Transvaginal Aspiration Research Articles

Safety of transvaginal aspiration of cysts in pregnancies complicated with ovarian endometrioma

Pregnancy complicated with ovarian endometrioma is a risk factor for preterm delivery and rupture or infection during pregnancy. This study aimed to clarify the effectiveness and safety of transvaginal aspiration during pregnancy for endometrioma diagnosed in the first trimester. This retrospective observational study included 8 pregnant women with endometrioma who underwent transvaginal cyst aspiration at 12-14 weeks (aspiration group) between March 2011-March 2018 and 23 pregnant women with endometrioma who refused aspiration during the same period (observation group). Characteristics of patients were compared in both groups. Safety, feasability and complications of transvaginal cyst aspiration were reported. Complications and obstetrical outcomes were reported and compared in both groups. The maximum cyst diameter was 8.9±1.5cm (mean ± standard deviation) in the aspiration group, which was significantly larger than that in the observation group (4.7±0.2cm). Four preterm deliveries (17.3%) occurred in the observation group and none in the aspiration group. The emergency cesarean section rate during delivery was 14.2% in the aspiration group and 43.7% in the observation group. The aspiration group tended to have lower rate of preterm deliveries and emergency cesarean sections, suggesting that cyst aspiration could be an effective, minimally invasive, and safe management option for endometrioma during pregnancy.

123 Efficiency of embryo production using ovum pickup oocytes recovered from stimulated and nonstimulated cows

Recovery of oocytes from live animals through ultrasound-guided transvaginal follicular aspiration (ovum pickup, OPU) is an essential element of modern livestock development. To increase the number of OPU oocytes retrieved, hormonal stimulation is broadly applied; however, the results are ambiguous. The aim of the present study was to compare recovery rate, quality of oocytes, and efficiency of invitro production of cattle embryos using OPU oocytes derived from hormonal treated and untreated cows. The study was performed in Simmental heifers at the age of 17 to 23 months. The heifers in the first group (n=7) were previously synchronized using prostaglandin F2α (Estrumate, MSD Animal Health) and gonadotrophin-releasing hormone (GnRH; Receptal, MSD Animal Health) and then underwent superstimulation with Stimofol (Reprobiol SPRL, Belgium) in a total amount of 0.25mg of FSH and 0.05mg of LH in a treatment of 6 injections on 3 consecutive days starting on Day −4 before OPU. The heifers of the second group (n=2) underwent OPU once a week for 5 weeks (10 OPU sessions). Groups of recovered cumulus–oocyte complexes (COCs) were matured in TCM-199 containing 10% fetal calf serum, 1mM Na-pyruvate, 50μg mL−1 gentamycin, 10μg mL−1 FSH, and 10μg mL−1 LH for 24h, fertilized in Fert-TALP (Tyrode’s-albumin-lactate-pyruvate) for 18 to 20h, and cultured in CR1aa medium for 7 days. All steps of IVP were performed at 38.5°C with 5% CO2 and 90% humidity. The cleavage and blastocysts rates were assessed on Day 2 and 7, respectively. In addition, Day-7 blastocysts were fixed, and the total cell number was determined using 4′,6-diamidino-2-phenylindole (DAPI) staining. The data were analysed by ANOVA using SigmaStat software package. As expected, the number of aspirated follicles per session in the first group of cows was higher than that in the second group: 14.6±1.2 vs. 6.8±0.5 (P<0.05). In total, 54 and 42 COCs were collected, which corresponds to recovery rates of 54.5±7.7 and 61.3±4.9% for stimulated and nonstimulated cows, respectively. After the quality evaluation, 36 COCs of SS heifers (67.2±1.9%) and 31 COCs of non-SS heifers (75.3±5.0%) were selected for IVP. We did not observe a significant difference in cleavage rate between two groups: 77.8% in the SS group vs. 71.0% in the non-SS group, whereas the rate of blastocyst production was higher (P<0.05) for SS heifers (25.0%) compared with non-SS animals (16.1%). The total number of cells in Day 7 blastocysts was significantly (P<0.01) higher in the first group (89.8±1.3) compared with the second group (70.6±3.2). Thus, superstimulation of heifers before OPU increases the efficiency of oocyte retrieval by ultrasound-guided transvaginal follicular aspiration as well as the developmental competence of OPU-derived bovine oocytes invitro. These studies were performed under financial support of the Russian Science Foundation (project No. 19-16-00115) and the Ministry of Science and Higher Education of Russia.

