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LncRNA Profiling and ceRNA Network Construction of Intrauterine Exosomes in Goats During Embryo Implantation.

Exosomes have been shown to play an important role in embryo implantation, but the mechanism is still unclear. This study aimed to investigate the functional roles of lncRNAs in intrauterine exosomes in goat pregnancy. We used RNA-seq to identify the lncRNA profiles of exosomes obtained from goat uterine rinsing fluid at 5, 15, and 18 days of gestation. In addition, we performed weighted gene co-expression network analysis based on differentially expressed mRNAs (DEMs) and lncRNAs (DELs). Functional enrichment analyses of gene modules were conducted using Gene Ontology classification (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. A lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) regulatory network was constructed based on predictive interaction derived from miRTarBase, miRDB and RNAhybrid databases. Altogether, 831 DELs were identified. GO and KEGG analysis showed that the target genes were enriched in processes associated with embryo implantation, such as signaling receptor activity, binding and immune response. Nine functional co-expression modules were enriched in various biological processes, such as metabolic pathways, protein transport, cell cycle and VEGF signaling pathway. Additionally, 12 lncRNA-mediated ceRNA networks were constructed. Our results demonstrate that exosomal lncRNAs in uterine flushing fluid exhibit dynamic changes across gestational stages and play an important role in regulating the uterine microenvironment during embryo implantation. These findings provide a foundational basis for screening exosome-derived lncRNAs that influence embryo implantation and contribute to elucidating the mechanistic roles of lncRNAs in exosome-mediated processes during early pregnancy.

embryo implantation

Accumulation of steroids in rabbit preimplantation blastocysts.

The concentrations of progesterone, androstenedione, testosterone, oestrone and oestradiol were measured by radioimmunoassay in blastocysts and uterine fluid flushings collected from rabbits 110 to 159 h post coitum (p.c.). None of the blastocysts or uterine flushings contained detectable levels of androstenedione, testosterone or oestrone. All uterine flushings contained large amounts of progesterone and some of the flushings also contained oestradiol. A small amount of progesterone (approximately 7-5 pg/blastocyst) was first detectable in some blastocysts at 135 h p.c.; progesterone levels/blastocyst then increased progressively, reaching levels of about 122-158 pg/blastocyst at 159 h p.c. Micropuncture of blastocysts at 159 h p.c. indicated that greater than or equal to 90% of the progesterone in the embryo was in the blastocoelic fluid. Blastocysts from rabbit uterine horns containing oestradiol also contained oestradiol but those in which oestradiol was detected were never observed in uteri lacking the hormone. It is inferred that rabbit blastocysts accumulate both progesterone and oestradiol from uterine fluid.

Androstenedione

Cyclic variation in concentrations of protein and hexose in human uterine flushings collected by an improved technique.

Uterine flushings were obtained from fertile women at various stages of the menstrual cycle. A technique was used which excluded contamination with cervical mucus and significantly lowered contamination with blood in comparison with an established technique. Contamination of the uterine flushings with seminal plasma and tubal fluid was also prevented. The concentrations of protein and hexose in the uterine flushings, corrected for contamination with plasma, were significantly lower in the secretory stages than in the proliferative stages of the cycle. It is concluded that proteins and carbohydrates are present within the uterine lumen not only after ovulation but also during the preovulatory period.

Adult

Protein patterns of human uterine flushings collected at various stages of the menstrual cycle.

Uterine flushings and plasma collected from normal, parous women at various stages of the menstrual cycle were subjected to gel electrophoresis. The protein profiles of the flushings differed in many instances from the plasma pattern by the presence of between one and eleven non-plasma proteins. The distribution of the major non-plasma proteins during the menstrual cycle was not statistically significant but that of a pretransferrin, observed in uterine flushings and in peritoneal fluid, was significant. However, the appearance of this protein band could not be related to a particular phase of the cycle, and hence to a specific hormonal condition.

Ascitic Fluid

Recovery of preimplantation blastocysts in the squirrel monkey by a laparoscopic technique.

