Intrauterine Infusion Research Articles

Platelet-Rich Plasma Intrauterine Infusion as Assisted Reproduction Technology (ART) to Combat Repeated Implantation Failure (RIF): A Systematic Review and Meta-Analysis.

Repeated implantation failure (RIF) is considered one of the major challenges facing clinician in assisted reproduction technologies (ART) despite the significant advances that have been made in this field. Platelet rich plasma (PRP), also known as autologous conditioned plasma, is a protein concentrate with anti-inflammatory and pro-regenerative characteristics. The use of PRP in women undergoing ART has been studied in the past, with varying degrees of success. The goal of this trial was to see if injecting PRP into the uterus improves pregnancy outcomes in women receiving ART. PubMed, Embase, Scopus, Web of Science, and the Cochrane Database of Clinical Trials were among the databases searched (CENTRAL), from 2015 to 2021. The pooled estimates were calculated using a meta-analysis with a random-effects model. There were 14 studies with a total of 1081 individuals (549 cases and 532 controls). There was no difference in miscarriage rates between women who got PRP and those who received placebo (P≤0.90). Chemical pregnancy (P≤0.00), clinical pregnancy (P ≤0.001), and implantation rate (P≤ 0.001) were all significantly higher in women. Endometrial thickness increased in women who got PRP vs women who received placebo after the intervention (P ≤0.001). PRP may be an alternate therapeutic approach for individuals with thin endometrium and RIF, according to the findings of this comprehensive study. To determine the subgroup that would benefit the most from PRP, more prospective, big, and high-quality randomized controlled trials (RCTs) are needed.

Induced endometrial inflammation compromises conceptus development in dairy cattle†.

Endometrial inflammation is associated with reduced pregnancy per artificial insemination (AI) and increased pregnancy loss in cows. It was hypothesized that induced endometritis alters histotroph composition and induces inflammatory signatures on conceptus that compromise development. In Experiment 1, lactating cows were assigned to control (CON; n = 23) or to an intrauterine infusion of Escherichia coli and Trueperella pyogenes (ENDO; n = 34) to induce endometritis. Cows received AI 26days after treatment, and the uterine fluid and conceptuses were collected on day 16 after AI. In Experiment 2, Holstein heifers were assigned to CON (n = 14) or ENDO (n = 14). An embryo was transferred on day 7 of the estrous cycle, and uterine fluid and conceptuses were recovered on day 16. Composition of histotroph and trophoblast and embryonic disc gene expression were assessed. Bacterial-induced endometritis in lactating cows altered histotroph composition and pathways linked to phospholipid synthesis, cellular energy production, and the Warburg effect. Also, ENDO reduced conceptus length in cows and altered expression of genes involved in pathogen recognition, nutrient uptake, cell growth, choline metabolism, and conceptus signaling needed for maternal recognition of pregnancy. The impact of ENDO was lesser on conceptuses from heifers receiving embryo transfer; however, the affected genes and associated pathways involved restricted growth and increased immune response similar to the observed responses to ENDO in conceptuses from lactating cows. Bacterial-induced endometrial inflammation altered histotroph composition, reduced conceptus growth, and caused embryonic cells to activate survival rather than anabolic pathways that could compromise development.

Use of platelet lysate for in-vitro embryo production and treatment of repeat breeding in cows

Platelet-rich plasma (PRP) is a biological hemocomponent derived from blood after the complete removal of red blood cells and the partial or complete removal of white blood cells to concentrate platelets in an appropriate volume of plasma. Platelets have important growth factors, cytokines, and active metabolites that improve the endometrial environment and positively affect implantation. This study evaluated the effect of the addition of activated PRP (platelets lysate; PL) on in vitro bovine oocyte maturation and embryonic development and the effect of intrauterine (IU) infusion of autologous PL in repeat breeder (RB) cows. Experiment 1 examined the effects of allogeneic PL, fetal calf serum (FCS), mixed PL + FCS, or platelet-poor plasma (PPP) supplementations to in vitro maturation and development media on in vitro oocyte maturation and embryo development in good- and poor-quality oocytes of Japanese Black cows. Experiment 2 examined the IU infusion of autologous PL, 24 h post-insemination, in 21 RB Holstein-Friesian dairy cows. The cleavage rate of good-quality oocytes was higher in the PL group (85.93 ± 2.50%) than in the PPP group (67.16 ± 3.41%) (P < 0.05), while the cleavage rate of the poor-quality oocytes was higher in the PL alone (76.13 ± 4.04%) and mixed PL + FCS treated (73.59 ± 4.22%) groups than in the PPP group (54.64 ± 2.93%) (P < 0.05). The blastocyst rate of the good-quality oocytes was higher in the PL group (40.97 ± 3.03%) than in the FCS (27.97 ± 3.31%) and PPP (25.33 ± 2.15%) groups (P < 0.05). The blastocyst rate of poor-quality oocytes and the hatching rates of both good and poor-quality oocytes showed no significant differences among all groups. The conception rate in the autologous PL-treated group was 41.67% (5/12), while it was 11.11% (1/9) in the control group. The platelets' count in the pregnant PL-treated cows (n = 5; mean ± SEM, 1.07 ± 0.10 × 109/mL) was higher than in the non-pregnant ones (n = 7; 0.67 ± 0.10 × 109/mL) (P < 0.05). In conclusion, allogeneic PL was effective in stimulating the in vitro oocyte maturation and embryonic development in both good and poor-quality bovine oocytes, and post-insemination IU infusion of autologous PL derived from high platelets’ count-PRP would be recommended for the treatment of RB cows.