10 Segmental cervical aplasia in a mare with mosaic X-chromosome aneuploidy

Segmental cervical aplasia is a congenital Müllerian abnormality characterised by the complete or partial failure of cervical development resulting from abnormal fusion of the Müllerian ducts to the urogenital sinus. In the present case report, we describe a congenital segmental cervical aplasia in a Colombian creole mare. The mare was presented to the Animal Reproduction Clinic of Universidad Nacional de Colombia for diagnosis because the external orifice of her cervix was not detected when a uterine lavage was attempted as a therapy for detectable uterine fluid accumulation. The mare had a history of 20-day oestrous cycles confirmed by receptivity to a mature stallion and had no history of natural service or artificial insemination. A complete breeding soundness evaluation of the mare including transrectal palpation, ultrasonography, vaginoscopy, endoscopy, transvaginal aspiration of the uterine fluid and cytogenetic analysis, and an oestrous cycle follow-up were performed. Clinical and ultrasonographic evaluation of the genital tract revealed normal-size ovaries with structures suggestive of regular ovarian activity. Ovulation was confirmed by the formation of a corpus haemorrhagicum followed by a mature corpus luteum in diestrus. In addition, granular free-floating fluid material distending the uterus was detected. Upon vaginal examination, the organ ended in a blind bag with a small papilla with no evident external os cervix. Cytology of the uterine fluid obtained by transvaginal aspiration showed predominant neutrophils with diplococcus bacteria and inflammatory cells compatible with inflammatory content. Cytogenetic analysis of 134 metaphase lymphocytes showed that the mare had an abnormal karyotype [64,XX]/[63,XO]/[65,XXX] with a ratio of 45%, 45%, and 10%, respectively. G- and C-banded analysis was conducted for the X chromosome. Mosaicism of the X chromosome was diagnosed, and the observed congenital segmental cervical aplasia was proposed as the clinical consequence of the mosaicism detected. To our knowledge, this is the first case of this reproductive pathology in a Colombian mare with regular ovarian activity and X chromosome aneuploidy in mosaic form. The cause of the persistently contaminated uterine content in this mare was not clear; it is possible that via the systemic or transcervical route, bacterial contamination could have colonized the uterus, resulting in chronic inflammation and fluid accumulation. This case report demonstrates the importance of performing an adequate routine gynaecological examination in mares to determine their reproductive health. In most cases, the diagnosis of congenital pathologies of the cervix is an incidental finding when performing breeding programs or therapeutic strategies for managing uterine inflammatory conditions. Furthermore, cytogenetic analysis is an important complementary tool for clinical reproductive examination, to accurately identify causes of congenital malformations, and to determine additional causes of reproductive failure in mares.

124 Effect of transvaginal oocyte aspiration on equine blood and peritoneal fluid parameters

Transvaginal aspiration of oocytes (TVA) in the equine industry has gained more relevance and become a valuable technique to produce offspring from subfertile mares. TVA is a semi-invasive procedure and requires handling the ovaries transrectally to position them closely to an ultrasound probe located in the mare’s vagina. Once the ovary lies in close apposition to the ultrasound probe, a 12G needle is inserted through the needle guide, puncturing, aspirating, and scraping each follicle to recover the oocyte. Potential complications described include rectal tears, puncturing of blood vessels, ovarian abscesses, and peritonitis. Occasionally, problems occur after uneventful procedures, such as colic, peritonitis, pain, and anorexia. However, the source of these complications is not fully known. We hypothesize that blood and peritoneal fluid parameters would differ pre- and post-TVA in mares. A few reports provide some parameters after TVA (e.g. peritoneal protein, neutrophils, nucleated cells) without reference to pre-TVA values. These studies have not identified an effect in peritoneal fluid variables due to multiple abdominocenteses. Therefore, our aim was to analyse blood and peritoneal fluid in mares pre- and post-TVA, and to identify changes in parameters of the procedure (duration, number of pokes, number of follicles) and the mares’ clinical responses. Ten healthy mares were selected to undergo the procedure. Thirty minutes before starting TVA, a blood sample was drawn for complete blood count (CBC) and blood chemistry, and abdominocentesis was performed to obtain abdominal fluid and assess the cytology. This same protocol was repeated 24 hours after TVA. Physical exams were performed pre- and post-TVA. Paired t-tests were used to identify differences between groups (pre- and post-TVA). Spearman correlations (ρ) were used to assess the relationship between variables. There was a significant increase in peritoneal lactate (5.65-fold), peritoneal total protein (2.4-fold), and total nucleated cells (46-fold) between pre- and post-samples. These parameters were not associated with operator, number of times the needle was introduced into the ovaries, or number of aspirated follicles. The remaining parameters evaluated in CBC and blood chemistry did not differ. A positive correlation between total peritoneal protein and blood albumin was found post-TVA (ρ=0.72, P=0.01) but not pre-TVA (ρ=−0.1, P=0.65), suggesting an increase in protein level due to bleeding. Clinically, 9 mares were healthy throughout the study except one that presented signs of pain (facial grimace, anorexia, hyperthermia) the day following TVA. In conclusion, we showed changes in the peritoneal fluid during uneventful TVA procedures. The information provided by this research gives further insight into changes potentially caused by a TVA in abdominal fluid parameters. Further studies are necessary to determine expected standards and the duration of the changes after TVA.

