Doses Of P4 Research Articles

Revealing the algicidal characteristics of Maribacter dokdonensis: An investigation into bacterial strain P4 isolated from Karenia mikimotoi bloom water.

Harmful algal blooms (HABs) are a global environmental concern, causing significant economic losses in fisheries and posing risks to human health. Algicidal bacteria have been suggested as a potential solution to control HABs, but their algicidal efficacy is influenced by various factors. This study aimed to characterize a novel algicidal bacterium, Maribacter dokdonensis (P4), isolated from a Karenia mikimotoi (Hong Kong strain, KMHK) HAB and assess the impact of P4 and KMHK's doses, growth phase, and algicidal mode and the axenicity of KMHK on P4's algicidal effect. Our results demonstrated that the algicidal effect of P4 was dose-dependent, with the highest efficacy at a dose of 25% v/v. The study also determined that P4's algicidal effect was indirect, with the P4 culture and the supernatant, but not the bacterial cells, showing significant effects. The algicidal efficacy was higher when both P4 and KMHK were in the stationary phase. Furthermore, the P4 culture at the log phase could effectively kill KMHK cells at the stationary phase, with higher algicidal efficacy in the bacterial culture than that of the supernatant alone. Interestingly, P4's algicidal efficacy was significantly higher when co-culturing with xenic KMHK (~90% efficacy at day 1) than that with the axenic KMHK (~50% efficacy at day 1), suggesting the presence of other bacteria could regulate P4's algicidal effect. The bacterial strain P4 also exhibited remarkable algicidal efficacy on four other dinoflagellate species, particularly the armored species. These results provide valuable insights into the algicidal effect of M. dokdonensis on K. mikimotoi and on their interactions.

Spatiotemporal expression and regulation of peptidase inhibitor 3 and secretory leukocyte protease inhibitor at the maternal-fetal interface in pigs.

Two serine protease inhibitors, peptidase inhibitor 3 (PI3) and secretory leukocyte protease inhibitor (SLPI), play important roles in protease inhibition and antimicrobial activity, but their expression, regulation, and function at the maternal-fetal interface in pigs are not fully understood. Therefore, we determined the expression and regulation of PI3 and SLPI in the endometrium throughout the estrous cycle and at the maternal-fetal interface in pigs. Endometrial tissues during the estrous cycle and pregnancy, conceptus tissues during early pregnancy, and chorioallantoic tissues during mid to late pregnancy were obtained, and the expression of PI3 and SLPI was analyzed. The effects of the steroid hormones estradiol-17β (E2) and progesterone (P4) on the expression of PI3 and SLPI were determined in endometrial explant cultures. PI3 and SLPI were expressed in the endometrium during the estrous cycle and pregnancy, with higher levels during mid to late pregnancy than during the estrous cycle and early pregnancy. Early-stage conceptuses and chorioallantoic tissues during mid to late pregnancy also expressed PI3 and SLPI. PI3 protein and SLPI mRNA were primarily localized to endometrial epithelia. In endometrial explant cultures, the expression of PI3 was induced by increasing doses of P4, and the expression of SLPI was induced by increasing doses of E2 and P4. These results suggest that the PI3 and SLPI expressed in the endometrium and conceptus tissues play an important role in antimicrobial activity for fetal protection against potential pathogens and in blocking protease actions to allow epitheliochorial placenta formation.

Assessment of progesterone levels on the day of pregnancy test determination: A novel concept toward individualized luteal phase support.

The main objective of the study is to define the optimal trade-off progesterone (P4) values on the day of embryo transfer (ET), to identify low P4-human chorionic gonadotropin (hCG), and to establish whether P4 supplementation started on the hCG day can increase the success rate of the frozen embryo transfer (FET) cycle. A single-center, cohort, retrospective study with 664 hormone replacement therapy (HRT)-FET cycles analyzed female patients who received vaginal 600 mg/day of P4 starting from 6 days before the FET, had normal P4 values on the day before ET, and whose P4 on the day of the pregnancy test was assessed. Of the 664 cycles, 69.6% of cycles showed P4 ≥ 10.6 ng/ml, while 30.4% showed P4 < 10.6 ng/ml on the day of the hCG. Of the 411 chemical pregnancies detected, 71.8% had P4-hCG ≥ 10.6 ng/ml (group A), while 28.2% had P4-hCG < 10.6 ng/ml. Of the cycles with P4-hCG < 10.6 ng/ml, 64.7% (group B) were supplemented with a higher dose of vaginal P4 (1,000 mg/day), while 35.3% (group C) were maintained on the same dose of vaginal micronized P4. The live birth rate was 71.9%, 96%, and 7.3% for groups A, B, and C, respectively. The likelihood to detect P4-hCG < 10.6 ng/ml decreased as the level of serum P4 the day before ET increased. The live birth rate (LBR) was shown to be significantly lower when P4 was low and not supplemented.