Local and systemic inflammatory response to the intrauterine infusion of enzymes during estrus in water buffaloes with subclinical endometritis

Our objective was to determine the effects of intrauterine infusion of proteolytic enzymes in buffaloes with subclinical endometritis (SCE) at estrus on the resolution of endometrial inflammation and reproductive performance. Buffaloes at spontaneous estrus (E1) were screened for SCE by endometrial cytology to identify SCE (≥5% PMN, n = 22) and non-SCE (<5% PMNs, n = 14) animals. All buffaloes underwent uterine ultrasonographic examination, low volume uterine lavage (cytokines and acute phase proteins) and blood sampling (cytokines and acute-phase proteins) at E1. On the same day (E1), SCE buffaloes were randomly selected either for intrauterine infusion of proteolytic enzymes (ENY, n = 11) or saline (PC, n = 11). Buffaloes without SCE were kept as untreated control (NC; n = 14). All buffaloes were re-examined and re-sampled during subsequent estrus (E2), inseminated during the following estrus (E3), and assessed for fertility related outcomes. Proteolytic infusion resulted a reduction in uterine PMN (P < 0.01) in SCE buffaloes. The concentrations of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in uterus, and TNF-α and IL-10 in serum were higher (P < 0.01) at E1 in buffaloes with SCE (PC and ENY) compared to NC. After treatment, uterine IL-1β and TNF-α (P = 0.02), and serum TNF-α and IL-10 were lower within the animals of ENY group (P < 0.01). Before treatment, buffaloes with SCE had higher concentrations (P < 0.01) of serum and uterine amyloid-A and haptoglobin, which decreased (P < 0.01) after treatment in the ENY group. None of the fertility outcomes differ between the treatment groups. In conclusion, intrauterine infusion of proteolytic enzymes reduced endometrial inflammation; however, did not improve reproductive outcomes.

Intrauterine colonization with Gardnerella vaginalis and Mobiluncus mulieris induces maternal inflammation but not preterm birth in a mouse model.

Preterm birth (PTB) remains a leading cause of childhood mortality. Recent studies demonstrate that the risk of spontaneous PTB (sPTB) is increased in individuals with Lactobacillus-deficient vaginal microbial communities. One proposed mechanism is that vaginal microbes ascend through the cervix, colonize the uterus, and activate inflammatory pathways leading to sPTB. This study assessed whether intrauterine colonization with either Gardnerella vaginalis and Mobiluncus mulieris alone is sufficient to induce maternal-fetal inflammation and induce sPTB. C56/B6J mice, on embryonic day 15, received intrauterine inoculation of saline or 108 colony-forming units of G. vaginalis (n=30), M. mulieris (n=17), or Lactobacillus crispatus (n=16). Dams were either monitored for maternal morbidity and sPTB or sacrificed 6h post-infusion for analysis of bacterial growth and cytokine/chemokine expression in maternal and fetal tissues. Six hours following intrauterine inoculation with G. vaginalis, M. mulieris, or L. crispatus, live bacteria were observed in both blood and amniotic fluid, and a potent immune response was identified in the uterus and maternal serum. In contrast, only a limited immune response was identified in the amniotic fluid and the fetus after intrauterine inoculation. High bacterial load (108 CFU/animal) of G. vaginalis was associated with maternal morbidity and mortality but not sPTB. Intrauterine infusion with L. crispatus or M. mulieris at 108 CFU/animal did not induce sPTB, alter pup viability, litter size, or maternal mortality. Despite inducing an immune response, intrauterine infusion of live G. vaginalis or M. mulieris is not sufficient to induce sPTB in our mouse model. These results suggest that ascension of common vaginal microbes into the uterine cavity alone is not causative for sPTB.

P-350 Evaluation of EndoSERA Autologous Platelet Derived Growth Factors for improving clinical pregnancy and live birth rate in FET cycles for women with refractory thin endometrium

