Transrectal Ultrasound Research Articles

DETERMINATION OF OVULATION TIME IN GAYO MARES BASED ON IMAGE OF PREOVULATORY FOLLICLE GROWTH

This study aimed to obtain the ultrasound image of preovulatory follicle growth in gayo mares to predict the ovulation time. This study used three Gayo mares and one gayo stallion for teaser aged 5-10 years and weighed 200-250 kg. Preovulatory follicle growth was observed using transrectal ultrasonography which was performed when the dominant follicle size was ≥ 3.5 cm. Observations were made for 3 consecutive estrous periods on gayo mares at relatively the same time. The absence of previous follicular appearance and the presence of hypoechoic area in the same ovary indicate that ovulation has occurred. The result revealed that the diameter of preovulatory follicle of gayo mares was 4.02±0.32 to 5.05±0.04 cm. There was no significant follicle growth in the last 48 hours before ovulation and the preovulatory follicle shape altered from spherical to pearshape. Many small follicles (2 cm in diameter) were observed in peripheral area of the ovary indicated the recruitment of follicles which form the first follicular wave. To conclude, the alteration in the size and pearshape of preovulatory follicle and the recruitment of small follicles can be used as a marker to determine the time of ovulation on gayo mares.

The Pattern of Prostate Cancer Screening and Diagnosis among Indonesian Urologists: A Questionnaire Survey

Background: Prostate cancer (PCa) screening and diagnosis are mandatory to deliver optimal management in the early phase. Even though it has been discussed in many guidelines, the implementation of PCa screening and diagnosis in Indonesia remains unknown. This study aims to evaluate the pattern of PCa screening and diagnosis among Indonesian urologists and their adherence to guidelines. Methods: This cross-sectional study was conducted between February and July 2019. Respondents were Indonesian urologists registered as members of the Indonesian Urological Association (IUA) and had already practiced for at least six months. Data were collected using questionnaires, which were distributed at a national urology symposium and electronically via Google Form. Data were presented descriptively, and all data were processed using SPSS version 23. Results: Of 458 urologists, 195 (42.6%) gave full responses. Most of the respondents, 181 (92.8%) urologists, used the IUA guidelines. Among the 103 (52.8%) respondents who performed screening, nearly half (42.7%) agreed to screen patients aged ≥ 50 years or ≥ 45 years with a family history of PCa. Moreover, 76.8% would repeat screening annually, and 35.6% would stop when the patient’s age reached 70 years old. Digital rectal examination (DRE) was frequently performed for screening (74.5%), while prostate-specific antigen (PSA) tests were only performed in 52.3% of cases. The PSA test was available in 74.8% of hospitals. Transurethral resection of the prostate (TURP) was still used by 67.2% of respondents for diagnosis. Only 52.3 % of participants used transrectal prostate biopsy for diagnosis, using anesthesia (78.1%) during the procedure, and increased PSA level (98%) as its indication. However, Transrectal Ultrasound (TRUS) was only available in 49% of hospitals. This study found that Indonesian urologist adherence level toward guidelines was 63.3% (9–100%). Conclusions: PCa screening and diagnosis are still varied among Indonesian urologists, which might arise due to the different availability of diagnostic modalities.

Comparative Prostate Cancer Detection Rate of Transrectal Ultrasonography Guided versus Finger Guided Prostate Biopsy

Background: Prostate biopsy was used to diagnose and establish a therapy for prostate cancer (PCa). Instead of using conventional finger-guided prostate biopsy (FGPB) to approximate prostatic architecture, transrectal ultrasonography-guided biopsies of the prostate (TRUSGB) have become more popular nowadays because of offer direct visualization. However, the lack of availability of transrectal ultrasound probes in less-developed regions raises concerns regarding the need to diagnose patients with PCa. Moreover, different conclusions have been found from prior studies that examined the efficacy of both methods. This study aims to compare the accuracy of TRUSGB to FGPB in prostate cancer.. Methods: This study was done retrospectively from 50 medical records of PCa in the Urology Division of Sardjito Hospital from January 2009 until December 2013. Patients’ age, PSA value, digital rectal examination, and histopathological examination were analyzed. Results: The mean age was 65.18 ± 7.76 years in FGPB and 67.52 ± 10.79 years in TRUSGB group. The median PSA was 65.01 (range: 16.33–114.72) ng/mL in FGPB and 71.75 (range: 19.86– 123.47) ng/mL in TRUSGB. Abnormal DRE was found in 75.75% of patients in FGPB group and 70.58% in TRUSGB. Comparable cancer detection rates were found in the FGPB and TRUSGB groups (45.45% vs. 52.94%) (p = 0.136).Conclusions: The cancer detection rates for FGPB and TRUSGB procedures are comparable. This supports using FGPB as the first-line diagnostic technique, especially in low-resource situations where ultrasonography is unavailable.

A novel multi-modality imaging phantom for validating interstitial needle guidance for high dose rate gynecological brachytherapy.

