Corresponding Author email: dale.kelley@okstate.edu
Abstract
Amnion-derived acellular bioscaffold (ADABP) products demonstrate interesting anti-inflammatory and healing properties which could be beneficial for intrauterine use. The objective of this study was to evaluate the safety of intrauterine injection of ADABP on systemic and uterine health. The study design randomly assigned subjects to one of two groups, control mares (n=3) which received 3mL injection of sterile saline in the base of each uterine horn, and treatment mares (n=9) which received 3mL of ADABP in the base of one uterine horn and 3mL injection of sterile saline in the base of the other uterine horn. The leukogram had no significant effect of group and no group by day interaction. The serum biochemistry panel had no effect of group on any of the parameters examined. There were no significant differences in uterine culture or uterine biopsy results. The data suggests intrauterine injection of ADABP has no negative systemic or uterine effects.
Keywords: Amnion, Mare, Endometrial biopsy, safety
- Introduction
Tissue damage can be caused by a variety of stimuli and with exaggerated and uncontrolled deposition of extracellular matrices result in the formation of scar tissue [1]. Tissue engineering aims to replace a patient’s diseased tissue with healthy autologous tissue using either a cell based construct or a cell-free matrix [2, 3]. Although these approaches are in their infancy, tissue engineering has been used in people clinically to slow disease progression and restore organ functionality [1].
Regenerative medicine is commonly used in cases of equine lameness [4] and skin wounds and utilizes stem-cell based therapies and scaffold based therapies [5]; however, it is less frequently utilized in reproductive tissue. Recent data in rats suggests that platelet-rich plasma injected into the uterus may stimulate endometrial regeneration and decrease fibrosis [6]. In mares, mesenchymal stem cells (MSC) injected into the uterus have been found to decrease endometrial fibrosis and type III collagen 60 days after treatment [7]. These therapies offer the potential to either prevent or treat endometrial fibrosis in mares, which has been shown to decrease fertility [8].
Amnion-derived acellular bioscaffold products (ADABP), a type of cell-free matrix, have been used to promote wound healing in human and veterinary medicine. Proteins and cytokines present in ADABP are reported to modulate the inflammatory response, decrease fibroblast formation and fibrosis [9] and could offer an additional therapy to treat or prevent endometritis, endometrial fibrosis and uterine adhesions in mares.
Amnion-derived acellular bioscaffold products demonstrate interesting anti-inflammatory and healing properties which could be beneficial for intrauterine use. Inflammatory cytokines in the uterus, such as TNF-α, IL-6 and Il-1β, have been found to be elevated in mares prone to endometritis [10]. Thus, it is plausible that reducing the expression of intrauterine inflammatory cytokines through immune modulation may reduce the severity of endometritis in mares. Safety of intrauterine injection in the mare of an ADABP has not yet been demonstrated. Due to the potential benefits of this product for modulation of inflammation and promoting uterine health, the objective of this study was to evaluate the safety of intrauterine injection of ADABP on systemic and uterine health.
- Materials and Methods
Twelve clinically healthy mares (mean age:11.5 years; range: 5-22 years), (9 light-breed type, 3 Warmblood) were the subjects of this experiment. All mares were clinically healthy and maintained on pasture full-time and supplemented with a complete pelleted feed. The study was conducted during the fall (October- November) in Central Kentucky, USA. Rectal temperature was monitored throughout the duration of the study and subjects were monitored for any signs of discomfort.
2.1 Study Design
This study was approved by the Kentucky Wildlife Center IACUC AUP 2024-01. The study design randomly assigned subjects to one of two groups, control mares (n=3) which received 3mL injection of sterile saline in the base of each uterine horn, and treatment mares (n=9) which received 3mL of ADABP (EquusCell StemWrap D™, AniCell Biotech) in the base of one uterine horn and 3mL injection of sterile saline in the base of the other uterine horn. The side the product was injected was randomly assigned and the initial site of biopsy procurement was randomly assigned (axial vs. abaxial)
On Day 0, blood was obtained for serum biochemistry, Complete Blood Count (CBC), Serum Amyloid A (SAA), Fibrinogen (FIB), and Progesterone analysis. Mares were sedated and restrained in stocks. The mares’ reproductive tracts were evaluated with palpation and transrectal ultrasonography and results of the ovarian findings, uterine edema, intraluminal uterine fluid and cervical tone were recorded. Endometrial swabs were submitted for cytology and aerobic culture using a double-guarded Kalajian swab technique previously described (11). Control mares (3) received 3mL injection of sterile saline in the base of both uterine horns during hysteroscopy, and treatment mares (9) received 3mL of ADABP (EquusCell StemWrap D™, Anicell Biotech) injection in the base of one uterine horn, and 3mL sterile saline in the base of the other horn, with side of ADABP injection randomly assigned. Each mare had a single biopsy obtained adjacent to each injection site (2 biopsies per mare) at the same level of the uterine horn where injection occurred about 2 cm abaxial or axial to the injection site (Fig. 1)
On Day 4 blood was obtained for serum biochemistry, CBC, SAA and FIB. All other procedures and progesterone analysis were performed on Day 21 and the mares’ reproductive tracts were evaluated with palpation and transrectal ultrasonography. Endometrial swabs were submitted for cytology and aerobic culture as previously described [11] and blood was obtained for Progesterone analysis. Hysteroscopy was performed and each mare had a single biopsy obtained at the same level of the horn adjacent to each injection site but on the opposite side (axial or abaxial to the previous biopsy site from Day 0 (Fig. 1)).