Abstract Study question Can Autologous Platelet Derived Growth Factors (hereafter EndoSERA) be used as promising coadjuvant therapy in assisted reproductive techniques to improve endometrial thickness and implantation rate? Summary answer Use of EndoSERA significantly increased endometrial growth, clinical pregnancy and Live Birth Rate In Patients With refractory thin endometrium during FET cycles. What is known already Inadequate endometrial thickness and receptivity are major causes for RIF and surrogacy is reasonable option when endometrium remains unresponsive to conventional treatments. Recent studies demonstrated platelet-rich plasma (PRP) improves pregnancy outcomes in thin endometrium and RIF patients. The mechanisms of PRP have not been completely elucidated, but laboratory studies have shown that the high concentration of growth factors in PRP can potentially speed up the healing process. Need for an optimized method to ensure right concentration of platelets and growth factors to maximize the therapeutic outcomes formed the basis for developing EndoSERA-implantation friendly platelet derived growth factors concentrate. Study design, size, duration In this prospective interventional self-controlled study, 55 women in the age group of 25-45 years from July 2018 to July 2022 with &amp;gt; 3 failed FET due to refractory thin endometrium, negative hysteroscopic screening for endometrial pathology, and negative bacteriologic screening and failed to get pregnant with multiple immune therapy regimens like intralipid infusions, granulocyte colony-stimulating factor infusions, steroid therapy, endometrial scratching were selected to undergo Endo-SERA treatment. Participants/materials, setting, methods After obtaining informed consent subjects were treated with intrauterine infusion of EndoSERA 3 times (Day7 and Day 12 of their menstrual cycle day and 2 days before ET). 54 patients underwent FET. Intrauterine infusion of 0.8 ml of EndoSERA was infused into uterine cavity in addition to standard HRT protocols. Clinical pregnancy was determined by positive serum β-HCG, 2weeks after ET and presence of fetal heart beat in trans-vaginal ultrasound 5weeks after ET. Main results and the role of chance Post EndoSERA administration endometrial thickness(ET) was significantly thicker (7.86 ± 0.22 vs 6.22 ± 0.31 mm; P &amp;lt;.05) in 98% of patients and optimal response to EndoSERA was considered to reach ET ≥ 7mm after 2nd EndoSERA dose administration. Only one patient’s cycle got canceled due to ET &amp;lt; 7mm. Out of 55 women, 42 became pregnant (76%) and 12 patients did not conceive (22%). 39 women (70.9%) had a clinical pregnancy and 5 women (9.1%) miscarried before 6-12 wks and 3 women (5%) had biochemical pregnancy. 33 women (60%) had delivered healthy full-term babies and one patient (2%) is in her 24th week of uneventful gestation. There were no adverse effects reported by the patients who were treated with EndoSERA. There are studies highlighting the need for an angiogenic and anti-inflammatory environment for successful implantation. EndoSERA is standardized to contain 6-9 folds higher amounts of growth factors than peripheral blood majorly implantation friendly and anti-inflammatory cytokines which restore impaired uterine environments and optimized for cyclical requirements of proliferation, secretion &amp; Implantation phases of Endometrium. It has also been shown to be safe, reproducible, and effective in mimicking the natural processes of tissue repair and regeneration. Limitations, reasons for caution This prospective self-controlled study with small sample size lacks a randomized control group. While the beneficial effects observed in this study are improved than published data, larger study with patients recruited based on inclusion criteria of refractory thin endometrium is proposed for recommending EndoSERA as routine adjuvant during IVF procedures. Wider implications of the findings EndoSERA improved implantation, pregnancy, and live birth rates (LBR) in refractory thin endometrium patients which indicates clearly endometrial thickness and receptivity improvement and cumulative 62% has motivated us to plan randomized controlled studies to confirm the results and provide opportunity for women with refractory thin endometrium to conceive without surrogacy. Trial registration number Not Applicable

P-472 Endometrial stimulation with culture medium containing granulocyte macrophage colony-stimulating factor promotes embryo implantation after vitrified-warmed blastocyst transfer