To design, manufacture, and validate a female pelvic phantom for multi-modality imaging (CT, MRI, US) to benchmark a commercial needle tracking system with application in HDR gynecological (GYN) interstitial procedures. A GYN needle-tracking phantom was designed using CAD software to model an average uterus from a previous patient study, a vaginal canal from speculum dimensions, and a rectum to accommodate a transrectal ultrasound (TRUS) probe. A target volume (CTVHR ) was designed as an extension from the cervix-uterus complex. Negative space molds were created from modeled anatomy and 3D printed. Silicone was used to cast the anatomy molds. A 3D printed box was constructed to house the manufactured anatomy for structural integrity and to accommodate the insertion of a speculum, tandem, needles, and TRUS probe. The phantom was CT-imaged to identify potential imperfections that might impact US visualization. Free-hand TRUS was used to guide interstitial needles into the phantom. The commercial tracking system was used to generate a 3D US volume. After insertion, the phantom was imaged with CT and MR and the uterus and CTVHR dimensions were verified against the CAD model. The manufactured phantom allows for accurate visualization with multiple imaging modalities and is conducive to applicator and needle insertion. The phantom dimensions from the CAD model were verified with those from each imaging modality. The phantom is low cost and can be reproducibly manufactured with the 3D printing and molding processes. Our initial experiments demonstrate the ability to integrate the phantom with a commercial tracking system for future needle tracking validation studies.

Rectovaginal endometriosis with nodular smooth muscle metaplasia diagnosed via transrectal ultrasound-guided fine-needle aspiration cytology: An underused minimally invasive diagnostic technique?

Rectovaginal endometriosis is a severe variant of deeply infiltrating endometriosis. Laparoscopic assessment with tissue sampling remains the gold standard for diagnosis of endometriosis. However, transvaginal (TVUS) and transrectal ultrasound (TRUS) have been shown to be especially helpful in the diagnosis of deep endometriosis. We present the case of a 49-year-old female with menorrhagia, dysmenorrhea, and constipation. Upon pelvic examination, an incidental mass was palpated. A computed tomography (CT) scan revealed an anterior rectal wall mass and colonoscopy was non-diagnostic. Further work-up with MRI showed a 3.9 cm mass centered within the upper rectovaginal septum. TRUS guided fine-needle aspiration (TRUS-FNA) revealed cohesive epithelial cell groups without significant cytologic atypia and a second population of bland spindle cells. Cell block slides showed glandular epithelium with associated stroma that exhibited endometrial morphology and immunophenotype. Nodular fragments of spindle cells with smooth muscle immunophenotype and fibrosis were also present. The overall morphologic findings were consistent with rectovaginal endometriosis with nodular smooth muscle metaplasia. Medical management with nonsteroidal aromatase inhibitor with radiologic follow-up was selected. Rectovaginal endometriosis represents a type of deep endometriosis usually associated with severe pelvic symptoms. Metaplastic smooth muscle cells are a frequent component of endometriosis in the rectovaginal pouch with nodular growth and may present diagnostic challenges. TRUS-FNA is a minimally invasive procedure that can provide an accurate diagnosis of endometriosis, even in this variant of deep infiltrating disease.

Correlation of resistive index of prostatic capsular artery to clinical symptoms in men with chronic prostatitis/chronic pelvic pain syndrome.

To evaluate the relation between resistive index (RI) of prostatic capsular arteries by transrectal Doppler ultrasonography in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) and its correlation with lower urinary tract symptoms, erectile dysfunction and premature ejaculation parameters of CP/CPPS. In total, we included 68 patients presenting with chronic prostatitis/chronic pelvic pain syndrome. We formed two groups, as Group 1 of 35 patients with a RI ≥ 0.7 and Group 2 of 33 patients with RI < 0.7. All patients were assessed with International Prostate Symptom Score (IPSS), International Index of Erectile Function (IIEF-5), premature ejaculation diagnostic tool (PEDT) and National Institutes of Health Chronic Prostatitis Symptom Index (CPSI). In addition, the RI of the prostate capsular artery was measured in all patients using Doppler ultrasound. Statistical analyses were performed with SPSS version 18. A p value < .05 was considered significant. Demographic characteristics were similar in the two groups. IPSS was 11.3 ± 6 in Group 1 and 9.7 ± 5.3 in Group 2. IIEF-5 was 18.6 ± 2 in Group 1 and 20.4 ± 2.3 in Group 2. PEDT was 12.4 ± 5.6 in Group 1 and 11.2 ± 4 in Group 2. CPSI (total) was 19.3 ± 12.3 in Group 1 and 10.6 ± 7.7 in Group 2. There was significant statistical difference in IPSS, IIEF-5, and CPSI between the two groups (p < .001, p < .001, p < .001 respectively). However, we found no significant difference in PEDT between the two groups (p = .19). There is a significant correlation between the lower urinary tract symptoms and erectile dysfunction parameters and the RI of the prostatic capsular artery in CP/CPPS and RI is an effective and noninvasive method to assess the severity of the disease.

