Effects of dexamethasone and indomethacin on estrogen-induced uterine growth

Certain aspects of estrogen-induced uterine growth are reminiscent of an inflammatory response. Dexamethasone (DEX) and indomethacin (IND), two anti-inflammatory agents that interfere with arachidonic acid metabolism, were examined with respect to their effects on several growth-associated responses of the uterus to estrogen. Ovariectomized rats were given a s.c. injection of either DEX (2 mg) or IND (8 mg) immediately prior to receiving a s.c. injection of estradiol (10 ωg). At 4 hr, DEX inhibited estrogen-stimulated uterine wet weight and ornithine decarboxylase (ODC) activity by 100% and 48%, respectively. At 24 hr, ³H-leucine incorporation into protein was inhibited 44% and ³H-thymidine incorporation into DNA was depressed 83%. Estrogen-stimulated increases in uterine protein/DNA ratio and epithelial microvilli density at 24 hr were not inhibited by DEX. IND inhibited estrogen-stimulated wet weight by 64% and ³H-thymidine incorporation into DNA by 42%, yet did not inhibit the increases in ODC activity, ³H-leucine incorporation into protein or protein/DNA ratio. These results suggest that the inflammation-like component of estrogen-induced uterine growth is mediated, at least in part, by arachidonic acid metabolites and is directed primarily toward stimulating cell division, and not cell growth.     

Effects of asynchrony on rabbit blastocyst development

The development of synchronously and asynchronously transferred rabbit morulae (recovered at Day 3 p.c.) and blastocysts (recovered at Day 4 p.c.) was investigated before the anticipated time of implantation. The results obtained with various techniques (evaluation of gross morphology, measurement of diameter, thymidine incorporation, light and electron microscopy) led basically to the same conclusions. Embryos being asynchronously transferred to the uterus of recipient rabbits survived, at least in terms of certain cellular functions like cell proliferation, for more than 2 days. Development, however, was clearly retarded and ultrastructural examination revealed substantial cell damage. Some blastocysts showed, even after 3 days, normal growth and cell proliferation indicating considerable differences between individuals in the ability to compensate for suboptimal developmental conditions before implantation. In general, this ability was greater in the transferred Day-3 morulae than in the Day-4 blastocysts. Embryonic growth and the ability to dissolve the zona pellucida, to synthesize crystalloid bodies and to differentiate extraembryonic endoderm indicated the maintenance of some developmental functions under asynchronous conditions. Blastocyst development was influenced by the progestational stage of the recipient. At 1 day after transfer into asynchronous older uteri, blastocyst diameters were larger and cell proliferation was increased compared with all other groups, suggesting an attempt of the blastocyst to adjust to the more advanced maternal milieu. Development in asynchronous younger uteri was delayed. No comparable differences in development were found in cultured embryos for which the media had been supplemented with flushings from the same progestational uterine stages as used for transfer. Thymidine incorporation in cultured embryos did not differ between the various supplements (P greater than 0.05) and was generally lower than in chronologically aged asynchronously transferred embryos (P less than 0.05 for Day-3 and P less than 0.001 or P greater than 0.05 for Day-4 embryos).     

Effects of colchicine and of emetine on capillary permeability]

Colchicine and emetine administered locally in the hind paw of the rats increased markedly the capillar permeability. The action may be strongly inhibited by methysergide (0.2 mug). Mepyramine (0.5 mug) is not so active and indomethacine (1 mg/kg per os), even less. As some prostaglandines release serotonine and histamine (8) it is possible that some liberation of these mediators may be attribuable to a previous release of prostaglandines, but the experiments suggest also the possibility of a direct and concomitant release of those two mediators by the alkaloids assayed.     