Abstract Study question Does endometrial stimulation with culture medium containing granulocyte macrophage colony-stimulating factor (GM-CSF) promote implantation potential of vitrified-warmed blastocysts? Summary answer Endometrial stimulation with culture medium containing GM-CSF prior to transfer of vitrified-warmed blastocysts may promote embryo implantation and improve pregnancy continuation. What is known already Human embryonic development and implantation in vivo is precisely regulated by various cytokines, including growth factors. GM-CSF is a cytokine that plays an important role in reproductive function. We reported the possibility that the use of culture medium containing GM-CSF in cleavage stage vitrified-warmed embryo transfers (ETs) promotes embryo implantation (ESHRE2018). However, it has been reported that endometrial stimulation with culture supernatant up to blastocyst stage (SEET method) improves embryo implantation, but the effect of endometrial stimulation with culture medium containing GM-CSF (G-SEET method) on embryo implantation is not clear. Study design, size, duration This study was conducted at a single in vitro fertilization (IVF) center from June 2020 to February 2022. The G-SEET procedure was performed at each patient’s request. All study participants provided informed consent, and the study design was approved by the ethics committee of IVF Nagata Clinic, Fukuoka, Japan. Participants/materials, setting, methods We examined 702 cycles of vitrified-warmed blastocyst transfer. Warmed blastocysts were incubated in recovery culture for several hours and transferred on day 5 (control group). For the G-SEET method, only culture medium containing GM-CSF was injected into the uterus two days before ET, which was performed as in the control group (G-SEET group). The human chorionic gonadotropin (hCG) positive, clinical pregnancy, miscarriage, and ongoing pregnancy rates were compared between the two groups. Main results and the role of chance Of the 702 cycles of single vitrified-warmed blastocyst transfers, the control and G-SEET groups had 459 and 243 cycles, respectively. The G-SEET group had a higher patient age (37.5±4.2 vs. 36.2±3.8, p &amp;lt; 0.01), a thinner endometrial thickness (10.3±1.9 vs. 10.9±2.0, p &amp;lt; 0.01), a lower percentage of good blastocyst transfers (65.4% vs. 79.5%, p &amp;lt; 0.01), and a higher cumulative number of implantation failures (3.6±2.5 vs. 1.9±1.9, p &amp;lt; 0.01) than the control group. The G-SEET method was performed at the patient’s request, so the number of cycles and patient background varied. Multivariate analysis was performed using female age, male age, number of miscarriages, endometrial thickness, presence of good blastocysts, and cumulative number of implantation failures as confounding factors. Compared with the control group, the G-SEET group exhibited the following: hCG positivity rate [odds ratio (OR): 1.14, 95% confidence interval (CI): 0.79–1.64, p = 0.481], clinical pregnancy rate (OR: 1.81, 95% CI: 1.21–2.71, p = 0.003), miscarriage rate (OR: 0.735, 95% CI: 0.30–1.76, p = 0.491), and ongoing pregnancy rate (OR: 1.79, 95% CI: 1.20–2.69, p = 0.004). Clinical pregnancy and ongoing pregnancy rates were significantly higher in the G-SEET group compared with the control group. Limitations, reasons for caution The study was limited by the study size and lack of data about live birth rates after ET. In addition, intrauterine infusion of GM-CSF-free culture medium should be used as a control, but this was difficult in the private clinic setting. Wider implications of the findings This study suggests that endometrial stimulation with culture media containing GM-CSF prior to transfer of vitrified-warmed blastocysts promotes embryo implantation and enhances the ongoing pregnancy. The G-SEET method may be clinically useful as a new ET method. Trial registration number not applicable

P-314 PlasmaShotting: a new method of treating “thin” endometrium

Abstract Study question Is a new method of subendometrial platelet-rich plasma (PRP) injection (PlasmaShotting) effective in patients with “thin” endometrium? Summary answer PlasmaShotting showed higher efficiency in the group of patients with “thin” endometrium compared to conventional intrauterine infusion of platelet-rich plasma What is known already The problem of “thin” endometrium is one of the urgent challenges of modern reproductive medicine. In patients suffering from this condition, there is a decrease in the pregnancy rate and an increase in miscarriage rates. “Thin” endometrium is less than 7 mm according to ultrasound. To correct this condition, many methods have been proposed: hormone replacement therapy with high doses of estrogens, aspirin, GnRH agonists, vitamin E, pentoxifylline, sildenafil, intrauterine infusion of colony-stimulating factors (G-CSF) and PRP, but none of the proposed methods has shown enough effectiveness. Study design, size, duration To evaluate the effectiveness of PlasmaShotting we organized a study that included 72 people. Inclusion criteria were age less than 40 years, endometrial thickness less than 6.5 mm on days 8-10 of hormone replacement therapy, and 3 or more unsuccessful embryo transfers of excellent quality embryos before. Participants/materials, setting, methods The patients were divided into 2 groups: Group 1 (23 patients) underwent hysteroscopy with submucosal injections of platelet-rich plasma (PRP - PlasmaShotting) on days 10-12 of the cycle. Group 2 (49 patients) underwent intrauterine infusion of platelet-rich plasma (PRP) on days 10-12 of the cycle Main results and the role of chance The clinical pregnancy rate in group 1 was 47.8% (n = 11), and in group 2 was 34.6% (n = 17). The live births rate in group 1 was 34.7% (n = 8), and in group 2 was 20.4% (n = 10). The miscarriage rate was in group 1 was 13.1% (n = 3), and in group 2 was 14.2% (n = 7) Limitations, reasons for caution The research on the effectiveness of PlasmaShotting should be continued Wider implications of the findings PlasmaShotting as a new method of treating “thin” endometrium can be used in patients who can't achieve pregnancy Trial registration number not applicable

The effectiveness and safety of intrauterine infusion of autologous regulatory T cells (Tregs) in patients with recurrent pregnancy loss and low levels of endometrial FoxP3+ cells: A retrospective cohort study.

Regulatory T cells (Tregs) are a specialized type of T cells that help maintain immune tolerance and homeostasis. The potential of Tregs cell-based therapies in treating diseases has been demonstrated in several clinical trials, which have shown promising outcomes and high safety in autoimmune diseases, transplant rejection, and graft-versus-host disease. However, their effectiveness and safety in improving endometrial receptivity and reducing pregnancy loss in human reproduction are unknown. The study used a retrospective design and included patients with recurrent pregnancy loss (RPL) and lower levels of endometrial FoxP3+ Tregs. Patients in the Tregs group (n=33) received intrauterine Tregs infusion three times during the follicular phase, while the control group (n=28) did not receive any intrauterine infusion. The intrauterine infusion of autologous Tregs increased the levels of FoxP3+ Tregs and CD56+ NK cells. Patients in the Treg group had higher live birth rates and lower miscarriage rates, especially early miscarriage rates. However, the two groups had no differences in the implantation rate, clinical pregnancy rate, and percentage of preterm delivery. The findings suggest that intrauterine Tregs infusion may be a potential therapeutic approach for RPL. Further research in larger clinical trials is needed to confirm these findings.