Intra-prostatic gold fiducial marker insertion for image-guided radiotherapy (IGRT): five-year experience on 795 patients

IntroductionProstate cancer is the second most commonly diagnosed cancer in males. The use of intra-prostatic fiducial markers (FM) for image-guided radiotherapy (IGRT) has become widespread due to their accuracy, relatively safe use, low cost, and reproducibility. FM provides a tool to monitor prostate position and volume changes. Many studies reported low to moderate rates of complications following FM implantation. In the current study, we present our five years’ experience regarding the insertion technique, technical success, and rates of complication and migration of intraprostatic insertion of FM gold marker.MethodsFrom January 2018 to January 2023, 795 patients with prostate cancer candidate for IGRT (with or without a history of radical prostatectomy) enrolled in this study. We used three fiducial markers (3*0.6 mm) inserted through an 18-gauge Chiba needle under transrectal ultrasonography (TRUS) guidance. The patients were observed for complications up to seven days after the procedure. Besides, the rate of marker migration was recorded.ResultsAll procedures were completed successfully, and all patients tolerated the procedure well with minimal discomfort. The rate of sepsis after the procedure was 1%, and transient urinary obstruction was 1.6%. Only two patients experienced marker migration shortly after insertion, and no fiducial migration was reported throughout radiotherapy. No other major complication was recorded.DiscussionTRUS-guided intraprostatic FM implantation is technically feasible, safe, and well-tolerated in most patients. The FM migration can seldom occur, with negligible effects. This study can provide convincing evidence that TRUS-guided intra-prostatic FM insertion is an appropriate choice for IGRT.

Time of insemination in relation to pregnancy rates in beef cattle after oestrus detection with automated activity monitoring system

BackgroundThe timing of artificial insemination is critical to achieve acceptable results in cattle production systems. Over the past 60 years the length and expression of oestrus in dairy cattle has altered. Recent studies have indicated the optimal timing for insemination after the commencement of oestrus may now be earlier than traditional recommendations in beef cattle, as is the case in dairy cattle. The aim of the current study was to evaluate the effect of time from onset of oestrus [as determined by an automated activity monitoring system (AAMS)] to artificial insemination (AI) on pregnancy outcome in Norwegian beef cattle. Five commercial beef suckler herds participated in a cohort study by providing data on the time of AAMS alarm and time of AI. Blood sampling on the day of AI was performed and serum progesterone concentration measured. Pregnancy detection was performed by transrectal ultrasonography and aging of the fetus performed when necessary. A mixed logistic regression model was fitted to study the effect of time from AAMS alarm to AI on pregnancy outcome. Time categories used in the model were < 12 h, 12–24 h, and > 24 h.ResultsAI periods (n = 229) with serum progesterone concentration < 1 ng/mL were available for analysis. Overall pregnancy risk per AI for the whole study period was 65.5%, with an inter-herd variation from 10 to 91%. Median time elapsed from AAMS alarm to AI was 17.75 h. Herd affected pregnancy outcome (P = 0.001), while breed and parity status (heifer/cow) did not. The time category closer to AAMS alarm 0–12 h showed a numerically lower pregnancy risk as compared to the baseline group which had AI 12–24 h after onset of oestrus.ConclusionThis study found no evidence to support a change in the recommended timing of AI in beef suckler cows.

A Prospective Comparison of Transrectal Standard, Cognitive, Transperineal Fusion, and Mapping Prostate Biopsy for Cancer Detection.

Purpose: The aim of this research was to compare the clinically significant prostate cancer (csPCa) detection rate (International Society of Urological Pathology [ISUP] ≥2) for the four biopsy methods: transrectal ultrasound-guided biopsy (TRUS-GB), cognitive transrectal biopsy (COG-TB), fusion transperineal biopsy (FUS-TB), and transperineal template mapping biopsy (TPMB). Materials and Methods: The inclusion criteria were as follows: prostate-specific antigen (PSA) >2 ng/mL, and/or positive digital rectal examination (DRE), and/or suspicious lesion on transrectal ultrasound (TRUS) and Prostate Imaging Reporting and Data System (Pi-RADS) v2.1 ≥ 3 score. In total, 102 patients were enrolled in the study. Biopsies were performed by two urologists. In a single procedure, the first urologist performed a FUS-TB and TPMB followed by second urologist who performed TRUS-GB and COG-TB. All specimens were obtained within a single procedure. Results: The csPCa detection rate and overall cancer detection rate (CDR) per patient were comparable among the respective biopsy methods (p > 0.05). Compared with other biopsy methods, a lower clinically insignificant prostate cancer (cisPCa) was detected using COG-TB (p = 0.004). The positive cores percentage ratio (p < 0.001) as well as positive cores containing csPCa percentage ratio (p < 0.001) significantly increased for the targeted biopsy methods. The median maximum cancer core length (MCCL; p = 0.52) as well the median for the MCCL of csPCa (p = 0.47) did not differ significantly among the respective biopsy methods. Concordance of the Gleason scores between biopsy and postprostatectomy pathology did not differ significantly among biopsy methods (p = 0.87). For TRUS-GB, FUS-TB, and TPMB, the common predictive factors for csPCa were positive DRE, suspicious lesion on ultrasound and Pi-RADS 5. As for COG-TB, the only predictor was Pi-RADS 5. Conclusion: The targeted methods did not show an increase in detection of csPCa and overall CDR over systematic ones in patients with Pi-RADS ≥3. A lower cisPCa was detected using COG-TB in comparison with the other methods. The sampling efficiency increased for the targeted biopsy methods, which used only a proportion of positive cores and cores containing csPCa. There was no statistical difference in histology concordance among the biopsies. One common predictive factor of increased csPCa detection for all biopsy methods was Pi-RADS 5.

Combination of fusion guided multiparametric MRI-transrectal US with systematic biopsy of the prostate for the detection of clinically significant prostate cancer: A prospective single-center study.