Surgical treatment for tubal disease in women due to undergo in vitro fertilisation.

Surgical treatment for tubal disease in women due to undergo in vitro fertilisation.

Spontaneous Ovarian Hyperstimulation Syndrome: Looking Beyond the Ovary

We report a case of a 22-year-old single female with spontaneous ovarian hyperstimulation syndrome (s-OHSS) referred for transvaginal aspiration of follicles. Investigations revealed primary hypothyroidism, mild hyperprolactinaemia and unelevated levels of both follicle stimulating hormone (FSH) and estradiol. Supplementation with L-thyroxine lead to euthyroid status and gradual resolution of signs and symptoms of ovarian hyperstimulation syndrome (OHSS) over 4 months.

Factors affecting in vitro blastocyst production after intracytoplasmic sperm injection (ICSI)

In vitro production of horse embryos via intracytoplasmic sperm injection (ICSI) is a useful clinical and research technique. Current rates of blastocyst production are typically sub-optimal, and few methods to increase the rate of equine blastocyst development have been reported. Factors that might improve blastocyst production in a horse embryo culture system were explored. Myo-inositol is found in the horse oviduct and improves blastocyst development in other species, thus Experiment 1 was conducted to assess the effect of 10 mM myo-inositol added to Day 0−5 embryo culture medium, using horse oocytes recovered by transvaginal aspiration. Experiment 2 was conducted to investigate effects of exclusion of a standard post-ICSI holding step (culture for 30−60 min in M199-based medium). Experiment 3 was conducted using oocytes recovered from abattoir-derived ovaries, to evaluate effects of earlier transition (Day 4 vs. Day 5) to the second-step medium and of media refreshment at different time points (Day 3 and/or Day 7) during embryo culture. In Experiments 1 and 2, there were no differences (P > 0.05) between groups in blastocyst development (Exp. 1, 36.7 % and 39.2 %; Exp. 2, 41.5 % and 44.6 %). In Experiment 3, blastocyst development was not different (P > 0.05) for embryos refreshed at both Day 3 and 7 (10.8 %) or only at Day 7 (26.6 %), or those transferred to second-step medium on Day 4 or Day 5 (20.6 % and 18.5 %). Knowledge of culture procedures compatible with blastocyst formation in vitro is valuable to laboratories starting to develop procedures for ICSI in horses.

Interleukins’ expression profile changes in granulosa cells of preovulatory follicles during the postpartum period in dairy cows