Concentrations of penicillin and gentamicin following intrauterine administration of two compounded formulations

The use of broad-spectrum antimicrobial therapy either by intrauterine or systemic route is commonplace in equine practice, however, much remains unknown about antibiotic efficacy in this environment. We recently demonstrated the activity of penicillin and gentamicin against common endometrial pathogens ex vivo.1 The objective of this study was to measure the concentration of gentamicin and penicillin in the uterine fluid of mares following intrauterine infusion of either a standard (PPGent) or long-acting (LA-PPGent) compounded formulation of procaine penicillin and gentamicin. We hypothesized that both drug formulations would result in therapeutic concentrations following administration, with total concentrations sustained for longer using the long-acting formulation (LA-PPGent). Ten reproductively sound mares in early estrus were administered 2378mg of procaine penicillin and 200mg of gentamicin via a single intrauterine infusion in either a standard (n=5) or a lyophilized formulation suspended in a slow-release matrix (n=5). Intrauterine fluid was collected at 0.5, 2, 4, 8, 24, 32, 48 and 72 h following infusion. A pre-weighed section of umbilical tape was introduced into the uterus through a double-guarded pipette for 30 seconds and then weighed to determine the absorbed fluid volume. Penicillin and gentamicin analyses were performed using high-performance liquid chromatography(HPLC) and ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), respectively. Penicillin and gentamicin concentrations were assessed between groups and over time using a repeated measures analysis with treatment, time, and their interaction in the model statement with significance set at p<0.05. Mean intrauterine drug concentrations of PPGent peaked at 0.5 hours (Penicillin: 10,123.0 ± 4,298.0 µg/mL, Gentamicin: 3,397.3 ± 1,338.5 µg/mL), well above concentrations previously shown to inhibit bacterial growth ex vivo ([1] Von Dollen et al. J Equine Vet Sci 2019;79:121-26), and exceeded MIC for relevant organisms for 72 hours (Penicillin: 2.59 ± 6.34 µg/mL, Gentamicin: 2.14 ± 2.4 µg/mL). Mean concentrations of LA-PPG were lower at peak (Penicillin: 2,213.8 ± 967.8 µg/mL – p<0.05, Gentamicin: 1,859 ± 2,413 µg/mL) and exceeded MIC for 24h and 32h, respectively. These results support the use of procaine penicillin and gentamicin as intrauterine antimicrobial therapeutics. The long-acting formulation was not found to extend the time over MIC of antibiotics in the uterus of estrus mares. Acknowledgements: LA-PPGent was prepared by SR Veterinary Technologies LLC, Windsor CO using previously lyophilized PPGent. This research was supported by the Grayson-Jockey Club Research Foundation.

Mesenchymal stromal cell conditioned media is immunomodulatory in the equine endometrium

Endometritis is a common and costly disorder in horses that results in poor reproductive performance. Current therapies are supportive, and the industry suffers from a paucity of immunomodulatory treatment modalities for this disease that is characterized by inflammatory dysregulation. Recently, mesenchymal stromal cell conditioned media (MSC-CM) is being explored as a treatment option for various inflammatory conditions due to its immunoregulatory, antimicrobial, biofilm disruption, and tissue repair properties. Our objective was to evaluate changes in the endometrial inflammatory response after treatment with MSC-CM in vitro. We hypothesized that endometrial cells in culture would have altered cytokine expression and production. Five estrous mares were biopsied to establish primary endometrial cell cultures. After cell dissociation, endometrial cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotic antimycotic solution (Sigma Aldrich) at 37°C with 5% CO2. At 80% confluence, cultures were hormonally supplemented with 1nM Estradiol-17β, 100 nM Progesterone, or no supplementation; and 24 hours later, treated with MSC-CM or DMSO as control. Supernatants from the cell cultures were analyzed for cytokine expression using an equine cytokine Multiplex assay (Luminex Corp. Austin, TX) quantifying inflammatory biomarkers including IFN-α, IFN-γ, IL-4, IL-17, IL-10, IL1-β, CCL2, CCL3, CCL5, CCL11, TNF-α, and sCD14. In parallel, cells were lysed and RNA extracted for Real-time PCR using the ΔCT method with ACTB as the reference gene for geneexpression of IL-6, IL-8, PTGS2, PTGES, PTGIS, TNFα, TGFB, and NFKb. Data analysis was performed with JMP Pro 16 with level of significant p<0.05. A restricted maximum likelihood model with treatment, supplementation and their interaction and mare as a random factor was used. There was no effect of hormone supplementation on supernatant cytokine concentrations, but treatment with MSC-CM altered their concentration. MSC-CM treatment increased the concentration of CCL2 pg/ml [median (interquartile)] [1862554 (32482, 240080) (p≤0.02); and decreased CCL3 [66 (0.1, 99)] (p≤ 0.01) and IL-10 [6.5 (0.1, 9.5)] (p≤0.007) compared to DMSO control [3114 (2398.5, 7483); 109 (0.1, 154); and 13 (0.1, 17)], respectively. Gene expression analysis showed that MSC-CM-treated cells had increased expression of IL-6 (p≤0.001), IL-8 (p≤0.001), PTGS2 (p≤0.002), PTGES (p≤0.0012), and NFKb (p≤0.01). Additionally, gene expression analysis revealed an effect of hormone supplementation, with increased expression of IL-8 (p≤0.027) and PTGS2 (p≤0.0443) in estrogen or progesterone supplemented cultures compared to no supplementation. Taken together, treatment with MSC-CM resulted in increased inflammatory marker production and greater gene expression of inflammatory cytokines and prostaglandin-related genes. These results are supportive of the potential role of MSC-CM in equine endometrial immunomodulation. Further studies in vivo are warranted to investigate the clinical outcome of this immunomodulatory effect after intrauterine infusion.