To investigate the diagnostic efficacy of fusion guided multiparametric MRI (mpMRI)-transrectal ultrasound (TRUS) biopsy versus systematic biopsy of the prostate in patients with suspicion of prostate cancer. A total of 185 patients with PI-RADS 3 lesions or higher underwent fusion guided targeted and systematic prostate biopsy. Histology of samples was correlated with PI-RADS score and biopsy method for each patient. A total of 81/185 (43.8%) cases positive for cancer were detected; 23/81 (28.4%) cases with clinically insignificant prostate cancer-insPCa and 58/81 (71.6%) cases with clinically significant prostate cancer-csPCa. There was a statistically significant difference in the overall detection of adenocarcinomas between methods (p = .035, McNemar test). Moreover, there was a statistically significant difference in the detection of insPCa between the two methods (p = .004, McNemar test). Systematic biopsy detected 13 patients with insPCa more (14.4%) than the targeted biopsy method. However, there is no statistical difference in the detection rate of csPCa between the two methods (p = 1, McNemar test). When both techniques were combined more cases of csPCa were detected. The combined implementation of fusion-guided targeted mpMRI-TRUS and systematic biopsy of the prostate provides higher detection number of csPCa, compared to each method alone. The potential of fusion-guided mpMRI-TRUS biopsy of the prostate needs to be further assessed since each method has its limitations; therefore, systematic prostate biopsy still plays an important role in clinical practice.

Effect of mid-diestrus oxytocin treatment on early pregnancy in the mare

Progesterone supplementation in early pregnancy is commonplace in equine reproduction. Recently, there has been some controversy questioning the beneficial effects of routine progesterone administration and rising concerns of medication exposure as an occupational hazard for humans. Although premature luteal regression and resulting low plasma progesterone concentrations are rare in the mare, select cases benefit from luteal support. Duration of luteal function can be prolonged following oxytocin administration during mid-diestrus in the non-pregnant mare (Vanderwall.et.al. Journal of Equine Veterinary Science. 2012; 32:536-541). The aim of this field study was to investigate the effects of oxytocin administration on early pregnancy in the mare. Thoroughbred mares (n = 39; age 5 to 19 years) were enlisted and bred over 55 estrous cycles. Prior to natural breeding, uterine cultures were obtained, and mares were assigned to 1 of 2 groups: Oxytocin treatment (OT); oxytocin 60 units, intramuscularly, once daily, days 7-14 post-ovulation; or Control (CT); no treatment. Mare reproductive cycles were monitored by transrectal palpation and ultrasonography. Ovulation was documented and defined as day 0. Pregnancy diagnosis was performed by transrectal ultrasonography day 14 post-ovulation. If pregnancy was confirmed, a blood sample was collected and assayed for plasma progesterone concentration. Mares that did not conceive were administered prostaglandin F2αanalogue (cloprostenol; 250mcg once intramuscularly) at day 16 post-ovulation. These mares were rebred and treatment was determined by the stage in the study; therefore, some mares underwent the same treatment more than once. Subsequent pregnancy examinations were performed on days 28 and 45 of pregnancy. Data were analyzed using two-tailed Fisher's Exact tests for small counts, with significance defined at p < 0.05. First and per-cycle pregnancy rates for OT and CT were 90.9 and 91.3% and 68.2 and 55.1%, respectively. Median progesterone concentrations for OT were 6.9 and 7.0 ng/ml for CT. There were no differences between OT and CT groups for first (p = 0.165) or per cycle (p = 0.289) pregnancy rates, days 28 and 45 pregnancy rates (p = 1.0), or progesterone concentrations (p = 1.0). In this population of mares, oxytocin administration during mid-diestrus in early pregnancy did not induce a negative effect on pregnancy rate nor a positive effect on progesterone concentration. Whilst the study design did not assess the effect of OT on the inter-ovulatory interval (IOI), the authors’ hypothesize that the IOI would be increased, due to prolonged luteal function, in the non-pregnant OT mare if prostaglandin F2α analogue was not administered. Further investigation in this area may reveal potential beneficial effects of oxytocin administration in early pregnancy.

Evaluation of cortisol, thyroxine (T3) and triiodothyronine (T4) concentrations in preovulatory follicles in jennies.

Reference values of follicular fluid components at different stages of follicular development in jennies are unknown. It is important to estimate such values because the biochemical composition of follicular fluid provides a suitable microenvironment for oocyte development, growth and maturation. The experimental protocol was carried out in the Equine Reproduction Clinic of the FMVZ-UNAM and was approved by an Institutional Sub-committee for the Care and Use of Experimental Animals (SICUAE.MMVZ-2020/4-2). Seven healthy female donkeys “Mexican burro” with average body condition score of 3 on a scale of 1 to 5 (Svendsen et al. The professional Handbook of the Donkey. 2008), were monitored by transrectal ultrasonography through the Veterinary Digital Diagnostic Ultrasound System 5 MHz (Model, DUS 60 VET) with a multi-frequency linear transducer. The ovum pick up (OPU) by transvaginal aspiration (TVA) was performed in a total of 70 preovulatory follicles, 10 for each jenny using a real- time B- mode ultrasound vaginal 6.5 MHz (Sonovet 600) with curvilinear transducer and 12 gauge (12 G) needle. Follicular fluid components were measured and analyzed by Luminex Multiplex Assays at the Universidad Nacional Autónoma de México. The relationship between cortisol, thyroxine (T3) and triiodothyronine (T4) concentrations in preovulatory follicles and follicular size was assessed. The preovulatory follicles are classified in three groups accordingto the diameter: small (27-30 mm), medium (31-35 mm) and large (36-38 mm). Concentrations of cortisol were lower in the large follicles group (18.8 ng/ml) compared with small (721.44 ng/ml) and medium follicles (361 ng/ml). The concentrations of thyroxine were higher in the medium follicle group (49 ng/ml) than in the small (21ng/ml) and large follicle groups (15.9 ng/ml). Finally, the concentration of triiodothyronine was higher in the small follicles group (1043 ng/ml) followed by the medium (545 ng/ml) and the lowest concentration was in the large follicles group (22 ng/ml). It is concluded that thyroxine concentrations are lower than T4 concentrations in preovulatory follicles irrespective of size as reported for human follicular fluid (Wakim. et. al. Fertil Steril 1993; 59(6) 1187–1190). Furthermore, no relationship was found between preovulatory follicular size and thyroxine hormone concentrations. It has been suggested that cortisol and triiodothyronine concentrations are related to preovulatory follicle size. Cortisol concentrations are higher in preovulatory follicles that in subordinate follicles and cortisol is a mediator of apoptosis in granulosa cells, differentiation, development and maturation of tissues, specifically in the oocyte (Acosta & Miyamoto. Anim Reprod Sci 2004; 82:127-140, Scarlett. et al. Dom Anim Endocrinol 2016; 59:11-22). The role of cortisol within the different sizes of preovulatory follicles is unknown.