2.2 Bloodwork
2.3 Reproductive tract evaluation
Mares were placed in stocks and sedated using detomidine hydrochloride (0.02-0.03mg/kg IV, Zoetis) and butorphanol tartrate (0.2-0.3 mg/kg IV, Zoetis). Transrectal palpation and ultrasonography of the reproductive tract was performed using a Sonosite Linear transducer (Edge 11 Sonosite; Japan) and the ovarian findings, endometrial edema, intraluminal fluid and cervical tone were recorded.
2.4 Hysteroscopy and Uterine Injection
The perineum and vulva were prepped aseptically. Endometrial cytology and aerobic culture specimens were collected and analyzed as previously described [11] using double-guarded cotton swabs (Kalayjian swabs).
Hysteroscopy was performed using an 1030mm endoscope, outer diameter 9.8mm and instrument channel inner diameter 2.8mm (Olympus H180, Olympus America Inc.). The endoscope was placed aseptically through the cervix and the ventral aspect of the mid uterine horn was visualized. Injection of 3mL of assigned product (sterile 0.9%NaCl or 1 vial of ADABP (EquusCell StemWrap D™, Anicell Biotech) reconstituted to a final volume of 3mL using sterile 0.9%NaCL) was performed using an aspiration needle passed through the instrument portal (WangÒ Transbronchial Cytology Needle. 15mm/1.9mm x 130 cm/ 21 G; ConMed).
The injection needle was primed prior to entering the scope and the full 3mL was injected followed by sterile saline to clear the line of an appropriate volume.
2.5 Uterine Cytology
Cytological specimens were air-dried then stained with Diff Quick stain and were evaluated microscopically under oil immersion (×100) by one of three medical technologists blinded to treatment. A minimum of 10 fields were evaluated on each slide and the number of neutrophils (PMNs) per high power field (hpf) counted and averaged. Uterine cytology was classified as normal (<2 PMNs per hpf), moderate (3-5 PMNs per hpf), or severe (>5 PMNs per hpf).
2.6 Uterine culture
Uterine culture swabs were submitted to Rood and Riddle Equine Hospital laboratory where they plated on blood agar and Levine Eosin-Methylene Blue plates within 6 hours of procurement. Plates were incubated at 37 °C and examined every 24 hours for 3 days. Bacteria were identified using the BBL crystals system (BD) . Antimicrobial sensitivities of all isolated organisms were determined by the Kirby Bauer method. Primary identification of yeast was performed by dark field microscopy.
2.7 Biopsy
Endometrial biopsies were obtained by endoscopic visualization of the desired site of acquisition and the procurement was achieved using endometrial biopsy forceps (Jackson Uterine Biopsy Forceps, cutting area 4mm x 28mm, 60cm Shaft, Jorgensen Labs) introduced through the cervix. The biopsy was obtained 1-2 cm axial or abaxial to the site of injection. These samples were placed immediately in Bouins solution for 24 hours then processed by Rood and Riddle Equine Hospital’s laboratory using H & E staining. Histologic evaluation was performed by a boarded Theriogenologist and Pathologist blinded to treatment. Histologic assessment of fibrosis (Trichrome) and Biopsy grading [12] was performed and recorded. Briefly, the biopsy grading categorizes the endometrial sample based on the degree of inflammation and fibrosis and assigns a score of either I (> 80% probability to carry a pregnancy to term), IIa (50 to 80% probability to carry a pregnancy to term), II (approximately 50% probability to carry a pregnancy to term), IIb(10 to 50% probability to carry a pregnancy to term), or III(< 10% probability to carry a pregnancy to term).
2.8 Statistics
Continuous data were analyzed to determine the main effects of group, day and their interaction using the SAS (version 9.4) MIXED procedure with a repeated statement for day and a random effects statement using mare as a covariate. When a significant (P ≤ 0.05) group by day interaction existed, a pairwise comparison was made between groups on the same day. Data are expressed as Least Squares means ± standard error.