Effects of euploid blastocyst morphological development on reproductive outcomes

Blastocyst culture and transplantation have now been widely used in most reproductive centers. Our aim was to explore the effects of euploid blastocyst morphological development on the reproductive outcomes of frozen–thawed euploid blastocysts on the fifth day or sixth day. The retrospective analysis included the clinical data of 849 patients who underwent euploid blastocyst transplantation between January 2014 and January 2018. The patients were divided into well-developed blastocysts (BB) or poorly developed blastocysts (BC, CB) groups according to blastocyst morphology grade. The intracytoplasmic sperm injection (ICSI) was carried out on the day of egg retrieval. Morphological evaluation of the resulting blastocysts was performed on Day5 or Day6 of in vitro culture. The pregnancy rate, cumulative pregnancy rate, live birth rate and cumulative live birth rate were the highest in the BB group (P ≤ 0.001). In addition, not only the pregnancy rate of CB group Day5 was lower than that of the BB (P = 0.005) and BC (P = 0.042) groups, but the live birth rate in CB group was lower than BB (P = 0.001) and BC (P = 0.007) groups. However, there was no difference in miscarriage rate among the three groups (P = 0.154). Furthermore, the miscarriage rate of BB (P = 0.048) and BC (P = 0.019) groups at the Day5 cycle was lower than the Day6 cycle. Blastocyst morphological development has positive effects on pregnancy rate and live birth rate, but has no effect on miscarriage rate.     

Effects of hyperoxia on alveolar permeability of neutropenic rabbits

We investigated whether neutrophil suppression would prevent the early hyperoxic injury to the rabbit alveolar epithelium. Rabbits received a single intravenous injection of either nitrogen mustard (2 mg/kg) or saline and were exposed to 100% C2 for 64 h. At the end of the hyperoxic exposure, there were 20 +/- 7 neutrophils/ml blood in the nitrogen mustard group vs. 5,935 +/- 1,174 in the control group (means +/- SE; P less than 0.05). The corresponding numbers in lung extravascular tissue, expressed per high-power field, were 0.37 +/- 7 and 5.9 +/- 0.35, respectively (P less than 0.05). At this time, the rate constants of solute flux for 57Co-vitamin B12 (r = 6.5 A) and 131I-cytochrome c (r = 17 A), across the alveolar epithelium, were 33 +/- 5 (min-1) and 7 +/- 2 for the nitrogen mustard and 29 +/- 5 and 6 +/- 1 for the saline group, respectively. These variables were ninefold higher than their corresponding values in animals breathing air. We concluded that neutrophils do not play a significant role during the early stages of sublethal hyperoxic injury to rabbit alveolar epithelium.     

Exosome-derived uterine microRNAs isolated from cows with endometritis impede blastocyst development

As a common disease of cows occurring during their perinatal period, endometritis is known to affect fertility. At present, the studies on endometritis mainly focus on preventing microbial invasion. However, the mechanism that uterine inflammation affects embryo activity and implantation is unclear. Mainly containing lipids, proteins, mRNAs, and microRNAs, exosomes widely exist in various tissues and body fluids. Exosome extractions were used by commercial kits and confirmed through morphological examinations and Western blot. After exosomes’ mRNA profiles were generated using RNA sequencing, it was investigated how uterine cavity fluid exosomes affect the developmental competence of in vitro fertilization (IVF) embryos in case of endometritis. In this study, the isolated exosomes were spherical particles with a diameter of 30–150 nm according to the transmission electron microscopy. Identified with Western blotting, positive CD63 and CD9 expressions showed that the isolated exosomes could be used for the subsequent tests. We found 118 differentially expressed miRNAs in the exosomes of the uterine cavity fluid in healthy cows and those with endometritis, among which, 52 miRNAs were down regulated and 66 up regulated. Furthermore, the qRT-PCR results confirmed the up-regulation of three miRNAs and down-regulation of six miRNAs, which were consistent with the deep sequencing results. IVF embryos co-incubated with the endometritis exosomes significantly decreased the blastocyst formation rate in comparison with those co-incubated with the healthy exosomes (21.84+3.17 vs. 32.37+2.69). Therefore, exosome miRNAs may be a cause of infertility in cows with endometritis.     