The effect of progesterone injection combined with prostaglandineF2α, GnRH and hCG administrations on the pregnancy and lambing rate of lactating and non-lactating fat-tailed ewes during the breeding season.

The objective was to compare the effect of human chorionic gonadotropin (hCG) and gonadotropin-releasing hormone (GnRH) on the pregnancy and lambing rate of treated ewes with a short-term progesterone (P4) injection during the breeding season. In Exp 1, non-lactating ewes (n=158) were used and received P4 , three times every 48h and received PGF2α along with the last dose of P4. Ewe received hCG (n=79, 3PHCG, IM) or GnRH (n=79, 3PGnRH, IM) 24 h after the last dose of P4 treatment. In the Exp 2, lactating ewes (n=62) received P4 and hCG (n=24, 3PHCG) or GnRH (n=24, 3PGnRH) similar to Exp 1, or considered as control (n=14) and received PGF2α 48 h before ram release. Rams were released into the studied ewe's flock 24 h after the hCG or GnRH administration, and pregnancy diagnosis was performed on day 50 after ram release. In Exp 1, all reproductive indices were not significant between groups except twin lambing rate, that was higher in the 3PHCG (11.3%) compared with the 3PGnRH (1.9%) group (p=.05). There were no significant differences in overall pregnancy and lambing rates between 3PHCG (70.9% and 67.1%) and 3PGnRH (69.6% and 68.4%) groups (p > .05). In Exp 2, all reproductive parameters were not significant between 3PHCG and 3PGnRH groups (p > .05). There was significant difference in overall oestrous rate between control (35.7%) and treatment (3PHCG, 70.9% and 3PGnRH, 79.2%) groups. To conclude, administration of GnRH can be a good alternative to hCG injection under field conditions.

Progesterone Induces Apoptosis and Steroidogenesis in Porcine Placental Trophoblasts.

Simple SummaryThis study investigated the effects of progesterone treatment in vitro on apoptosis and steroidogenesis in porcine placental trophoblasts and the underlying molecular mechanisms. Trophoblasts were treated with different concentrations of progesterone for 48 h. Cell counts, steroidogenesis, and relevant gene and protein expression levels were measured. Progesterone inhibited trophoblast proliferation in a dose-dependent manner. High doses of progesterone significantly altered the expression levels of apoptosis-related and steroidogenesis-related genes and proteins, while low doses had a less pronounced effect. Thus, increased progesterone induces the apoptosis of porcine placental trophoblasts and induces abnormal steroidogenesis in the placenta. We believe that our study makes a significant contribution to the literature because it elucidates the effects of progesterone on porcine placental trophoblast functions.Placentation and placental steroidogenesis are important for pregnancy and maternal–fetal health. As pregnancy progresses, the main site of progesterone (P4) synthesis changes from the corpus luteum to the placenta, in which placental trophoblasts are the main cell type for P4 synthesis. Therefore, this study investigated the effects of P4 on apoptosis and steroidogenesis in porcine placental trophoblasts and the underlying molecular mechanisms. Porcine placental trophoblasts were treated with different concentrations of P4 for 48 h in a serum-free medium in vitro. Cell number, steroidogenesis, and relevant gene and protein expression levels were detected. A high dose of P4 (10.0 μM) significantly increased P4 (p < 0.01), androstenedione (p < 0.05), testosterone (p < 0.05), and estradiol (p < 0.05) production in porcine placental trophoblasts compared with that in control cells, while a low dose of P4 (1 × 10−3 μΜ) had no marked impact on steroid production. The mRNA expression of apoptosis-related genes (CASP3, CASP8, and Bax) (p < 0.05) and steroidogenesis-related genes (CYP11A1, CYP19A1, and StAR) (p < 0.01) was upregulated, and the expression of HSD3B and HSD17B4 was inhibited (p < 0.05) in the porcine placental trophoblasts treated with high doses of P4. Low doses of P4 had a lighter effect on gene expression than high doses. The expression of apoptosis-related proteins CASP3 (p < 0.05), and Bax (p < 0.01) and steroidogenesis-related proteins CYP19A1 (p < 0.05) and StAR (p < 0.01) was raised, but the proliferation-related protein CCND2 (p < 0.01) was downregulated in the pTr cells treated with high dose of P4. In comparison, a low dose of P4 inhibited the expression of Bax, CYP11A1 (all p < 0.01), and CCND2 (p < 0.05), but the expression of CASP3 (p < 0.05) and StAR (p < 0.01) was upregulated. In summary, excessive P4 can induce the apoptosis of porcine placental trophoblasts and lead to abnormal steroidogenesis in the placenta and hormone imbalance.