Obtaining follicular fluid through ovum pick up to determine steroidal concentrations of preovulatory follicles in donkeys

There is currently no evidence of reference values for steroid hormones in the follicular fluid of the species Equus asinus during the estrous cycle. The present study aimed to determine the hormonal concentrations of estradiol and progesterone in the follicular microenvironment to establish a better understanding of the role they play in the donkey and their importance in reproductive physiology of this species.. The study was carried out at the FMVZ-UNAM Equine Reproduction Clinic authorized by the Institutional Sub-committee for the Care and Use of Experimental Animals (SICUAE.MMVZ-2020/4-2). Seven Creole donkeys aged between 3 and 9 years, clinically healthy, weighing between 128 and 206 kg and with a body condition of 3 on a scale of 0 to 5, were used. In order to identify preovulatory follicles with a diameter from 27 to 38 mm (depending on the specimen), each donkey was monitored by ovarian palpation and transrectal ultrasonography. Each follicle was standardized based on its size and previous records over three estrous periods where the optimal follicular diameter was observedprior to ovulation. Subsequently, the follicular fluid was recovered using the transvaginal ultrasonography-guided follicular aspiration technique and stored at -20°C until determination of hormones by ELISA (Estradiol ELISADRG, EIA-2693 and Progesterone ELISA DRG, EIA-1561). Prior to the analysis of each sample, follicular fluid was diluted in PBS 0.1M, pH 7.2 from 1:1000 to 1:10,000. Each sample was measured in duplicate. A total of 70 follicular aspirations were obtained during 10 estrous cycles from each donkey, with an average interval of 15 to 17 days. data in Table 1 represent the mean ± standard error of the mean of 10 observations per animal. The results show for first-time concentrations of estradiol and progesterone in the donkeys, which will serve as a reference in subsequent studies.

Utilizing an immunostimulant (MCWF; Settle) to hasten the degradation of endometrial cups

Placentation in the horse begins at approximately 35 days of gestation, when the chorionic girdle leads to the development of endometrial cups. If embryonic loss occurs in the presence of endometrial cups, return to cyclicity is delayed due to the retainment of these structures, continued production of equine chorionic gonadotropin (eCG), and prolonged progesterone output from accessory corpus lutea. It is believed that natural degradation of endometrial cups is governed by IFNγ-producing lymphocytes at 100-120 days of gestation, but external stimulation of this process has been relatively unsuccessful. Recently, we observed an increase in IFNγ following the use of an immunostimulant (Mycobacterium cell wall fraction (MCWF); Settle) in the postpartum mare. Therefore, we hypothesize that MCWF administration following embryonic loss will increase IFNγ production, hasten the degradation of the endometrial cups, and decrease the time to return to cyclicity in mares. To investigate this, 16 mares were aborted with PGF2α at 40-45d of gestation. Following abortion, mares were divided into groups: MCWF-Treated (6mg MCWF in 20mL LRS; n=10) and Control (20mL LRS; n=6). Between 45-50 days of gestation, mares were assessed via trans-rectal ultrasonography for ovarian function and endometrial cup distribution. Hysteroscopic-guided injection of endometrial cups was performed, with 1mL placed into eachvisible endometrial cup, and the remaining volume injected peripherally. This treatment was repeated 7 days later. Mares were monitored for rectal temperature and serum was taken to assess eCG and IFNγ concentrations. Additionally, transrectal ultrasonography was performed to monitor cyclicity. Statistics were performed utilizing SAS9.4 with a general linear model of repeated measures applied for all temperature, eCG, and cytokine data. A chi-squared test was performed to assess return to cyclicity. An overall decrease in eCG concentrations was noted in the MCWF-treated group (P<0.0001) when compared to controls. This suppression was significant 7 days after onset of treatment and remained suppressed 35 days following treatment. This coincided with an increase in peripheral IFNγ in the MCWF-treated group compared to controls (P<0.01). A higher percentage of MCWF-treated mares returned to cyclicity in comparison to controls (8/10 vs. 2/6; P=0.06), and a pre-ovulatory follicle was noted 23±4 days after onset of treatment. To our knowledge, this is the first report of a successful treatment for degradation of endometrial cups following abortion, by hastening the natural immune response to these structures. This caused mares to return to cyclicity within a month following treatment onset, allowing for repeat attempts at a successful pregnancy within the confines of the breeding season.