A laparoscopic uterine flushing technique has been developed for the recovery of preimplantation embryos from the squirrel monkey. Fourteen adult female squirrel monkeys were induced to ovulate with 4 or 5 days' treatment with follicle-stimulating hormone (1 mg) followed by human chorionic gonadotropin (HCG) (500 IU). Natural mating with a fertile male or artificial insemination was performed near the time of ovulation. Thirty-six hours and eleven days after HCG administration, the females were laparoscoped to determine whether ovulation had occurred. Under laparoscopic control, 1.2 to 3 ml of warmed saline were flushed through the uterine lumen 4 to 7 and 15 to 17 days after HCG administration. Flushed fluid was recovered with a plyethylene catheter or a Pasteur pipette inserted into the cleaned vagina. Fifty-eight uterine flushings were performed, with an average of 65.4% fluid recovery. Six nonfertilized ova and two preimplantation blastocysts were recovered by this technique.

Animals

The culture of mouse blastocysts in the presence of uterine flushings collected during normal pregnancy, delayed implantation and pro-oestrus.

When day-3 mouse embryos were cultured in a simple medium supplemented with uterine fluids of mice autopsied on day 4 of pregnancy, 48 h after administration of oestradiol, or during pro-oestrus, the percentage of blastocysts hatching from the zona pellucida was significantly greater than in unsupplemented medium. In the presence of uterine fluids recovered during delayed implantation this stimulation of blastocyst hatching was not observed. When the culture medium was supplemented with dialysed uterine flushings containing 20 or 30 microgram protein/ml, both 'day 4' and 'delay' uterine proteins were equally effective in enhancing hatching frequency (P less than 0.05). The results suggested that 'delay' uterine fluids may contain a dialysable inhibitor of blastocyst activity. The putative inhibitor was not effective in the presence of serum, since uterine fluids recovered both on day 4 of pregnancy and during delayed implantation significantly increased the size attained by blastocyst outgrowths in the presence of foetal calf serum (P less than 0.01). The percentage of blastocysts exhibitng giant cell transformation and outgrowth was also increased (P less than 0.02) by these uterine fluids when the concentration of FCS in the medium was minimal (0.25%).

Animals

Cervicectomy with uterovaginal anastomosis in the rhesus monkey: a new surgical model for reproductive studies.

Cervicectomy with uterovaginal anastomosis in the rhesus monkey allows easy and non-traumatic access to the uterine cavity through the vagina for such purposes as biopsy, flushing, aspiration of fluid, and placement of IUDs. Endometrial and tissue samples cannot be contaminated by cervical mucus. The surgical procedure and results on eleven animals are described. Neither uterine nor ovarian function appeared to be significantly altered. The surgical model seems well suited to long-term studies on IUDs.

Animals

Factors in mouse uterine fluid that inhibit the incorporation of [3H]uridine by blastocysts in vitro.

Fluid flushed from the uteri of "delayed implanting" and "implanting" mice was fractionated by gel-filtration. The fractions were freeze-dried, individually resuspended in culture medium containing [3H]uridine, and used for incubating "implanting" blastocysts. Several factors were found that reduced incorporation of [3H]uridine by the blastocysts. The amount of inhibitory activity in corresponding factors was generally similar in flushings from both types of uteri, but there was significantly more inhibitory activity in the void volume fractions of flushings from "delayed implanting" animals and it is suggested that this factor may be responsible for the metabolic dormancy of embryos during the diapause associated with delayed implantation.

Animals

The progesterone content of rabbit uterine flushings.

A detailed study has been made of the progesterone content of uterine flushings taken from rabbits of different endocrine states. A high content of progesterone was recovered from the uterine lumen of pregnant and pseudopregnant rabbits. The amount of progesterone recovered in the flushings was not simply dependent on the concentration in the plasma, because plasma progesterone concentrations rose before detection of progesterone in the flushings and remained high after uterine progesterone content had fallen. Progesterone did not appear in the uterine fluid unless uterine protein was present. This was particularly evident in rabbits treated with progesterone in which, despite abnormally high plasma concentrations, little progesterone was found in uterine fluid until the protein content increased. After treatment of rabbits with oestrogen at the time of ovulation, uterine progesterone was present in higher amounts concomitant with an elevation of protein content although the peak values of progesterone and protein were delayed by 2--3 days. Plasma progesterone concentrations of about 5 ng/ml were necessary before progesterone appeared in the uterine lumen.