The postpartum period in dairy cows is associated with a state of temporary negative energy balance and could induce functional changes into ovarian granulosa cells (GC) resulting in significant impact on the ovarian function and fertility. Yet, the regulation of interleukin receptors (ILRs) in GC as well as ILs expression profile during the postpartum period have not been fully investigated. We hypothesized that the postpartum period is associated with changes in ILs expression profile that could affect follicular development and ovulation rate. First, we aimed to investigate the expression and regulation of different IL and IL receptors in GC at different stages of follicular development and then analyse the changes in target ILs expression profile induced during the postpartum period. In the first objective, normal cycling cows were selected and GC were collected from small follicles (SF), dominant follicles at day 5 of the estrous cycle (DF), and ovulatory follicles, 24 h following hCG injection (OF). In the second objective, dairy cows between 50 and 70 days postpartum were randomly selected, and β-hydroxybutyrate (BHB) concentrations were measured in blood samples in order to assign cows to the BHB+ group (>1.4 mmol/L) or BHB− group (<1.2 mmol/L). GC were collected from preovulatory follicles by transvaginal aspiration. Total RNA was extracted from GC of all groups for analysis of target ILs and ILRs expression. Steady-state mRNA levels of IL4R was strongest in the DF, while IL15R expression was greatest in the OF, and IL21R showed increased steady-state mRNA levels in the corpus luteum as compared to the different groups of follicles. Overall, expression of IL1A, IL1B, IL8, IL15, IL23 and TNFα was stronger in OF as compared to DF, while IL4 and IL10 expression was stronger in SF than in DF. Similarly, expression of IL1A, IL1B, IL8, IL15, IL23, and TNFα were significantly stronger in GC of BHB+ cows than in the control, while IL4 expression was significantly reduced in BHB+ as compared to control cows. We have established an IL expression profile, which suggest a correlation with BHB levels during the postpartum period. Additionally, we have demonstrated a differential regulation of target ILRs in GC at different stages of follicular development. Overall, these data provide a better understanding of the changes that could affect follicular development and ovulation during the postpartum period and lay the ground for further investigations.

Voltage-Gated Proton Channels Exist in the Plasma Membrane of Human Oocytes

Voltage gated proton channels (HV1) are highly expressed in human (but not mouse) sperm where they contribute to capacitation and motility (Lishko et al. 2010. Cell. 140:327; Musset et al. 2012. J. Biol. Chem. 287:9376). We investigated whether HV1 are also present in human oocytes. Frozen, thawed human metaphase II oocytes were obtained from material donated to the gamete repository at the Rush Center for Advanced Reproductive Care. Oocytes were retrieved by transvaginal ovarian follicle aspiration following treatment with recombinant follicle stimulating hormone and purified urinary gonadotropins with GnRH antagonist for controlled ovarian hyperstimulation. After sufficient 18-20 mm follicles were visualized by transvaginal ultrasound, human chorionic gonadotropin was administered to initiate the ovulatory cascade. Transvaginal aspiration of the follicles under ultrasound guidance was performed 36 h later. Human oocytes are large ∼110 μm diameter spheres surrounded by a thick extracellular glycoprotein matrix (the zona pellucida). Their size and zona pellucida make oocytes difficult to study by conventional patch-clamp techniques. To obtain an electrically tight seal we exposed bare oolemma by removing sections of the zona pellucida using a laser, followed by repeated pipetting to further separate the oocyte from the zona pellucida. Patch-clamp studies were performed using whole-cell configuration on oocytes, vesicles derived from oocytes, or inside-out patches of membrane, under conditions optimized for voltage-gated proton currents. Proton currents were present at significant levels in human oocytes where they exhibit properties similar to those reported in other human cells, as well as those in heterologous expression systems transfected with the HVCN1 gene. The gating kinetics, voltage dependence, and selectivity were indistinguishable from those of hHV1 in heterologous expression systems. Possible functions for voltage gated proton channels in human oocytes, most likely involving pH regulation, may now be contemplated.

Successful Transvaginal Aspiration of Cervical Pregnancy after Failed Methotrexate Treatment: A Case Report

Background: Cervical pregnancies are infrequent and fatal. Systemic methotrexate (MTX) treatment is conventionally administered to preserve fertility and prevent hemorrhage. However, some patients do not respond to therapy. Methods: Transvaginal aspiration because MTX treatment failed. Results: Two cases were successfully treated by transvaginal aspiration. Serial ultrasound exams and blood beta-hCG confirmed resolution of cervical pregnancy. Conclusion: Our results suggest that transvaginal aspiration should be considered an alternative upon failure of systemic MTX treatment or in the presence of contraindications for MTX injection.

Pain Relief during Oocyte Retrieval by Transcutaneous Electrical Acupoint Stimulation: A Single-Blinded, Randomized, Controlled Multicenter Trial.