Post-breeding intrauterine infusion of dehydrated amnion product produces similar pregnancy rates as antibiotics

Results of two large studies by Pascoe and then Pycock first demonstrated the value of postbreeding treatments in mares. When combined, these authors analyzed pregnancy data from over 2200 mares. In their studies, those treated with antibiotics (ABX) and plasma or oxytocin had a higher conception rate than any other group. However, a Cochrane-style review by Barter and Barrelet (Veterinary Evidence 2018;3(1):1-11. of these and numerous smaller studies didn't convincingly confirm a treatment benefit of any specific therapy. In a 2-part multicenter clinical study involving three veterinary practices, we aimed to determine whether postbreeding intrauterine (IU) infusion with a novel dehydrated amnion-derived allograft1 (DAA) would enhance pregnancy rates in a clinical setting. To achieve this, we first performed a targeted prospective study of postbreeding IU DAA compared to ABX in first-cycle barren Thoroughbred mares. Sixty mares that had either failed to get pregnant on two or more cycles in the previous year or which lost a pregnancy were randomly assigned to either a DAA or ABX treatment group at the onset of the breeding season. Mares with no bacterial growth and <5mm IU fluid prior to breeding were included in the study and administered IU either1 vial LAP diluted in 60 ml LRS or 7.2 million IU procaine penicillin and 600mg gentamicin, extended to 60 mL with LRS within 24 hours postbreeding. Mares with intra-uterine fluid were lavaged or received oxytocin based on clinical presentation. Statistical analysis was performed using a publicly available chi square calculator with significance set at P<0.05. There was no difference in the first cycle pregnancy rate of the DAA group compared to ABX (65% vs 64%, respectively). Based on these results, clinicians used DAA as a component of their reproductive treatment protocols during the 2022 breeding season. Retrospective analysis of over 500 breeding cycles from 386 mares was performed using the same methods as above to compare pregnancy rate in cycles with no treatment, DAA alone, DAA/lavage, ABX alone and ABX/lavage based on the clinical judgement of the attending veterinarian. Pregnancy rates were not statistically different between groups (69%, 57%, 73%, 64%, 62%), however pregnancy rates were numerically highest in cycles treated with DAA/lavage. The above data suggest that the use of IU DAA is safe and support its use as a postbreeding infusion to optimize pregnancy rates in broodmares.

Mitigation of post breeding endometritis in mares treated with platelet rich plasma

Persistent breeding-induced endometritis (PBIE) is one of the major causes of infertility in mares, characterized by a lingering inflammatory response in the uterus, which leads to accumulation of uterine fluid and polymorphonuclear cells (PMN), harming the reproductive efficiency of the animal. The aim of this study was to determine if the presence and volume of intrauterine fluid and the percent of endometrial neutrophils decreased after intrauterine application of blood platelet-rich plasma (PRP) in mares with post-mating endometritis. Healthy estrous crossbreed mares (n = 9) aged between 6 to 12 years, with optimal body condition and under the same nutritional management, had two cycles randomly assigned to a crossover trial. Each received either intrauterine infusions of lactate ringer solution (LRS) or autologous PRP 4h after artificial insemination (AI) with fresh semen of low sperm quality from the same stallion. All the animals went through all the treatments. A total of 100 mL of citrated whole blood was collected for PRP production and was centrifuged at 2315g for 15 minutes then 579g for 10 minutes. The presence of intrauterine fluid (IUF) was analyzed by ultrasonographic examination and the percentage of endometrial neutrophils was determined through endometrial cytology, both before insemination (in the presence ofa preovulatory follicle ≥35mm and endometrial edema) and 24h after treatment application. Statistical analysis was performed by a Mixed model (SAS, 2023) for repeated measures with treatment and time as between subject factors. The presence of intrauterine fluid had an effect of time (p ≤ 0.049), where fluid size was smaller before AI than 24h after treatment; however, there was no treatment effect (p ≥ 0.264) over intrauterine fluid. The percentage of endometrial neutrophils had an effect of time (p ≤0.001), less inflammatory cells were found before AI than 24 hours after treatment; there was an effect of treatment (p ≤0.001) as well, where PRP decreased the number of endometrial polymorphonuclear cells compared to the control group infused with LRS. The increase in intrauterine fluid with time suggests that semen itself increases blood perfusion that generates intrauterine fluid accumulation. After induction of physiological endometritis, endometrial polymorphonuclear cell numbers were reduced successfully with PRP treatment. PRP is rich in peptides and signaling proteins that suppress the expression of proinflammatory cytokines such as IL-1 and TNF, inhibits polymorphonuclear neutrophil migration, and thus regulates intrauterine inflammation through decreased endometrial COX-2 expression.