Endometrial immune cells at the time of implantation in mares – preliminary results

Implantation in mares occurs approximately between days 35 when the chorionic girdle starts to invade endometrium and day 45 when the allantochorion is more firmly attached to endometrium. Immune cells are known to be present in the endometrium over this period but their phenotype and role in implantation in the mare is poorly understood. The aim of this study was to phenotypically characterize subpopulations of immune cells in the endometrium around the time of implantation using single cell RNAseq. Clinically healthy mares were monitored via transrectal ultrasonography. Ovulation was induced with hCG when the follicle diameter was ≥35mm and uterine edema was present. Mares were inseminated with cooled semen. Morphologically normal embryos were flushed by non-surgical lavage at day 33 (n=2) and day 42 (n=2) post ovulation, endometrial biopsies obtained, and serum samples collected for progesterone assays. Biopsies were digested to obtain single cell suspensions, PBMC enriched over Ficoll and then 10 000 cells per sample were processed for scRNA-Seq on the 10X Genomics Chromium platform (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1; Dual Index). Sequencing was performed at the initial depth of ∼20 000 reads/cell. Data was analyzed with Seurat R package v4.0 and the EquCab 3.0 as the reference. Clusters were annotated using established markers of immune cells . (Patel et al. BMC biology. 2021;19:1-13).Unsupervised graph-based clustering of 53909 cells detected 17 clusters of cells that expressed 17667 genes. Cluster 3 was annotated as NK-like cells based on expression of LY49F, LY49C, GNLY, GZMA, CTSW and CD96. Cluster 4 was annotated as heterogenous T cell populations based on the expression of CD3 genes, CCL5, CD2 and CD5. Cluster 7 was assigned to macrophages due to expression of C1Q transcripts, CD68, APOE, DQ and DR transcripts, CXCL10 and CCL8. Both cluster 12 and 16 showed transcripts characteristic for neutrophils including CXCL8, IL1RW, IL1B, IL1BP, TG, RGS2, CCRL2 and PLEK. Cells in cluster 15 expressed genes characteristic both for γδT cells like CCL3, IL2RB, GLNY, GZMA, CTSW, but also Tregs as IL2R and CD80. Cells in cluster 17 expressed markers characteristic for plasma cells and B cells such as ENSECAG00000039599, TP53INP1, CD79A, MEF2C, PRDX4. Some clusters could not be annotated (cluster 1, 2, 5, 6, 8, 9, 10, 11) and are currently being screened for markers for endometrial cells and fibroblasts. In conclusion, these results indicate scRNAseq is a viable approach to simultaneously identify a large number of immune populations present in the endometrium. Deeper sequencing is needed to further differentiate and annotate subpopulations of cells within the clusters and to compare these subpopulations prior to and after chorionic girdle invasion.

Impact of ovulation inducers on ovarian artery in mares: pulsatility and resistivity indexes

A failure of vascular development in reproductive organs may be the cause of several dysfunctions during the estrous cycle and gestation. Therefore, it is important to evaluate the in vivo influences of ovarian vaso- irrigation on ovulation. The ovarian follicles need an adequate blood supply for oxygen, nutrients, and hormones, besides eliminating CO2 and other metabolites (Bruno et al. Animal Reproduction. 2009; 6, 2:371-379). Transrectal ultrasonography (US) with color, power, and spectral Doppler US has been used to study blood flow and ovarian perfusion in mares (Acosta et al. Biology of Reproduction. 2004b; 71, 2: 502-507). Studies have shown a daily increase in vascularization of the wall of the dominant follicle as it approaches the day of ovulation (Gastal et al. Animal Reproduction Science. 2007; 102, 3-4: 314-321); However, a few hours before ovulation, an abrupt decrease in blood perfusion of the follicular wall has been detected (Ginther et al. Journal of Equine Veterinary Science. 2007a; 27, 1: 20-27). The drugs most used as ovulation inducers in horses are human chorionic gonadotropin (hCG) and analogues of gonadotropin-releasing hormone (GnRH), such as deslorelin (Chavatte & Palmer Equine Veterinary Education. 1998; 10, 26-30). Using different ovulation inducers, this study aimed to evaluate changes in ovarian arteryblood flow after induction of ovulation in mares with follicles ≥ 35mm diameter. Twenty-eight estrous cycles were followed, randomly and equally distributed: GI: control mares without treatment; GII: 1000 IU IV of hCG; GIII: 750µl IV of deslorelin; GIV: 250µg IM of hysterelin. The ovarian artery pulsatility and resistivity indexes (PI and RI) were measured. Thereafter, estrous cycles were followed every 12 hours from time 0 (induction) until ovulation using B-mode US and spectral mode Doppler US of ovarian arteries. Statistical analysis was performed with ANOVA, a p-value of <0.05 was considered significant. No statistical difference was found in PI between groups (p=0.1297) of at the different evaluation times. When the times of analysis were compared separately between the groups, no statistical difference was found between 0h (p= 0.7092), 12h (p= 0.5148), 24h (p=0.2642), and 36h (p=0.582). Similarly, the RI did not differ between the groups analyzed (p= 0.1397), regardless of the time of evaluation, nor between the timepoints analyzed separately between 0h (p= 0.5049), 12h (p= 0.7334), 24h (p=0.3589) and 36h (p=0.1621). It can be concluded that although an increase in vascularization of the wall of the dominant follicle occurred, the PI and RI of the ovarian artery were not influenced by any ovulation inducer of the pre-ovulatory follicles.