Effect of probiotics addition on total organic matter and survival rate of catfish (Clarias sp.) maintenance using recirculating aquaculture system (RAS)

Most catfish farmers do not make efforts to manage water quality. This causes high organic matter. Therefore, water management is carried out through the Recirculating Aquaculture System (RAS). Continuous use of water in the RAS system causes filter performance to decrease, so technological improvements are needed with the addition of probiotics. The research was conducted on 22 February – 23 March 2021 at the Chemistry and Analysis Laboratory, Faculty of Fisheries and Marine, Universitas Airlangga to determine the influence of probiotic additions on the RAS system total organic matter and the survival rate of catfish.The study was conducted experimentally using RAL by adding different doses of probiotics, namely P0 (Control), P1 (0.5 ml/L), P2 (1 ml/L), P3 (1.5 ml/L) and P4 (2 ml/L ) with four replays. Data analysis using ANOVA and DMRT showed that the addition of probiotics in the RAS system has an effect (p<0.05) on total organic matter and survival rate. Organic matter was highest at P2 (46.89 mg/L) and lowest at P2 (34.07 mg/L). Survival rates were highest at P2 (85%) and lowest at P0 (71.25%).

Reproductive efficiency of treated Karakul ewes with short-term progesterone and hCG injections during the non-breeding and breeding seasons

The aim of this study was to evaluate reproductive efficiency of ewes following a short-term treatment with progesterone (P4) plus human chorionic gonadotropin (hCG) injections. In Exp 1, ewes (n = 65) were used during the non-breeding season and received P4, three times every other day (n = 22, 3PHCG, IM) or a single dose of P4 (n = 21, PHCG). We injected hCG in all ewes 24 h after the P4 treatment, and distilled water in control group (n = 22). Blood collection were performed on days −13, −6 and + 8 relative to ram release. In Exp 2, during the breeding season, ewes (n = 66) were assigned to the 3PHCG (n = 33) and control (n = 33) groups described for Exp 1, and subsequently received PGF2α 48 h before ram release. Pregnancy diagnosis was performed on day 42 after the ram release which was done 24 h after the hCG injection. In Exp 1, Estrus, pregnancy rate at induced estrus, and lambing rates at the first cycle were greater in 3PHCG (81.8%, 59.1% and 54.5%) group compared with the PHCG (38.1%, 23.8% and 19%) and control (0%, 0% and 0%) groups (P < 0.05). In Exp 2, Estrus, pregnancy rate at induced estrus, and lambing rates at the first cycle were greater in 3PHCG (75.8%, 57.6% and 51.5%) group compared with the control (39.4%, 30.3% and 27.3%) groups (P < 0.05). To conclude, hCG injection 24 h after P4 administration is an effective protocol to induce fertile estrus during non-breeding season, and synchronize estrus and lambing of ewe in the breeding season.

Efektivitas Penambahan Astaxanthin pada Pakan Komersil untuk Meningkatkan Kecerahan Warna, Pertumbuhan dan Sintasan Ikan Komet (Carassius auratus)

This study aims to determine the effectiveness of adding Astaxanthin to commercial feed to increase color brightness, growth and survival rate of comet fish. This study used a completely randomized design (RAL) with 4 treatments and 3 replications. The treatment given was (P1) = without Astaxanthin; (P2)= 250 mg Astaxanthin /1/2 kg of feed; (P3)= 500 mg Astaxanthin /1/2 kg feed; (P4) 750 mg Astaxanthin/1/2 kg feed. Parameters observed were color brightness, absolute length growth rate, absolute weight growth, daily growth rate, survival rate, and FCR. The results showed that the effectiveness of adding Astaxanthin to commercial feed did not have a significant effect (Fhit&lt;Ftab) on increasing color brightness, absolute length growth, daily growth rate, survival rate and had a significant effect (Fhit&gt;Ftab) on absolute weight growth, FCR. The additional dose of Astaxanthin P4 (750 mg) was the best dose for absolute weight growth and FCR.&#x0D;

Progesterone dose during synchronization treatment alters luteinizing hormone receptor and steroidogenic enzyme mRNA abundances in granulosa cells of Nellore heifers