Categorical data was analyzed using the SAS LOGISTIC procedure using generalized logits with treatment group and day as independent variables and their interaction in the model. When an interaction was present, the SAS FREQ procedure was used to compare proportions on a given day using a chi square test. Significance was set at P ≤ 0.05.
- Results
The leukogram found a tendency (P = 0.06) for control mares to have an elevated SAA compared to treatment mares; however, there was no significant interaction between group and day. The remainder of the parameters on leukogram had no significant difference between treatments and no treatment by day interaction (Table 1). The serum biochemistry panel had no effect of treatment on any of the parameters examined. There was a significant treatment by day interaction for creatine kinase (P = 0.03) and a pairwise comparison found no significant difference on Day 0 and on Day 4, a tendency (P = 0.09) for creatine kinase to be increased in the control group compared to treatment group. There was a significant treatment by day interaction for calcium (P = 0.04) and a pairwise comparison found no significant difference between treatments on Day 0 and Day 4. There was no significant treatment by day interaction for the other parameters examined (Table 2). Additionally, there was no significant difference between treatments and no significant treatment by day interaction for serum Progesterone concentrations.
There was no significant difference (x 2 > 0.25) between treatments in the frequency of a CL being present at any time point and there was no significant difference on the diameter of the largest follicle at any time in the study, despite mares being used at various time points in the estrous cycle. There was no significant difference between treatments in edema score, presence of fluid or cervical score and no significant treatment by day interaction for any of these parameters.
Cytology performed on Day 0 of the study found all control mares (n = 3) and all treatment mares (n = 9) had a normal cytology (< 2 PMNs/hpf). Cytology result from Day 21 found all control mares (n= 3) had a normal cytology, while 8 treatment mares had a normal cytology and 1 mare had a cytology classified as severe (> 5 PMNs/hpf).
Culture performed on Day 0 found one control mare grew a mixed culture of Beta-Streptococcus and E. coli. All other mares had a negative Day 0 culture. On the Day 21 culture, one control mare had slight growth of an unidentified bacteria and one treatment mare grew a Beta-Streptococcus, all other mares had a negative culture.
There was no significant difference between treatments (P = 0.99) in biopsy category and no treatment by day interaction (P = 1.00; Table 3). When the biopsy was examined for the presence of inflammation, there was no effect of treatment; however, there was a treatment by day interaction (P = 0.0347). There was no difference in the frequency of histologic uterine inflammation between groups (x 2 = 0.21) on Day 0. There was a tendency towards significance (x 2 = 0.07) in an increased frequency of histologic uterine inflammation in control mares compared to ADABP treated mares at Day 21. No significant differences were found between groups or interaction of group by day for the pattern of inflammation, presence of periglandular fibrosis, pattern of periglandular fibrosis, number of cell layer surrounding fibrotic glands, number of fibrotic nests, the presence of edema or lymphatic dilation on endometrial histology.
- Discussion
Our study found no negative systemic or uterine effects from intrauterine injection of ADABP. There was a tendency for lower creatinine kinase in treatment mares compared to control mares, while all other parameters on the leukogram and serum biochemistry panel were not different. Adipose derived mesenchymal stem cells have been found to decrease serum creatinine kinase-myocardial band in rats subjected to ischemic reperfusion injury [13]. Whether ADABP influenced creatine kinase is unclear since it is unknown how much ADABP could enter circulation from an intrauterine injection site. The tendency for lower creatine kinase in treatment mares raises the question, does ADABP diffuse into circulation and affect myocytes.
There was no difference in cytology or culture result between groups; however, these are relatively insensitive measures of inflammation and endometrial biopsy is a better method of evaluation [14]. While there was no difference in cytology score, the one treatment mare with a severe cytology classification was the mare which grew a Beta-strep on culture. Since this was on Day 21, it is possible this could have been introduced during a prior procedure. There was no difference between groups on categorization of biopsies; however, there was a tendency for ADABP mares to have less uterine inflammation present on Day 21. This is consistent with research examining the immunomodulatory potential of MSC. Mesenchymal stem cells have been found to interact with all cells of the immune system, where they gently steer the immune reaction towards an anti-inflammatory response [15]. Amnion-derived acellular bioscaffold products appear to produce similar local responses. In diabetic mice skin wound models, cellular amniotic membrane incorporating exosomes accelerated wound healing while decreasing inflammation [16]. The reduced inflammation in endometrial tissue in treated mares is consistent with observations in other organ systems in different species.