164 Intrauterine infusion of honeybee product in healthy postpartum dairy cows is safe

164 Intrauterine infusion of honeybee product in healthy postpartum dairy cows is safe

165 Intrauterine infusion of TNF as possible treatment of metritis in cows

165 Intrauterine infusion of TNF as possible treatment of metritis in cows

Induced endometritis in early lactation compromises production and reproduction in dairy cows

Objectives of this experiment were to study the effect of infusing utero-pathogenic bacteria to induce endometrial inflammation on productive performance in early lactation and subsequent reproduction. Although endometritis is associated with perturbed reproduction, numerous factors may contribute to the observed association. It was hypothesized that induced endometrial inflammation, resulting in localized and systemic inflammatory responses, compromises production and reproduction. Holstein cows without clinical disease and with less than 18% polymorphonuclear leukocytes (PMN) in endometrial cytology on d 31 ± 3 postpartum had their estrous cycle synchronized. Cows were blocked by parity and genomic breeding value for cow conception rate and, within block, assigned randomly to remain as untreated controls (CON; n = 37) or to receive an intrauterine infusion of 5.19 × 108 cfu Escherichia coli and 4.34 × 108 cfu Trueperella pyogenes during the luteal phase to induce endometrial inflammation (INF; n = 48). Endometrial cytology was taken on d 2 and 7 after treatment to evaluate the proportion of PMN. Rectal temperature, dry matter intake, and yields of milk and components were measured in the first 7 d after treatment. Blood serum was analyzed for concentration of haptoglobin. Leukocytes were isolated from blood on d 2 and 7 after treatment and on d 19 after artificial insemination (AI) and mRNA was quantified for a select group of genes. Cows received AI and reproduction was followed for 300 d postpartum. Bacterial infusion induced endometrial inflammation with increased proportions of PMN in the endometrial cytology on d 2 (4.4 ± 0.7 vs. 26.3 ± 2.8%) and 7 (10.9 ± 1.7 vs. 17.4 ± 2.1%) after treatment, resulting in increased mean prevalence of subclinical endometritis (>10% PMN; 23.3 ± 6.3 vs. 80.9 ± 5.1%). Rectal temperature did not differ between CON and INF, but the concentration of haptoglobin in serum tended to increase in INF compared with CON (113 ± 14 vs. 150 ± 16 µg/mL). Induced endometrial inflammation reduced yields of milk (44.9 ± 0.8 vs. 41.6 ± 0.8 kg/d), protein (1.19 ± 0.03 vs. 1.12 ± 0.03 kg/d), and lactose (2.17 ± 0.04 vs. 2.03 ± 0.04 kg/d) and tended to reduce dry matter intake (20.7 ± 0.5 vs. 19.4 ± 0.6 kg/d) in the first 7 d after treatment. Indeed, the reduction in milk yield lasted 4 wk. However, treatment did not affect yields of energy-corrected milk or fat because treatment with INF increased the concentration of fat in milk (3.54 ± 0.10 vs. 3.84 ± 0.10%). Induced endometrial inflammation reduced pregnancy per AI at all inseminations (33.4 ± 5.1 vs. 21.6 ± 3.7%) and the hazard of pregnancy (0.61; 95% CI = 0.36-1.04), which extended the median days open by 24 d. Blood leukocytes from INF cows had increased mRNA expression of the pro-inflammatory gene IL1B on d 2 and 7 after treatment, but reduced expression of the IFN-stimulated genes ISG15 and MX2 on d 19 after AI. Induced endometrial inflammation depressed production and caused long-term negative effects on reproduction in lactating dairy cows.

Intrauterine Infusion of Leukocyte-Poor Platelet-Rich Plasma Is an Effective Therapeutic Protocol for Patients with Recurrent Implantation Failure: A Retrospective Cohort Study.