The objective was to investigate effects of progesterone (P4) dose on abundance of luteinizing hormone receptor (LHCGR), aromatase (CYP19A1), 3β-hydroxysteroid dehydrogenase (HSD3B1), and other steroidogenic mRNA transcripts in granulosa cells from dominant follicles. Nellore heifers were assigned to one of six groups: new, first-use controlled internal drug release device (CIDR1) inserted for 5 days (Large-P4-dose-D5; n = 7) or 6 days (Large-P4-dose-D6; n = 8), prostaglandin (PG)F2α administered on D0 and 1 previously-used CIDR (CIDR3) inserted for 5 days (Small- P4-dose-D5; n = 8) or 6 days (Small-P4-dose-D6; n = 8), CIDR1 inserted on D0 and removed plus PGF2α on D5 (Large-P4-dose-proestrus (PE); n = 7), and CIDR3 and PGF2α on D0 and 1, CIDR3 removed plus PGF2α on D5 (Small-P4-dose-PE; n = 7). Duration of P4 treatment (D5 compared to D6) affected abundances of CYP19A1 mRNA transcripts, with there being greater abundances on D6 than D5 (P ≤ 0.05). Heifers treated with the large dose of P4 had a smaller dominant follicle, less serum and intra-follicular estradiol (E2) concentrations (P ≤ 0.05) and lesser LHCGR, CYP19A1, and HSD3B1 transcript abundances (P ≤ 0.05). Heifers treated to induce PE had a larger follicle diameter (P = 0.09), greater intra-follicular E2 concentrations and larger abundances of CYP19A1 mRNA transcript (P ≤ 0.05) than heifers of the D6 group. Overall, treatment with larger doses of P4 resulted in lesser abundances of LHCGR, HSD3B1, and CYP19A1 mRNA transcripts; thus, potentially leading to development of smaller dominant follicles and lesser E2 concentrations.

The Effect of P4 + eCG Estrus Induction Protocol during the Deep and the Transition Anestrous Period on the Reproductive Performance of Crossbred Dairy Goats.

Simple SummaryThe effect of an ultra-short progesterone (P4) plus equine chorionic gonadotropin (eCG) based estrus induction protocol during deep seasonal anestrous, or the reproductive transition period upon reproductive performance in an arid environment (26° N) was assessed. Results confirm a multidimensional response of goats regarding the effectiveness of P4 + eCG estrus induction protocols, mainly modulated by a specific time within the anestrous season (June) or even by specific management or a particular environment at herd level (herd 1 in this study), although, it is quite remarkably non–dependent on the animal’s body weight or body condition score.Seasonal reproduction restricts the offering of goat commodities across the year. Therefore, it is crucial to improve diverse strategies to induce the reproductive response in goats during the anestrus stage. The effectiveness of a short P4 + eCG-based estrus induction protocol during both the deep anestrous (March) or the reproductive transition period (June) upon the reproductive performance of crossbred dairy goats was assessed. Adult, anestrous, 24–30-month- old dairy crossbred (Saanen–Alpine–Nubian × Criollo) goats (n = 123) from two commercial herds and 10 sexually active goat bucks were used. Before the trials, the anestrous status of goats was confirmed. Then, goats were randomly allocated into two different experiments. In Exp. 1, we tested the effect of different doses (D) of intramuscular progesterone (P4; 10 or 20 mg + eCG (100 UI)) and type of breeding (TB), natural mating (NM), or artificial insemination (AI), on two commercial goat herds (H1 & H2), in March (deep anestrous). In Exp. 2, we evaluated the effect of D (P4; 10 or 20 mg + 100 UI eCG) in goats subjected to NM, and either during deep anestrous (March; M) or transitional anestrous (June; J), in two commercial herds. After breeding, conception and pregnancy were diagnosed with ultrasound scanning. The response variables were estrus induction (EI; %), estrus latency (EL; h), ovulation (OVP; %), ovulation rate (OR; units), fertility (FERT; %), and pregnancy (PREG; %). No differences (p > 0.05) in live weight (LW) and body condition score (BCS) occurred between herds in both trials. In Exp. 1, EI, EL, OVP, OR, FERT, and PREG were affected (p < 0.05) by the H–TB–D interaction, whereas in H1 + P4–20 combination had the highest (p < 0.05) EI, EL, and OVP values. Irrespective of TB, H1 had the largest (p < 0.05) OR, independently of TB or D. Also, the lowest (p < 0.05) OVP occurred in the AI + P4–10 group, while the AI had the lowest (p < 0.05) FERT, irrespective of D. FERT and PREG were two-fold higher (p < 0.05) in NM compared with AI. In Exp. 2, EI, EL, OVP, OR, FERT, and PREG were affected (p < 0.05) by the H–M–D interaction. In general, H2 + P4–10 had the lowest (p < 0.05) reproductive outcomes in March, whereas H1 had the largest (p < 0.05) values in either month. No differences (p > 0.05) between P4 doses occurred for EI, OVP, OR, FERT, and PREG. Yet, the largest (p < 0.05) EL occurred with P4–20 in June. No correlations (p > 0.05) occurred between LW and all the reproductive variables. BCS was positively correlated (p < 0.05) with EI (0.34), OVP (0.44), OR (0.58), and PREG (0.20). Also, positive correlations (p < 0.05) occurred between EI with EL (0.83), OVP (0.80), OR (0.64), and PREG (0.56); EL with OVP (0.58), OR (0.44), and PREG (0.42); OVP with OR (0.79) and PREG (0.70), as well as OR and PREG (0.63). Results of these studies confirm a multidimensional response regarding the effectiveness of P4 + eCG for estrus induction in goats mainly modulated by a specific time within the anestrous season, or even by specific management or a particular environment at the herd level (H1), although quite remarkably independent of the animal’s LW or BCS at herd level. Moreover, the best reproductive outcomes occurred with NM in June. The most reproductive variables were similar using either 10 or 20 mg P4 + 100 IU eCG, giving the possibility to lessen the scale in the use of exogenous hormones while obtaining acceptable out of season reproductive response.