There are limitations with this study, namely the small sample size which could result in insufficient power to detect differences between treatments. A power analysis was not performed as this study was to gain preliminary data.
Although further research is needed to determine if intrauterine injection of ADABP can reduce endometrial fibrosis or improve endometrial healing, this study suggests that intrauterine injection of ADABP has no deleterious local or systemic effects.
Author’s Declaration of Competing Interests
None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.
Ethical Animal Research
The authors have made a full and honest effort to adhere to all the ethical “Duties of Authors” as listed on the “Elsevier publish ethics website”.
Sources of Funding
Funding for the product, and horse care and hysteroscopy was provided by AniCell Biotech.
References
[1] Fernández-Colino A, Iop L, Ventura Ferreira MS, Mela P. Fibrosis in tissue engineering and regenerative medicine: treat or trigger? Advanced Drug Delivery Reviews. 2019;146:17-36.
[2] Langer R, Vacanti JP. Tissue Engineering. Science. 1993;260:920-6.
[3] Wissing TB, Bonito V, Bouten CVC, Smits AIPM. Biomaterial-driven in situ cardiovascular tissue engineering—a multi-disciplinary perspective. npj Regenerative Medicine. 2017;2:18.
[4] Ribitsch I, Oreff GL, Jenner F. Regenerative Medicine for Equine Musculoskeletal Diseases. Animals. 2021;11:234.
[5] Koch TG, Berg LC, Betts DH. Current and future regenerative medicine – principles, concepts, and therapeutic use of stem cell therapy and tissue engineering in equine medicine. Can Vet J. 2009;50:155-65.
[6] Jang HY, Myoung SM, Choe JM, Kim T, Cheon YP, Kim YM, Park, H. Effects of Autologous Platelet-Rich Plasma on Regeneration of Damaged Endometrium in Female Rats. Yonsei Med J. 2017;58:1195-203.
[7] Segabinazzi L, Carmo C, Fonseca-Alves C, Landim F, Canisso I, Alvarengo M. Endometrial injection of mesenchymal stem cells may revert endomertiosis in mares. Society for Theriogenology. Bellvue, WA2022. p. 266.
[8] Lehmann J, Ellenberger C, Hoffmann C, Bazer FW, Klug J, Allen WR, Sieme, H, Schoon, H-A. Morpho-functional studies regarding the fertility prognosis of mares suffering from equine endometrosis. Theriogenology. 2011;76:1326-36.
[9] Mamede AC, Carvalho MJ, Abrantes AM, Laranjo M, Maia CJ, Botelho MF. Amniotic membrane: from structure and functions to clinical applications. Cell Tissue Res. 2012;349:447-58.
[10] Fumuso E, Giguère S, Wade J, Rogan D, Videla-Dorna I, Bowden RA. Endometrial IL-1β, IL-6 and TNF-α, mRNA expression in mares resistant or susceptible to post-breeding endometritis: Effects of estrous cycle, artificial insemination and immunomodulation. Veterinary Immunology and Immunopathology. 2003;96:31-41.
[11] Riddle WT, LeBlanc MM, Stromberg AJ. Relationships between uterine culture, cytology and pregnancy rates in a Thoroughbred practice. Theriogenology. 2007;68:395-402.
[12] Kenney R, Doig P. Equine endometrial biopsy. In: Morrow D, ed. Current Therapy in Theriogenology: Diagnosis, Treatment, and Prevention of Reproductive Diseases in Small & Large Animals. 2nd ed. Saunders, 1986:723–729.
[13] Cui X, He Z, Liang Z, Chen Z, Wang H, Zhang J. Exosomes From Adipose-derived Mesenchymal Stem Cells Protect the Myocardium Against Ischemia/Reperfusion Injury Through Wnt/β-Catenin Signaling Pathway. J Cardiovasc Pharmacol. 2017;70:225-31.
[14] Diel de Amorin M, Gartley C J, Foster R A, Hill A, Scholtz E L, Hayes A, Chenier T S. Comarison of clinical signs, endometrial culture, endomterial cytology, uterine low-volume lavage and unterine biopsy and combinations in the diagnosis of Equine endometritis. J Eq Vet Sci. 2016; 44: 54-61.
[15] van den Akker F, Deddens JC, Doevendans PA, Sluijter JPG. Cardiac stem cell therapy to modulate inflammation upon myocardial infarction. Biochimica et Biophysica Acta (BBA) – General Subjects. 2013;1830:2449-58.
[16] Xiao S, Xiao C, Miao Y, Wang J, Chen R, Fan Z, et al. Human acellular amniotic membrane incorporating exosomes from adipose-derived mesenchymal stem cells promotes diabetic wound healing. Stem Cell Research & Therapy. 2021;12:255.