Obesity and age on post-breeding endometritis in the mare

Obesity has been associated with uterine dysfunctions in the pregnant mare (Sessions-B et al, Biology of Reproduction, 2018: 99(4), 761-772). This study hypothesized that obesity itself (body condition score, BCS) affected parameters associated with post-breeding endometritis. Mares (n=17) were classified by their BCS (Henneke et al., 1983) in obese (>7, n=6) and normal-weight (<7, n=11) and age group in mature (5 to 14 years, n=9) and aged mares (15 to 21 years, n=8). Transrectal ultrasonography was performed every day and oedema score and intrauterine fluid diameter were determined at insemination and 8 hours afterwards when a low volume (50 mL) uterine flush with PBS was performed (lavage). Neutrophils were counted using a Neubauer camera and the flush was centrifuged; the supernatant was frozen for metabolite determination by spectrophotometry. Blood samples were taken at this moment also for metabolite determination. Data were analyzed with PROC MIXED (SAS) including BCS and age as fixed effects. Data not normal was log-transformed. Obese mares tended to present greater neutrophil counts than normal-weight mares (43.6±8.3 vs 28.8±5.7 × 106/mL P=0.08); aged mares had greater counts than mature mares(46.7±7.7 vs 21.0±6.3 × 106/mL P=0.02). No effect of age or BCS was found on oedema and IUF at insemination or lavage. In serum, glucose, triglycerides, albumin, and urea concentrations were unaffected. Serum cholesterol concentrations were affected by BCS (P<0.01) and tended to be affected by age (P<0.09). Serum globulin concentrations were not affected by BCS, but aged mares had greater concentrations than mature mares (P=0.05). In the intrauterine fluid, BCS did not affect albumin or glucose concentration nor did age affect them. No effect was observed in cholesterol or triglyceride concentrations in the IUF. Although urea concentrations in the IUF were not affected by BCS, aged mares had greater concentrations than mature mares (1.22±0.23 vs 0.37±0.19 mM, P=0.016). Although the number of animals is limited, data shows that obesity and age are associated with greater neutrophil counts in the uterine lumen 8 hours after insemination, suggesting an altered uterine clearance in these mares. As far as we know this is the first description of metabolites in IUF according to obesity and age, showing significantly greater urea concentrations in the aged group.

Usefulness of pulsatility and resistive index for detection of mares with and without endometritis

Endometritis, a condition which causes inflammatory or degenerative alterations of the endometrium, is one of the most serious causes of infertility or subfertility in equine reproduction, whether associated or not with the presence of infectious agents (Mattos et al. Revista Brasileira de Reprodução Animal. 2003; 27:150-156). A complementary diagnosis, carried out with the use of Doppler ultrasonography (US), will enable an assessment of local blood flow and a more accurate analysis of the physiology of the ovaries and uterus (Ginther et al. Animal Reproduction Science. 2007; 102:314-321; Ferreira et al. Ciência Rural. 2011; 41:1063-1069). The main purpose of the present study was to assess potential alterations in the uterine vasculature of mares with clinical or subclinical endometritis and mares with no endometritis. Twenty-one mares were organized into three groups: CG: without endometritis; GCE: with clinical endometritis; GSE: with subclinical endometritis. In the latter group, mares had no alteration in the B-mode US but had positive cytology and/or bacterial and/or fungal culture. Based on this, the estrous cycle was closely observed until the preovulatory follicle appeared and subsequently, samples were collected for cytological examination, bacterial and fungal culture. After the induction and detection of ovulation, B-mode transrectal US and spectral-mode Doppler US of the left and right uterine arteries near the bifurcation with internal iliac arteries was undertaken.These ultrasound examinations were performed on a daily basis, until the next ovulation so that the complete estrous cycle could be assessed. Statistical analysis was performed by means of ANOVA using the Tukey test for one factor. According to the results of this study, no statistical difference was found in the evaluation of the pulsatility index between groups of mares without endometritis (158.71±38.27) and with subclinical endometritis (166.60±41.68) and between groups with subclinical (166.60±41.68) and clinical endometritis (171.27±44.75). However, a significant statistical difference was observed in the pulsatility index between groups of mares without endometritis (158.71±38.27) and mares with clinical endometritis (171.27±44.75) (p<0.01). As for the evaluation of the resistive indices, no statistical difference was observed between the groups of mares with subclinical (0.72±0.08) and clinical (0.72±0.08) endometritis. However, a statistically significant difference was identified in the resistive index between groups of mares without endometritis (0.69±0.07) and subclinical endometritis and clinical endometritis (p<0.01). Thus, the indexes evaluated were inversely proportional to vascular perfusion. It is concluded that spectral mode Doppler ultrasonography can be used as a complementary diagnostic tool in the identification of subclinical and clinical endometritis, by evaluating vascular perfusion of the uterine arteries.