Supplementation of 17β-Estradiol Normalizes Rapid Gastric Emptying by Restoring Impaired Nrf2 and nNOS Function in Obesity-Induced Diabetic Ovariectomized Mice.

Gastroparesis (Gp) is a multifactorial condition commonly observed in females and is characterized by delayed or rapid gastric emptying (GE). The role of ovarian hormones on GE in the pathogenesis of obesity induced type 2 diabetes mellitus (T2DM) is completely unknown. The aims of our study are to investigate whether supplementation of 17β-estradiol (E2) or progesterone (P4) restores impaired nuclear factor erythroid 2-related factor 2 (Nrf2, an oxidative stress-responsive transcription factor) and nitric oxide (NO)-mediated gastric motility in ovariectomized (OVX) mice consuming a high-fat diet (HFD, a model of T2DM). Groups of OVX+HFD mice were administered daily subcutaneous doses of either E2 or P4 for 12 weeks. The effects of E2 and P4 on body weight, metabolic homeostasis, solid GE, gastric antrum NO-mediated relaxation, total nitrite levels, neuronal nitric oxide synthase (nNOSα), and its cofactor expression levels were assessed in OVX+HFD mice. HFD exacerbated hyperglycemia and insulinemia while accelerating GE (p < 0.05) in OVX mice. Exogenous E2, but not P4, attenuated rapid gastric emptying and restored gastric nitrergic relaxation, total nitrite levels, nNOSα, and cofactor expression via normalizing Nrf2-Phase II enzymes, inflammatory response, and mitogen-activated protein kinase (MAPK) protein expression in OVX+HFD mice. We conclude that E2 is beneficial in normalizing metabolic homeostasis and gastric emptying in obese, diabetic OVX mice consuming a fat-rich diet.

Progesterone at high doses reduces the growth of U87 and A172 glioblastoma cells: Proteomic changes regarding metabolism and immunity.

While pregnancy may accelerate glioblastoma multiforme (GBM) growth, parity and progesterone (P4) containing treatments (ie, hormone replacement therapy) reduce the risk of GBM development. In parallel, low and high doses of P4 exert stimulating and inhibitory actions on GBM growth, respectively. The mechanisms behind the high‐dose P4‐suppression of GBM growth is unknown. In the present study, we assessed the changes in growth and proteomic profiles when high‐dose P4 (100 and 300 µM) was administered in human U87 and A172 GBM cell lines. The xCELLigence system was used to examine cell growth when different concentrations of P4 (20, 50, 100, and 300 µM) was administered. The protein profiles were determined by two‐dimensional gel electrophoresis in both cell lines when 100 and 300 µM P4 were administered. Finally, the pathways enriched by the differentially expressed proteins were assessed using bioinformatic tools. Increasing doses of P4 blocked the growth of both GBM cells. We identified 26 and 51 differentially expressed proteins (fc > 2) in A172 and U87 cell lines treated with P4, respectively. Only the pro‐tumorigenic mitochondrial ornithine aminotransferase and anti‐apoptotic mitochondrial 60 kDa heat shock protein were downregulated in A172 cell line and U87 cell line when treated with P4, respectively. Detoxification of reactive oxygen species, cellular response to stress, glucose metabolism, and immunity‐related proteins were altered in P4‐treated GBM cell lines. The paradox on the effect of low and high doses of P4 on GBM growth is gaining attention. The mechanism related to the high dose of P4 on GBM growth can be explained by the alterations in detoxification mechanisms, stress, and immune response and glucose metabolism. P4 suppresses GBM growth and as it is nontoxic in comparison to classical chemotherapeutics, it can be used as a new strategy in GBM treatment in the future.