Acupuncture has pain-relief effects, but no data were available on the use of transcutaneous electric acupoint stimulation (TEAS) in pain relief during oocyte retrieval. This study was designed to examine the effect of TEAS for pain relief in women undergoing transvaginal ultrasound-guided oocyte aspiration. This single-blinded, multicenter, randomized controlled trial was performed in China between May 2013 and May 2015. The subjects were randomized to mock TEAS and TEAS. TEAS or mock TEAS was administered 30 min before oocyte retrieval until the end of the operation. The primary and secondary endpoints were the pain measured using the visual analog scale (VAS) within 1 min and 1 hour after oocyte retrieval, respectively. Serum β-endorphin levels were tested in the first 50 patients/group. 390 women were undergoing oocyte retrieval. Pain levels evaluated using VAS within 1 min (18.6 ± 1.3 vs. 24.4 ± 1.7, P < 0.01) and 1 h after oocyte aspiration (4.6 ± 0.7 vs. 6.8 ± 0.8, P < 0.05) were lower in the TEAS group than in the mock TEAS group. Nausea assessment revealed a significantly lower VAS score in the TEAS group within 1 min (1.2 ± 0.4 vs. 2.9 ± 0.7, P < 0.033). Serum β-endorphin levels were significantly higher in the TEAS group than in the mock TEAS group (11.4 ± 0.5 vs. 9.1 ± 0.4, P < 0.001) after retrieval. Serum β-endorphin levels were higher in the TEAS group after the procedure than baseline (11.4 ± 0.5 vs. 9.1 ± 0.3, P < 0.001). Oocyte retrieval causes pain and discomfort, but TEAS is effective and safe for suppressing the pain and alleviating nausea associated with the operation.

Dimethyl sulfoxide and glycerol as cryoprotectant agents of stallion semen: effects on blastocyst rates following intracytoplasmic sperm injection of IVM equine oocytes.

Numerous variables affect invitro blastocyst development following intracytoplasmic sperm injection (ICSI). The paternal factor is affected by initial semen quality, processing techniques and final selection of individual spermatozoon for injection. This study investigated whether there was an effect of sperm cryoprotectant agent (CPA) on equine invitro blastocyst production, and reviews recent developments examining how processing equine semen affects ICSI outcomes. Single ejaculates from five stallions were collected and processed in a freezing extender containing either 1M dimethyl sulfoxide (DMSO) or 3.5% glycerol. Immature equine oocytes were obtained from ovarian follicles of mares during diestrus by transvaginal aspiration (n=128). After invitro maturation, MII oocytes (n=90) were fertilised by ICSI with thawed stallion spermatozoa (n=45 in both the DMSO and glycerol groups). The embryo cleavage rate was greater in the DMSO than glycerol group (73.3% vs 46.7% respectively; P=0.0098), but the blastocyst development rate per fertilised oocyte was similar between the two groups (28.9% vs 15.6% respectively; P=0.128), as was the blastocyst production rate per cleaved embryo (39.4% vs 33.3% respectively; P=0.653). In this study, cryopreservation of equine spermatozoa in 1M DMSO was correlated with significantly higher cleavage rates in IVM oocytes fertilised by ICSI compared with spermatozoa cryopreserved using 3.5% glycerol. Although not statistically significant in this small number of stallions, increased blastocyst production and individual stallion variability was observed among CPA treatments. This warrants further critical examination of cryoprotectants used in equine sperm subpopulations used for ICSI in a larger number of stallions.

Equine fetal genotyping via aspiration of yolk-sac fluid at 22–28 days of gestation

Fetal genotyping has important applications in the horse, but currently necessitates embryo recovery and biopsy. We investigated whether fetal genotyping could be performed on yolk-sac fluid recovered from pregnant mares via transvaginal aspiration. Fluid was collected before Day 30 to provide results before establishment of the endometrial cups (Day 37). Genotyping and assessment of maternal DNA contamination was performed by analyzing histograms of PCR results for 19 loci. In Exp. 1, mares underwent yolk-sac aspiration on Days 22–28 of gestation. Fluid (0.56–1.02 mL) was recovered from five of seven mares. Four of the five mares maintained pregnancy. One pregnancy was electively terminated at Day 75; the other three mares delivered healthy foals. Extraction of DNA from the fluid sample followed by direct PCR allowed the highest rate of determination of fetal alleles. Fetal genotype was correctly determined in three samples, and for 14/19 alleles in one sample. In Exp. 2, we evaluated whether recovery of more fluid (up to 1.6 mL), and fractionation of the sample, would minimize maternal DNA contamination. One of four mares maintained pregnancy. Evaluation at informative loci showed no difference in maternal contamination among fractions. We determined that mares can maintain pregnancy after aspiration of yolk-sac fluid, and that fetal genotype can be accurately determined from the sample obtained. Further work is needed on factors affecting maintenance of pregnancy after the procedure. The ability to access the yolk sac in early pregnancy opens the door to novel potential clinical and research applications.