The clinical application of autologous leukocyte-poor platelet-rich plasma (LP-PRP) in patients with recurrent implantation failure (RIF) is rare. This retrospective observational cohort study aimed to evaluate the efficacy of LP-PRP intrauterine infusion in patients with RIF. Patients with RIF undergoing frozen embryo transfer (FET) from January 2019 to December 2021 (n = 118) were enrolled, with those undergoing LP-PRP intrauterine infusion as the PRP group (n = 64), and those receiving no LP-PRP treatment as the control group (n = 54). The beta-human chorionic gonadotropin (β-hCG)-positive rate, clinical pregnancy rate (CPR), live birth rate (LBR), and miscarriage rate (MR) per ET cycle were compared. The β-hCG-positive rate (57.8% vs. 38.9%, p = 0.041), CPR (45.3% vs. 24.5%, p = 0.022), and LBR per ET cycle (42.2% vs. 18.5%, p = 0.009) were higher in the PRP group than in the control group, and the three variables (62.5% vs. 41.2%, p = 0.040, 47.5% vs. 23.5%, p = 0.033, and 47.5% vs. 20.6%, p = 0.027) in the PRP group transferred with the blastocyst-stage embryos were also higher than those in the control group. The MR was similar in all groups. The LP-PRP treatment could improve the β-hCG-positive rate, CPR, and LBR in RIF patients undergoing FET cycles.

Elimination of Cefquinome Sulfate Residue in Cow's Milk after Intrauterine Infusion.

As set in the maximum residue limit regulations of the European Commission, this study aimed to obtain the residual parameters in milk with optimized UPLC-MS/MS conditions and to determine the conclusive drug withdrawal period to ensure food safety. In this research, an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to study cefquinome sulfate's residue elimination in milk and to calculate cefquinome's withdrawal period. Twelve healthy cows free of endometritis were selected for the experiment. Before using the drug, the vaginal orifice and perineum of each cow was disinfected. One dose of intrauterine perfusion was used for each cow, followed by an additional dose after 72 h. Before administration and 12 h, 18 h, 24 h, 36 h, 42 h, 48 h, 60 h, 66 h, 72 h, 84 h, 90 h, and 96 h after the last dose, milk (10 mL) was gathered from each cow's teat and pooled. For the measurement of cefquinome concentrations in milk, UPLC-MS/MS was performed. A calibration curve was generated using linear regression as follows: Y = 250.86X - 102.29, with a correlation coefficient of 0.9996; the limits of detection and the limits of quantitation were 0.1 μg·kg-1 and 0.2 μg·kg-1, respectively. The average recovery of cefquinome was 88.60 ± 16.33% at 0.2 μg·kg-1, 100.95 ± 2.54% at 10 μg·kg-1, and 97.29 ± 1.77% at 50 μg·kg-1. For 5 consecutive days at the three spiking levels, the intra and inter-day relative standard deviations (RSD) were 1.28%-13.73% and 1.81%-18.44%, respectively; the residual amount of cefquinome was less than the maximum residue limit of 20 μg·kg-1, 36 h after administration; and the residual amount was less than the limit of detection (0.1 μg·kg-1) 48 h after administration. The withdrawal time of cefquinome in cow's milk was 39.8 h, as calculated using WTM1.4 software. In terms of clinical practical use, the withdrawal period of milk was temporarily set at 48 h after the administration of the cefquinome sulfate uterus injection to cows, in accordance with the recommended dose and course.

O4-4 - Does intrauterine infusion of PRP affect the window of implantation?

O4-4 - Does intrauterine infusion of PRP affect the window of implantation?

Impact of intrauterine infusion of Platelets-Rich plasma on endometritis and reproductive performance of Arabian mare.

Equine endometritis is one of the most common causes of reproduction failure. To achieve better treatment outcomes, different diagnostic methods should be combined. In the current study, 39 repeat breeder mares were subjected to ultrasonography examination to detect excessive accumulation of intrauterine fluids and an abnormal oedema pattern, which revealed that 61.5% of mares were positive. Combined with endometrial cytology by low-volume uterine flush, 47.7% of smears contain neutrophils (more than 2-3 per HPF X100), and microbial culture. 92.3% of mares were infected with different bacterial isolates, such as Escherichia coli, Streptococci, Staphylococcus, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter freundii, Providencia alcalifaciens, and Proteus mirabilis. All mares were given saline solution and gravity withdrawal before being given 20 IU of oxytocin (as ecbolic agents). Mares were divided into three groups; Group one (n=15) received an intrauterine infusion of 20 mL of freshly prepared autologous platelet-rich plasma (PRP) 6 h after breeding, Group two (n=15) was treated with three doses of systemic Enrofloxacin 5% during the estrus period, and Group three (n=9) received only uterine lavage and 20 IU of oxytocin. PRP and Enrofloxacin resulted in a significant (p < .05) reduction in endometrial thickness (oedema; 5.05 and 6.74 mm, respectively) and disappearance of intrauterine fluids compared to the control (10.98 mm). Furthermore, PRP (days) and Enrofloxacin (17.89 days) reduced the days to the next oestrus compared to the control (18.58 and 17.89 vs. 21.19 days, respectively). Furthermore, the pregnancy rate improved to reach 70% in the PRP group and 60% in the Enrofloxacin group, while the control remained low at 22%. In conclusion, autologous PRP can be used as a low-cost alternative therapy for modulating the inflammatory process and effectively treating mares' endometritis.