Aminophylline and progesterone prevent inflammation-induced preterm parturition in the mouse†.

Although progesterone (P4) supplementation is the most widely used therapy for the prevention of preterm labor (PTL), reports of its clinical efficacy have been conflicting. We have previously shown that the anti-inflammatory effects of P4 can be enhanced by increasing intracellular cyclic adenosine monophosphate (cAMP) levels in primary human myometrial cells. Here, we have examined whether adding aminophylline (Am), a non-specific phosphodiesterase inhibitor that increases intracellular cAMP levels, to P4 might improve its efficacy using in vivo and in vitro models of PTL. In a mouse model of lipopolysaccharide (LPS)-induced PTL, we found that the combination of P4 and Am delayed the onset of LPS-induced PTL, while the same dose of P4 and Am alone had no effect. Pup survival was not improved by either agent alone or in combination. Myometrial prolabor and inflammatory cytokine gene expression was reduced, but the reduction was similar in P4 and P4/Am treated mice. There was no effect of the combination of P4 and Am on an ex vivo assessment of myometrial contractility. In human myometrial cells and myometrial tissue explants, we found that the combination had marked anti-inflammatory effects, reducing cytokine and COX-2 mRNA and protein levels to a greater extent than either agent alone. These data suggest that the combination of P4 and Am has a more potent anti-inflammatory effect than either agent alone and may be an effective combination in women at high-risk of PTL.

Uterine bleeding rates with hormone therapies in menopausal women with vasomotor symptoms

Introduction: Bleeding and spotting are major reasons why women discontinue use of hormone therapy (HT) taken to treat menopausal vasomotor symptoms (VMS). The REPLENISH trial (NCT01942668) evaluated TX-001HR (TherapeuticsMD, Boca Raton, FL), a single oral capsule containing bioidentical 17β-estradiol (E2) and progesterone (P4), for treating moderate to severe menopausal VMS in women with a uterus; the 1 mg E2/100 mg P4 dose was recently approved as Bijuva™ by the US Food and Drug Administration (FDA; October 2018). This report compares the incidence of uterine bleeding with TX-001HR to other FDA-approved menopausal HT and unapproved compounded bioidentical HT (CHT).

SAT-LB003 Nonsmokers Benefit from Lower Doses of an Estradiol/Progesterone Combination: Results of the REPLENISH Trial

Smoking has previously been reported to result in decreases in estradiol exogenous metabolism1,2,3 and may influence the efficacy of hormone therapy (HT) in postmenopausal women2,3. Importantly, differences in smoking rates among clinical trial populations may also contribute to differences in effects of HT observed across the trials.2 The REPLENISH trial (NCT01942668) was a 12-month, phase 3, randomized, double-blind, placebo-controlled, multicenter trial that evaluated 4 doses of TX-001HR (17β-estradiol and progesterone [E2/P4]; 1/100, 0.5/100, 0.5/50, 0.25/50) combined in a single, oral softgel capsule vs placebo in 1835 menopausal women (40−65 y; intact uterus) for the treatment of menopausal, moderate to severe vasomotor symptoms (VMS). The 1 mg E2/100 mg P4 dose is FDA approved as BIJUVATM (TherapeuticsMD, Boca Raton, FL). In this trial, statistically significant improvements in the frequency and severity of moderate to severe VMS were observed for the two highest doses of TX-001HR (1/100 and 0.5/100) and is reported in detail elsewhere.4 The objective of this analysis was to assess the impact of smoking on estradiol metabolism by analyzing estradiol and estrone concentrations measured throughout the study and to assess the potential impact on efficacy. The overall percentage of current smokers was higher in the Replenish trial (26%) than in many other trials of HT, making analysis of the impact of smoking on estrogen levels and efficacy possible. Estradiol and estrone concentrations at baseline were similar between current smokers and nonsmokers (former/never smokers) for all treatment groups (active and placebo). However, at Weeks 4 and 12, Months 6, 9, and 12, estradiol (25%-32%) and estrone (30%-36%) concentrations were statistically significantly lower in smokers than in nonsmokers for all active estradiol/progesterone treatment groups (1/100, 0.5/100, 0.5/50, 0.25/50). There were no significant differences at any time point in the placebo group. Efficacy in reducing the frequency and severity of VMS was greater in nonsmokers. When evaluating nonsmokers, the 0.25/50 dose also demonstrated statistically significant improvements at Weeks 4 and 12, with a weekly reduction of greater than 14 hot flushes compared to placebo beginning at Week 4 and sustained through week 12. Results from this large Phase 3 clinical trial demonstrated a significant impact of current smoking on estradiol and estrone concentrations and indicate that non-smokers may benefit from lower doses of estradiol and progesterone than smokers. 1. Jensen J, et al. NEJM. 1985;313(16):973-975. 2.Tansavatdi B, et al. Minerva Ginecol. 2004;56:105-114. 3. Ruan X and Mueck AO. Climacteric. 2015;18:38-46. 4. Lobo RA, et al. Obstet Gynecol. 2018;132:161-170. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