Physiological aspects of blastocyst uterine interaction

An interaction between the blastocyst and the uterus is essential for establishment of pregnancy. Because maternal estrogen is not an absolute requirement, estrogen of embryonic origin has been implicated in this process in the pig and the rabbit. Furthermore, estrogen forming capacity has been documented in the blastocyst of these species. However, while the complete machinery for steroid synthesis in the pig balstocyst has been demonstrated, the issue is still unresolved for the rabbit blastocyst. In the present communication we have shown that 17α-hydroxylase and C17–20-lyase, enzymes involved in the formation of androgens (C¹⁹-steroids) from C²¹-steroids (progestins), are present in day-6 rabbit blastocysts. C17–20-lyase activity was undetectable to low in day-5 and increased in day-6 balstocysts. The activity was further increased in day-6 blastocysts cultured for 24 h. Because prostaglandins have been implicated in uterine vascular changes at about the time of implantation and pregnancy establishment, and because catechol estrogens are more potent than phenolic estrogens in stimulating prostaglandin synthesis in the blastocyst and the uterus, we determined catechol estrogen forming capacity in the rabbit and pig blastocyst. Catechol estrogen forming capacity (estrogen-2/4-hydroxylase) in the pig blastocyst appears on day 10 of pregnancy, peaks on day 12 and then declines. Our preliminary experiments also indicate that day-6 rabbit blastocysts have catechol estrogen forming capacity. On the basis of our present findings and of others, we propose that catechol estrogens of embryonic origin mediate the stimulatory effect of estrogens on prostaglandin synthesis in the embryo and/or the uterus and thus participate in the process of establishment of pregnancy.     

Effects of indomethacin, prostaglandin E2 and prostaglandin F2 alpha on mouse blastocyst attachment and trophoblastic outgrowth in vitro

A study was performed in order to investigate the participation of prostaglandins (PGs) during implantation. The effects of indomethacin on mouse blastocyst attachment and trophoblastic outgrowth were examined in vitro. Studies were also carried out on cultures supplemented with PGE2 and/or PGF2 alpha along with indomethacin. (1) Blastocyst attachment and trophoblastic outgrowth were inhibited by indomethacin dose-dependency. (2) In the cultures supplemented with indomethacin and PGE2 or PGF2 alpha, respectively, the inhibitory effects of indomethacin were reduced. (3) In the cultures supplemented with all three substances with treatment (1) and (2), inhibition of indomethacin was partially reversed, but still lower than control group without indomethacin. The above results indicate that both PGE2 and PGF2 alpha have a promoting effect on implantation, and PGF2 alpha was more effective than PGE2.     

Effect of indomethacin on stretch-induced uterine activity in the post-partum

The uterus at 48 hours after normal delivery was mechanically stretched by the intra-uterine application of an inflated rubber balloon. Inauguration of regular and marked uterine activity was noted subjectively in all 16 healthy subjects and recorded by an external tocometer. The inaugurated uterine activity was significantly suppressed by the rectal application of 100 mg of indomethacin (p less than 0.01), but was not abolished entirely. The uterine activity ceased gradually with the discontinuation of stretching. These results strongly indicate that the purely stretch-induced uterine contractions are mediated by prostaglandins (PGs) which are released by stretching and/or thereby induced uterine contractions. In this study the possible source of PGs appeared to be by the myometrium itself.     

Effect of indomethacin on stretch-induced uterine activity in the post-partum

The uterus at 48 hours after normal delivery was mechanically stretched by the intra-uterine application of an inflated rubber balloon. Inauguration of regular and marked uterine activity was noted subjectively in all 16 healthy subjects and recorded by an external tocometer. The inaugurated uterine activity was significantly suppressed by the rectal application of 100 mg of indomethacin (p < 0.01), but was not abolished entirely. The uterine activity ceased gradually with the discontinuation of stretching. These results strongly indicate that the purely stretch-induced uterine contractions are mediated by prostaglandins (PGs) which are released by stretching and/or thereby induced uterine contractions. In this study the possible source of PGs appeared to be by the myometrium itself.