. Therefore, this alternative technique could be recommended to replace the radioactive methods in immunoassays to improve the reproductive performances and an efficient tool for reproductive management of dairy cattle.

Preeclampsia (PE) threatens the safety of mothers and fetuses, and its pathogenesis is still unclear. Our previous study has found the relationship between PE and serum Clusterin (CLU). This study aimed to investigate the role of CLU on PE. Firstly, levels of CLU in serum and placental tissue from PE patients and healthy pregnancies were compared. Then, RNA sequencing, cell counting kit-8, matrigel invasion, cell apoptosis, and angiogenesis assay were performed to evaluate the role of CLU on primary isolation trophoblast cells. We found the expression of CLU was increased before the clinical syndrome occurred, whereas its level was positively related to the severity of PE. CLU significantly inhibited the expression of matrix metalloproteinase-9 and Vimentin and enhanced E-cadherin to inhibit epithelial-mesenchymal transition of trophoblast cells, further reducing its migration and invasion. Our results suggested that CLU may play a role in regulating trophoblast invasion and migration during placental development, which may be one of the risk factors for PE.

Objective There is evidence that maternal immunity changes from type 1 (cell-mediated response) to predominantly type 2 (antibody-mediated immune response) during pregnancy. Lack of this shift in maternal immune response may contribute to unsuccessful pregnancy in animals. Changes in estradiol and progesterone during the estrous cycle affect immune function and potentially change the type 1–type 2 balance. The objective of this study was to examine the relationship between relative balance of type 1 and type 2 immunity at the time of insemination and fertility in beef cows, using a nanoparticle probe-enabled blood test. Materials and Methods Angus-Hereford cows (n = 394) were synchronized with a 5-d CO-Synch controlled internal drug release (CIDR) fixed-time AI protocol. Estrus behavior was monitored using estrus detection aids and visual observation from CIDR removal until the day of AI. At the time of AI, a blood sample was collected from all animals to determine relative ratio between type 1 and type 2 and serum progesterone concentrations. A rapid blood test (D2Dx; Tsai et al., 2021 ) was used to assess the type 1–type 2 immunity balance. Pregnancy was determined by ultrasound 48 to 63 d after AI. Results and Discussion Overall pregnancy per AI (P/AI) was 51%. Mean relative type 1–type 2 immunity balance as determined by the D2Dx immunity test was different (P Implications and Applications The results indicate that the measurement of the relative type 1–type 2 immunity balance at the time of AI may be used as a potential marker for pregnancy prediction in beef cows.

We studied the effect of extracellular vesicles of the follicular fluid on morphofunctional characteristics of human spermatozoa using CASA (computer-assisted sperm analysis) analytical system. The vesicles were obtained by sequential centrifugation at different rotational speeds and frozen at -80°C in the Sydney IVF Gamete Buffer medium. The sperm fraction was isolated from the seminal fluid of 21 patients aged 27-36 years by differential centrifugation in a density gradient. The precipitate was suspended in Sydney IVF Gamete Buffer to a concentration of 106/ml and incubated with vesicles (1:2) at 37°C in a CO2 incubator for 30 min and 1 h. Sperm fraction incubated without vesicles served as the control. After incubation, some sperm samples were centrifuged at 700g for 5 min and fixed in 2.5% glutaraldehyde in 0.1 M buffer for transmission electron microscopy. After 30-min and 1-h incubation, the progressive and total sperm motility improved, the curvilinear and linear velocity of spermatozoa did not change significantly. Incubation with vesicles significantly changed the trajectory of sperm movement, which can attest to an increase in their hyperactivation and, probably, fertilizing capacity. Analysis of the effect of extracellular vesicles of follicular fluid on sperm motility will help to improve the effectiveness of assisted reproductive technology programs with male infertility factor by improving sperm characteristics in patients with asthenozoospermia and increasing the fertilizing ability of the sperm.

With continued improvements in blastocyst culture, cell sampling approaches, and genetic analysis platforms, the resulting improvements in embryo development and the resolution and accuracy of chromosome analysis have provided valuable insights into the preimplantation embryo. This includes the impact of in vitro culture conditions on chromosomal dynamics. Specifically, through analysis of embryo aneuploidy and mosaicism, a growing number of reports indicate that rates of chromosomal abnormalities can vary between IVF centers. Because differences in mosaicism reflect mitotic errors, this endpoint analysis suggests that IVF laboratory-controlled variables during embryo development may be influencing chromosome separation and segregation. A growing body of literature suggests that culture media may be one variable influencing preimplantation embryo aneuploidy and mosaicism. However, these data are far from definitive in demonstrating cause-and-effect. Whether reported differences may be due to media formulation, use of sequential media or single-step media, or uninterrupted culture approaches is unknown. Importantly, variables directly impacting media performance and embryo development, including pH, temperature, osmolality, and oxygen concentration, must also be considered and make it difficult to isolate the impact of culture media as the sole factor responsible. These IVF laboratory variables will be reviewed and literature suggesting a possible link to mitotic aneuploidy/mosaicism will be discussed.