In Vitro Effect of Estradiol and Progesterone on Ovine Amniotic Epithelial Cells.

Amniotic epithelial cells (AECs), an emerging source of extrafoetal stem cells, have recently attracted attention for their great regenerative potential. Since AEC amplifications are accompanied by the loss of their native epithelial phenotype and by the progressive reduction of relevant biological properties, the issue to be addressed is the development of effective culture protocols. In this context, recently, it has been demonstrated that progesterone (P4) supplementation during ovine AEC (oAEC) expansion could prevent the undesirable epithelial-mesenchymal transition (EMT). In contrast, there is no information to date on the role of the other pregnancy steroids in culture. With this aim, the present study has been designed to clarify the impact of estradiol (E2), alone or in combination with P4 (12.5 μM and 25 μM), during oAEC amplification. Steroid supplementations were assessed by testing oAEC proliferation, stemness, EMT, and osteogenic or chondrogenic plasticity. The results indicated that EMT can be prevented exclusively in the presence of high doses of P4, while it occurred rapidly in cells exposed to E2 as denoted by protein (cytokeratin-8 and alpha-SMA) and gene expression (vimentin and snail) profiles. Moreover, steroid exposure was able to influence highly oAEC plasticity. Particularly, P4-treated cells displayed a precommitment towards osteogenic lineage, confirmed by the upregulation of OCN, RUNX2, and the greater deposition of calcium nodules. Conversely, P4 exposure inhibited oAEC chondrogenic differentiation, which was induced in E2-treated cells as confirmed by the upregulation of chondrogenesis-related genes (SOX9, ACAN, and COL2A1) and by the accumulation of Alcian blue-positive extracellular matrix. Simultaneously, E2-treated cells remained unresponsive to osteogenic inductive stimuli. In conclusion, media supplementation with high doses of steroids may be adopted to modulate phenotype and plasticity during oAEC amplification. Relevantly, the osteo or chondro steroid-induced precommitment may open unprecedented cell-based therapies to face the unsolved orthopaedic issues related to osteochondral regeneration.

Assessing bioavailability of ruminally protected methionine and lysine prototypes

Met and Lys are essential AA that can limit lactational performance in dairy cattle fed protein-sufficient diets. Thus, there is industry demand for ruminally protected (RP) sources of Met and Lys. One method of providing ruminal protection for Met and Lys is lipid encapsulation. The objective of this work was to assess 3 lipid-encapsulated Met prototypes (P1, P2, and P3) and 1 Lys prototype (P4) to determine ruminal protection, small intestine absorption (experiment 1), and animal production responses (experiment 2). Ruminal protection was estimated from 8-h in situ retention during ruminal incubation and intestinal absorption from plasma appearance after an abomasal bolus of the in situ retentate. Blood samples were collected over time to determine plasma Met and Lys concentration responses compared with unprotected Lys and Met infused abomasally. The prototypes were not exposed to the total diet or subjected to typical feed handling methods before evaluation. The bioavailability of P1, P2, and P3 Met prototypes was found to be 14, 21, and 18% of the initial AA material, respectively. The RP-Lys prototype had a bioavailability of 45%. To evaluate production responses, 20 Holstein cows were randomly assigned to 2 trials (n = 10 each) in a replicated Latin square design with 14-d periods. The base diet was predicted to be deficient in metabolizable Met (-14.8 g/d) and Lys (-16.1 g/d) per the Cornell Net Carbohydrate and Protein System (version 6.55). In the Met trial, the base diet was supplemented with RP-Lys to meet Lys requirements, and treatments were as follows: no added RP-Met (NCM), NCM plus Smartamine M (SM; Adisseo, Alpharetta, GA), and NCM plus P1, P2, or P3 at 148% of the Met content of SM. In the Lys trial, the base diet was supplemented with RP-Met to meet the Met requirement, and treatments were as follows: no added Lys (NCL), NCL plus AjiProL (AL; Ajinomoto Heartland Inc., Chicago, IL), and NCL plus P4 at 55, 78, or 102% of the reported absorbed Lys in AL. All products were top dressed on the diet without prior mixing or extended exposure to the rest of the diet. Milk protein concentration significantly increased when diets were supplemented with P2, P3, or SM (3.12, 3.12, and 3.11%, respectively) compared with NCM (3.02%). Only P1 (3.04%) was significantly lower than SM. Prototype P2 had the greatest numerical milk protein output response among the 3 RP-Met prototypes, suggesting that it may have had the greatest efficacy when supplemented into these rations. There was a numerical milk protein concentration response to AL and a linear increase in milk protein concentration for P4. The P4 and AL treatments resulted in comparable milk protein production regardless of P4 dose.