The effect of seminal plasma \u03b2-NGF on follicular fluid hormone concentration and gene expression of steroidogenic enzymes in llama granulosa cells

BackgroundNerve growth factor (β-NGF) from llama seminal plasma has been described as a potent ovulatory and luteotrophic molecule after intramuscular or intrauterine infusion in llamas and alpacas. We tested the hypothesis that systemic administration of purified β-Nerve Growth Factor (β-NGF) during the preovulatory stage will up-regulate steroidogenic enzymes and Vascular Endothelial Growth Factor (VEGF) gene expression in granulosa cells inducing a change in the progesterone/estradiol ratio in the follicular fluid in llamas.MethodsExperiment I: Female llamas (n = 64) were randomly assigned to receive an intramuscular administration of: a) 50 μg gonadorelin acetate (GnRH, Ovalyse, Pfizer Chile SA, Santiago, Chile, n = 16), b) 1.0 mg of purified llama β-NGF (n = 16), or c) 1 ml phosphate buffered saline (PBS, negative control group, n = 16). An additional group of llamas (n = 16) were mated with a fertile male. Follicular fluid and granulosa cells were collected from the preovulatory follicle at 10 or 20 h after treatment (Time 0 = administration of treatment, n = 8/treatment/time point) to determine progesterone/estradiol concentration and steroidogenic enzymes and VEGF gene expression at both time points. Experiment II: Granulosa cells were collected from preovulatory follicles from llamas (n = 24) using ultrasound-guided transvaginal follicle aspiration for in vitro culture to determine mRNA relative expression of Steroidogenic Acute Regulatory Protein (StAR) and VEGF at 10 or 20 h (n = 4 replicates) and progesterone secretion at 48 h (n = 4 replicates) after LH or β-NGF treatment.ResultsExperiment I: There was a significant increase in the progesterone/estradiol ratio in mated llamas or treated with GnRH or purified β-NGF. There was a significant downregulation in the mRNA expression of Aromatase (CYP19A1/P450 Arom) for both time points in llamas mated or treated with GnRH or llama purified β-NGF with respect to the control group. All treatments except β-NGF (20 h) significantly up regulated the mRNA expression of 3-beta-hydroxysteroid dehydrogenase (HSD3B) whereas the expression of StAR and Side-Chain cleavage enzyme (CYP11A1/P450scc) where significantly up regulated only by mating (20 h), or β-NGF at 10 or 20 h after treatment. VEGF was up regulated only in those llamas submitted to mating (10 h) or treated with purified β-NGF (10 and 20 h). Experiment II: Only β-NGF treatment induced an increase of mRNA abundance of StAR from llama granulosa cells at 20 h of in vitro culture. There was a significant increase on mRNA abundance of VEGF at 10 and 20 h of in vitro culture from granulosa cells treated with β-NGF whereas LH treatment increases VEGF mRNA abundance only at 20 h of in vitro culture. In addition, there was a significant increase on progesterone secretion from llama granulosa cells 48 h after LH or β-NGF treatment.ConclusionsSystemic administration of purified β-NGF from llama seminal fluid induced a rapid shift from estradiol to progesterone production in the preovulatory follicle. Differences in gene expression patterns of steroidogenic enzymes between GnRH and mated or β-NGF-treated llamas suggest local effects of seminal components on the preovulatory follicle.

Morphological characteristics of the oocyte–cumulus complexes bos taurus and bos indicus of different purpose productivity

In the technology of in vitro embryo production in bovines, qualitative and quantitative indicators of the initial population of viable oocytes determine the successful output of blastocysts suitable for native embryotransfer or vitrification Therefore the study of reproductive potential of cows Bos taurus and Bos indicus (zebu cattle) of different directions of productivity by the method of assessing the quality and quantity of oocytes is of scientific and practical interest. The aim of this study was to conduct a comparative analysis of Russian and Brazilian cattle on the yield of oocyte–cumulus complexes (OCC) and their morphological characteristics in the populations of dairy and beef cattle productivity. The following samples of cows were formed: Aberdeen–Angus breed n=82 (Russian Federation); black–and–white breed n=62 (Russian Federation); Nelore n=149 (Brazil); Gir n=63 (Brazil). The material of the study was oocyte–сumulus complexes obtained in the result of transvaginal puncture of follicles (transvaginal aspiration of oocytes TAO, ovum pick–up OPU). The quality of OCC was assessed by the following morphological parameters: the presence of cumulus layer and its quality, oocytes size, homogeneity and color of ooplasm, turgor. The number of viable OCC from the total amount is: Aberdeen Angus 67.15%; black–mottled 42%; Nelore 83.9%; Gir 71,4%. Thus, Bos indicus (zebu cattle) is more promising for the technology of production embryos in vitro, as it exceeds Bos taurus by more than 2 times in the output of oocytes, both in the group of beef cattle and in the group of dairy.