Animals

3H2O volume and exchange in uterine cavity of monkeys.

A technique was developed to measure water exchange in the endometrical canal (uterine cavity). This type of determination should provide information necessary to understanding intrauterine pharmacokinetics. In the study described, measurements were performed on one cynomologus and four rhesus monkeys. Endometrial fluid in the uterine cavity was replaced with a 3H2O solution of known specific radioactivity (dpm/ml). Radioactivity remaining in the cavity at measured intervals of approximately 0.5, 1, 2, and 3 min following replacement was determined by flushing out the cavity. By extrapolating the 3H2O disappearance curve versus time to t = 0 (zero time), the apparent cavity volume (aVc) was derived. Values for aVc and t 1/2 3H2O were used to calculate endometrial fluid-water turnover rate. Mean values determined in four rhesus monkeys were as follows: aVc, 0.032 ml; t 1/2 H2O, 1.17 min; 24-h H2O turnover, 19.5 ml. In general, this value for H2O turnover was considerably higher than values of endometrial fluid production quantitated by other techniques. Water measurements in human uteri similar to those reported here appear feasible and are anticipated in future work.

Animals

Cyclic changes in the concentration of prostaglandin F2alpha in human uterine flushings.

Prostaglandin F2alpha (PGF2alpha) was measured by radioimmunoassay in uterine flushings obtained from 41 women at various stages of the menstrual cycle. The concentration of PGF2alpha changed significantly during the cycle, with highest values in the mid secretory phase of the cycle (mean 9.1 ng/ml of uterine flushing, range 3.9 to 16.0 ng/ml, n = 9). The still unresolved role of intrauterine PGF2alpha is discussed in relation to luteolysis, myometrial contractility and implantation.

Body Fluids

Effect of human uterine flushings collected at various stages of the menstrual cycle on mouse blastocysts in vitro.

Mouse blastocysts were cultured in vitro in a defined medium supplemented with uterine flushings (containing 500 microgram protein/ml) obtained from normal women at various stages of the menstrual cycle. With one exception (uterine flushing collected on the last day of a menstrual period) blastocyst hatching and attachment were not impaired by flushings collected before or after ovulation.

Animals

Hormonal control of uteroglobin secretion in rabbit uterus: inhibition of uteroglobin synthesis and messenger ribonucleic acid accumulation by oestrogen and anti-oestrogen administration.

Investigations were conducted to quantify activity of uteroglobin mRNA and secretion of uteroglobin in rabbit uterus after administration of progesterone and 5alpha-dihydrotestosterone, either alone or concomitantly with oestradiol-17beta and tamoxifen, a non-steroidal anti-oestrogen. Poly(A)-containing mRNA was isolated from the uterine tissue by extraction with phenol/chloroform, precipitation with ethanol and chromatography on oligo(dT)-cellulose. Cell-free translation in vitro of the poly(A)-containing mRNA was carried out in a wheat-germ lysate, and the product isolated by specific immuno-precipitation with anti-uteroglobin antiserum purified by affinity chromatography. Radioimmunoassay was utilized to determine uteroglobin content in the uterine flushings and tissue preparations. When given for 5 days, both progesterone (1mg/kg per day) and 5alpha-dihydrotestosterone (25mg/kg per day) elicited a marked induction of uteroglobin secretion, which was accompanied with accumulation of uteroglobin mRNA in the tissue. Concomitant administration of oestradiol-17beta (50mug/kg per day) or tamoxifen (12.5mg/kg per day) significantly decreased both progesterone- and 5alpha-dihydrotestosterone-induced uteroglobin secretion, with a parallel decrease in the uteroglobin-mRNA activity. The decline in the uteroglobin content of the uterine flushes brought about by oestradiol-17beta or tamoxifen administration was not due to inhibition of secretion of this protein by the endometrial cells, since a simultaneous decrease occurred in the tissue uteroglobin content. After a 5-day pretreatment with progesterone (1mg/kg per day), administration of oestradiol-17beta (50mug/kg per day) during the ensuing 4 days greatly accelerated the decay of the uteroglobin content in the uterine fluid.

Animals