Efeito de dispositivos vaginais impregnados com alta ou baixa concentração de progesterona na taxa de prenhez de novilhas submetidas à inseminação artificial em tempo fixo

This study aimed to evaluate the use of low- or high-dose intravaginal progesterone (P4)-releasing devices on pregnancy rates of Zebu heifers submitted to fixed-timed artificial insemination (TAI). Pubertal Bos indicus Nellore heifers were injected at day 0 with 2 mg estradiol benzoate (EB) and assigned randomly to three groups. Anintravaginal device containing 1.9 or 0.75 g P4 was inserted in animals of groups 1 (n=80) and 2 (n=79), while group 3 (n=76) received no device. The intravaginal devices were removed on day 8 and one luteolytic dose (125 μg) of d-cloprostenol was administered. Half of the animals in each group additionally received 300 IU of eCG. Twenty-fourhours later, the heifers received a second injection of 1 mg EB and TAI was performed 54 hours after the injection of cloprostenol. Pregnancy was diagnosed 40 days after TAI by transrectal ultrasonography. Data were analyzed by logistic regression. Pregnancy rates were 27.5%, 30.4% and 28.9% in groups 1, 2 and 3, respectively (P>0.05). This resultwas not influenced by body score, farm, eCG, or presence of CL at the beginning of the treatments. In conclusion, the use of intravaginal devices containing either a high (1.9 g) or low (0.75 g) dose of P4 resulted in similar pregnancy rates after TAI in pubertal Nellore heifers.

PSX-9 Sexual Behavior of Rams Exposed to Anovulatory Ewes Receiving Different Doses of P4

PSX-9 Sexual Behavior of Rams Exposed to Anovulatory Ewes Receiving Different Doses of P4

Analysis of stage-specific expression of the toll-like receptor family in the porcine endometrium throughout the estrous cycle and pregnancy

Toll-like receptors (TLRs) play critical roles in innate immunity by regulating antimicrobial responses in mucosal tissues. The expression and function of TLRs in female reproductive tissues have been studied in several species, but the expression and function of TLRs and MYD88, an adaptor molecule in the TLR signaling pathway, at the maternal-conceptus interface are not well understood in pigs. Thus, we determined the expression of TLR1 - TLR10 and MYD88 in the endometrium, conceptus, and chorioallantoic tissues of pigs. TLR1 - TLR10 and MYD88 mRNAs were expressed in the endometrium during the estrous cycle and pregnancy in a stage-dependent manner. TLR and MYD88 mRNAs were also detected in early stage conceptuses and chorioallantoic tissues from Day 30 to term pregnancy. The expression of TLR2, TLR4, TLR5, and TLR7 was localized to epithelial and stromal cells in endometrial and chorioallantoic tissues. Increasing doses of P4, but not E2, induced the expression of TLR4, TLR5, TLR6, TLR7, and TLR8, while interferon-γ increased the expression of TLR2 and TLR7 in endometrial explant tissues. Expression of TLR3, TLR5, TLR6, TLR7, and MYD88 was higher in the endometrium with somatic cell nucleus transfer-derived conceptuses than conceptuses derived from natural mating on Day 12. These results indicate that the expression of TLR1 - TLR10 and MYD88 is dynamically regulated at the maternal-conceptus interface in pigs, suggesting that TLRs expressed in the endometrium and the placenta may play a critical role in regulating mucosal immune responses to support the establishment and maintenance of pregnancy.