Assisted reproductive technologies in cattle reproduction and selection

In vitro technology is one of the most dynamically developing and more and more stable biotechnological methods today accelerating selection, intensifying reproductive and genetic potential of breeding animals, allowing to increase breeding young animals production by one champion cow up to 5–10 calves per year, reduce generation interval and significantly accelerate process of updating and qualitative improvement of livestock. However, obtaining oocytes competent for in vitro development is one of the critical factors determining success of the method and depending on a number of biological and technical factors. This paper presents results of studies on effect of biological factors of direct and indirect impact on efficiency of obtaining oocytes in the system of transvaginal aspiration for the first time conducted in the Republic of Belarus. Yield of excellent and good quality oocytes increased during aspiration during the luteal phase of estrous cycle and remained almost unchanged during aspiration into the follicular phase. Presence of follicles with diameter over 8 mm in the ovaries during aspiration reduced yield of excellent and good quality oocytes averagely by 9.4 percentage points. Removing the dominant follicle 72 hours prior to aspiration allowed increasing the number of aspirated follicles by 41 %, and yield of oocytes – by 22.9 %. Microstimulation of ovaries prior to aspiration by follicle-stimulating hormones FSG-super and Plusset increased efficiency of aspiration in terms of the main indicators by 19.2–45.9 %. Follicular cyst or persistent corpus luteum in one of the ovaries reduced both quantitative and qualitative indicators of aspiration. The data obtained are of practical importance for development of technology for in vitro embryo production in the system of transvaginal aspiration of oocytes which will help to accelerate breeding process and increase efficiency of breeding work in livestock production in general.Acknowledgments. The research was conducted within the two state research programs: “Biotechnology”, subprogram “Development of biological science, biological education and biological industry for 2007–2011 and for the period up to 2020”, “High technologies and equipment for 2016-2020”, subprogram 1 “Innovative biotechnologies–2020”.

Stem cells from follicular fluid present multilineage differentiation capacity and form oocyte-like structures under the influence of FSH hormone

Background & Aim Recent studies highlight the existence of putative stem cells derived from human ovarian follicular liquid collected after routine procedures for in vitrofertilization techniques (Riva F et al, 2009; Polimeni M et al, 2012).The aim of this study was to isolate, characterize and evaluate the pluripotency of this heterogeneous cell population of stem cells isolated from follicular fluid. For this purpose human ovarian follicular fluids were collected from women during the IVF procedure at the Embryoclinic Assisted Reproduction Unit (Thessaloniki, Greece). Methods, Results & Conclusion The collection was performed during the retrieval process of oocytes by transvaginal ultrasound-guided aspiration needle. After removal of the cumulus oophorus-oocyte complex the remained follicular fluid was centrifuged and the pellet was analyzed by flow cytometry for total immunophenotypic characterization. Cells were seeded on plastic surfaces in DMEM medium supplemented with fetal bovine serum, penicillin/streptomycin and gentamycin for incubation at 37οC/5% CO2. After 10-day cultivation the cells were analyzed for the expression of mesenchymal and pluripotent stem cell markers with flow cytometry. The induced differentiation capacity of isolated cells towards bone, adipose and endothelium in specific culture mediums was estimated via alizarin red, oil red staining and flow cytometry respectively. Cultures of follicular stem cells were checked for their ability to form oocyte like structures with the addition of FSH hormone in different concentrations. Real time PCR was performed for primordial germ stem cells markers Sox-2 Nanog and OCT4. A population of CD34-/CD45+/CD90+/CD105+/CD133-/SSEA4-/OCT4-/CD44+ stem cells were isolated. Osteogenic, adipogenic and endothelial multilineage differentiation capacity of cells derived from follicular liquid after culture was successfully confirmed. Oocyte like structure were observed in different FSH concentrations in the culture medium. After the presence of FSH hormone, cells were positive for the primordial stem cells markers Sox-2, Oct4 and Nanog while expression was gradually decreased in larger FSH concentrations. Conclusively, stem cells from follicular fluid show multilineage potential capacity and express primordial stem cell markers in specific conditions. Follicular fluid could be used as an alternative source of pluripotent stem cells and it might be a hopeful cure to women suffer from premature ovarian failure.