Effects of sub-physiological plasma progesterone concentration in the early luteal phase on embryonic and fetal development, pregnancy outcome and foal characteristics

High progesterone concentration in the early luteal phase supports pregnancy and postovulatory progestin treatment stimulates conceptus growth (Willmann et al., Theriogenology 2011;75,421) whereas reduced progesterone concentration changes histotroph composition (Beyer et al., Theriogenology 2019;125:236). Embryos obtained on day 34 from mares with reduced progesterone concentration were less developed than embryos from control pregnancies (Okada et al., Reprod Fert Develop 2020;32:1156). Sub-physiological progesterone concentrations in early pregnancy thus delay embryonic development at least until placentation. In this study, subsequent fetal growth, pregnancy loss and foal characteristics were investigated in pregnancies with reduced progesterone concentrations in the early luteal phase (PGF; n=7) and control pregnancies (CON; n=7) in the same mares. Estrous mares were inseminated every 48h until ovulation (day 0). Group PGF received 62.5µg of the prostaglandin F2α analogue cloprostenol daily on days 0-3 to induce sub-physiological progesterone concentrations whereas CON pregnancies remained untreated. Mares were assigned to PGF and CON treatments in alternating order and received the opposite treatment in the following year. Blood was obtained and conceptus size determined by transrectal (≤day 100) and transabdominal (>day 100) ultrasonography at regular intervals. IgG concentration was determined in colostrum at foaling and in foal plasma at 18-22 hrs after birth. Foals were weighed,measured, submitted to a clinical examination and the neutrophil / lymphocyte (N / L) ratio was determined as an indicator of maturity. Time from birth to first standing and first suckling was recorded. Data were analyzed with the SPSS statistics software, using two-way repeated measures ANOVA and χ2 analysis. Progesterone concentration was reduced in the first two weeks of PGF pregnancies (p<0.001; day 7 CON 35±3, PGF 11±2 ng/ml). All foals from CON pregnancies were healthy and mature, whereas 3/7 PGF pregnancies were lost (one embryonic death, one abortion, one non-viable foal, p=0.05). Size of the conceptus (day 49: PGF 6.6±07, CON 7.7±07 cm, p=0.006), embryo proper (day 54; PGF 4.4±0.8, CON 5.8±0.6 cm, p=0.015) and fetal orbita diameter (day 73 PGF 0.7±0.1, CON 1.0±0.1 cm, p=0.007) were smaller in PGF than in CON pregnancies. Size differences between PGF and CON pregnancies decreased over time and were no longer present at foaling. The viable PGF foals also did not differ in N / L ratio and time to standing and first suckling from CON foals In conclusion, reduced plasma progesterone concentration in the early luteal phase delays fetal growth beyond placentation and increases pregnancy loss. When viable foals are born, however, they are not compromised. Results indicate that pregnancies with sub-physiological progesterone concentrations will benefit from early progestogen substitution.

Ultrasonographic and Doppler characteristics of corpus luteum luteogenesis in Remonta Argentino jennies

The corpus luteum (CL) plays a crucial role in maintaining pregnancy and preparing the uterus for implantation of the fertilized egg. Transrectal Doppler ultrasonography of the ovary can be used as a diagnostic tool to assess the reproductive status and functionality of the CL. Our aim was to characterize size, echogenicity and degree of vascularization of the corpus luteum over five days after ovulation in Remonta Argentino jennies. Five healthy cycling jennies aged 9 to 14 years were monitored transrectally using the MyLabTMOne ultrasound (ESAOTE, InMed, Argentina) with a 5-10 MHz linear array transducer. After a preovulatory follicle was detected, jennies were monitored daily for ovulation (day 0) and ultrasonographic evaluation of the CL until day 5. Two ovulations and the respective CL formation were analyzed in each jenny (n=10 CL). Size and echogenicity were evaluated using B mode and CL vascularization using the Doppler-color function, with a pulse repetition frequency of 1.2 MHz. All ultrasound examinations were video recorded for analysis. Adobe Premiere Pro CS6® was used to collect three images from each video where the largest area of the CL and the highest Doppler signal were detected. Image J. version 1.52i software (National Institute of Health, USA) was used to analyze images and to determinethe area, number of gray pixels (echogenicity) and color area within the CL in Doppler mode. The average value from the three images was used to determine CL size (area in cm2), echogenicity (average gray pixel intensity) and the proportion of vascularized area (AV; area with blood flow/total CL area x 100). Spearman's test was used to correlate the variables and the changes recorded between days 1 and 5 of diestrus. The results obtained on each day were the following (values expressed as mean±SEM): Day 0: area 3.26±0.5; echogenicity 84.2±4.86; AV 5.54±1.75; Day 1: area 4.86±0.32; echogenicity 91.25±7.42; AV 1.84±0.26; Day 2: area 4.83±0.41; echogenicity 88.72±7.89; AV 3.51±0.81; Day 3: area 4.64±0.43; echogenicity 89.69±4.9; AV 4.54±0.67; Day 4: area 4.38±0.48; echogenicity 97.78±7.61; AV 5.8±1.06; Day 5: area 4.24±0.49; echogenicity 89.18±9.25; AV 8.66±1.15. No statistical correlation was observed either between variables or with the day of diestrus (p>0.05). Blood samples were collected daily between day 0 and day 5 of diestrus and progesterone determinations are ongoing. Our results contribute to a better understanding of CL formation in Remonta Argentino donkeys that could potentially be used to determine CL age and function.