Correlation of Transvaginal USG and Endometrial aspiration findings with various menstruation patterns and histopathological examination of Uterus

Correlation of Transvaginal USG and Endometrial aspiration findings with various menstruation patterns and histopathological examination of Uterus

Ultrasound Guided Transvaginal Aspiration and Mechanical Destruction with Local Methotrexate Injection is a Promising Primary Treatment Approach for Caesarean Scar Ectopic Pregnancy (CSEP)

Caesarean scar ectopic pregnancy (CSEP) is considered an extremely rare entity, but life threatening, however, with the increase in C/S rates it is becoming a more common finding. To explore the possibility of utilizing ultrasound guided transvaginal aspiration and injection of 50 mg methotrexate followed by mechanical disruption as a promising primary treatment approach for CSEP. In a series of 11 cases at Tawam Hospital (UAE) diagnosed with CSEP, an ultrasound guided transvaginal aspiration of the amniotic fluid and gestational tissue, followed by injection of 50 mg Methotrexate into the remaining chorionic sac and then disruption of the sac with the needle. B-hCG level were measured on: day of procedure, day 1 or 2 after the procedure and then weekly. Final resolution was considered when B-hCG returned to &lt; 5mIU/mL. All cases presented and diagnosed in the 1st trimester, mean gestational age at diagnosis was 6.6 weeks (range 5-10). The presenting symptoms were mainly lower abdominal pain and vaginal bleeding in 8/11 patients (73%) while three women were asymptomatic (27%) and CSEP was diagnosed incidentally at viability scan. Nine out of eleven cases showed cardiac activity (82%). B-hCG day 1 or 2 post procedure dropped in all cases but by variable levels, ranging from 1.1% to 74.7%, while the drop during days 7-10, was more significant and reassuring; ranged from 45.7% to 92.5 %. No complications were reported with 100% success rate. Ultrasound guided transvaginal aspiration and injection of 50 mg Methotrexate followed by needle mechanical disruption is a promising primary treatment approach for CSEP. However, more studies with more numbers are needed to further support this treatment method.

Effect of the interval from follicle aspiration to initiation of lengthened FSH treatment on follicular superstimulatory and superovulatory responses and embryo production in lactating Simmental cows

The present study evaluated follicular superstimulatory (FSS) and superovulatory (SOV) responses and in vivo embryo production in lactating Simmental cows treated with FSH starting 1 or 2 days after follicle aspiration (FA). The performance of a lengthened superovulation program, named 6dFSH-P36-hCG60, is described. At random stages of the estrous cycle, cows (n = 52) were subjected to ultrasound-guided transvaginal aspiration of all follicles ≥ 5 mm. After FA, cows were randomly assigned to one of two groups in which FSH treatments started 1 or 2 days after FA (groups FA-1D and FA-2D, respectively). Cows were superstimulated with a total of 500 μg pFSH over 6 days on a decreasing dose schedule and were pre-treated with a single dose of 400 IU of eCG 24 h before the start of FSH treatments. Follicular superstimulatory (the mean numbers of follicles ≥ 8 mm on the day of hCG treatment) and SOV responses (the mean numbers of CL and cows with ≥ 3 CL at the time of collection) were similar in FA-1D and FA-2D groups. However, when compared to FA-1D group, the number of unfertilized ova tended to decrease (0.4 vs 1.7; P = 0.065) and percentage of fertilized ova tended to increase (95.8% vs 84.6%; P = 0.066) in FA-2D group. Moreover, the mean numbers and percentages of both transferable embryos (8.0 and 77.6% vs 6.4 and 57.7%) and freezable embryos (5.3 and 51.5% vs 3.5 and 31.1%) were numerically higher in FA-2D group than FA-1D group.The results of the study suggest that starting a lengthened superovulation programs in Simmental cows 2 days after FA has potential to increase percentage of fertilized ova and the number of transferable and freezable embryos, although new studies may be needed to confirm this findings.