Transvaginal Aspiration Research Articles

43 MASS VITRIFICATION OF GERMINAL-VESICLE STAGE EQUINE OOCYTES

Oocyte cryopreservation has the potential to preserve female genetics. In addition, equine oocytes are not readily available in some areas, and vitrification could be used to accumulate oocytes at remote locations to provide material for research. To preserve large numbers of oocytes, a method for rapid vitrification of multiple oocytes is needed. First, we determined whether immature equine oocytes could be held overnight before vitrification, and we tested the use of a mesh+capillary-action media-removal vitrification platform. Oocytes were collected via ultrasound-guided transvaginal follicle aspiration and randomly allotted to either immediate vitrification or overnight holding (24 to 27 h in 40% M199-Earle’s salts, 40% M199-Hanks’ salts, 20% fetal bovine serum, and 0.3 mM pyruvate) then vitrification. Oocytes were vitrified using different times (1 or 4 min) in vitrification solution and first warming solution: 1v1w, 1v4w, 4v1w, and 4v4w. The base solution was MH (80% M199-Hanks’ salts and 20% fetal bovine serum). Cryoprotectant concentration (vol/vol) was increased in 3 steps until reaching 7.5% dimethyl sulfoxide and 7.5% ethylene glycol. The oocytes were then held in vitrification solution (MH with 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose) for either 1 or 4 min, according to treatment, and 3 to 10 oocytes were transferred to a 75-μm sterile stainless steel mesh. The mesh was placed on sterile paper to absorb excess medium, then plunged in LN. The oocytes were warmed in MH solution with 1.25 M sucrose for either 1 or 4 min, then placed in 0.62 M and 0.31 M sucrose solutions for 5 min each and undetermined time in MH. After warming, oocytes were cultured for maturation (in vitro maturation) in M199-Earle’s salts, 5 mU mL–1 FSH, and 10% fetal bovine serum. After 30 to 36 h, the oocytes were denuded and stained with Hoechst 33258. Data were analysed by Fisher’s exact test. There were no significant differences (P > 0.05) in rates of meiotic resumption among timing treatments (35, 24, 26, and 39% for 1v1w, 1v4w, 4v1w, and 4v4w, respectively), nor between immediately vitrified (17/55, 31%) and overnight held-vitrified groups (18/56, 32%). In the second experiment, all oocytes were held overnight. They were vitrified and warmed using only the 1v1w and 4v4w schedules, then subjected to in vitro maturation, intracytoplasmic sperm injection, and embryo culture. The MII rate of the control group (27/37, 73%) was higher (P < 0.05) than that for 1v1w (12/33, 36%) or 4v4w treatments (10/35, 29%). The cleavage rate for control (25/27, 93%) was higher than that for 1v1w (5/9, 56%) but not than that for 4v4w (6/9, 67%). Blastocyst rates were 19% (5/27), 11% (1/9), and 0% (0/9) for control, 1v1w, and 4v4w, respectively (P > 0.05). These results indicate that blastocysts may be produced from equine immature oocytes vitrified en masse; however, both the maturation and blastocyst production rates were relatively low. Additional studies are required to improve the efficiency of this technique. This work was supported by the Clinical Equine ICSI Program, Texas A&M University.

63 EQUINE FOLLICLES MODULATE CORTISOL LEVELS AND CAPABILITY OF OOCYTES TO ADAPT TO STRESS SITUATIONS

Glucocorticoids are mediators of the systemic stress response. Acute or chronic stress characterised by high cortisol concentrations in the periphery impairs reproductive function in a variety of species and therefore may affect fertility. The ovary has been shown to be a target tissue for glucocorticoids in many species, including the mare. This study hypothesised that the equine ovary possesses internal mechanisms to modulate cortisol activity and that supraphysiologic levels of glucocorticoids do not affect oocyte IVM rates. Light horse mares (n = 9) were used in this study. Growing follicles from an induced follicular wave were divided into the following groups: G1: 5–9 mm, G2: 10–14 mm, G3: 15–19 mm, G4: 20–24 mm, and G5: ≥25 mm. Follicular fluid (FF) and compact cumulus‐oocyte complexes (COCs) were obtained by ultrasound-guided transvaginal aspiration. Blood samples were collected at the beginning and the end of every aspiration session. Cortisol (DE1887, Demeditec, Kiel-Wellsee, Germany), progesterone (ADI-901–011, Enzo Life Sciences, Farmingdale, NY, USA), and corticosteroid binding globulin (CBG, MBS047353, MyBioSource, San Diego, CA, USA) concentrations were determined by ELISA. COCs (n = 80) were randomly distributed to either the control group (DMEM-F12+ medium) or the following hydrocortisone treatment groups: 0.1 µg mL–1, 1 µg mL–1, 5 µg mL–1, 10 µg mL–1. Maturation rate was assessed 30 h after incubation. Statistical analysis was performed with the SPSS Statistics 22 software. Data were analysed using one-way ANOVA, Pearson correlation, and chi-squared test. Cortisol (115.4 ± 13.3 ng mL–1) and progesterone (22.1 ± 3.1 ng mL–1) FF concentrations were higher (P < 0.05) in G5 follicles than in all other groups, and were positively correlated (r = 0.8; P < 0.001). Plasma concentrations of cortisol (118.6 ± 7.8 v. 120.3 ± 12.2 ng mL–1), progesterone (2.4 ± 0.5 v. 2.5 ± 0.4 ng mL–1), and CBG (11.1 ± 5.1 v. 9.9 ± 3.2 µg mL–1) did not differ before and after follicle aspiration. However, plasma CBG and progesterone were negatively correlated (r = –0.56; P < 0.01). Maturation rates did not differ among groups, regardless of the hydrocortisone concentration added to the culture medium. Our results demonstrated higher cortisol concentrations in preovulatory follicles in vivo, suggesting its importance for oocyte maturation. The greater unbound cortisol available in the FF of preovulatory follicles can be indicative of the displacement of cortisol from CBG in favour of progesterone. Furthermore, equine oocytes were capable of surviving cortisol concentrations 100 times higher than those physiologically present in preovulatory follicles. This finding suggests the ability of equine oocytes to modulate cortisol levels and adapt to stress situations.

Comparing transabdominal and transvaginal ultrasound-guided follicular aspiration: A risk assessment formula

ObjectiveWe sought to identify patients at risk of incomplete transvaginal oocyte retrieval, develop a risk assessment formula to identify patients who would benefit from a transabdominal approach, and compare complication and pregnancy rates between these two approaches. Materials and MethodsIn this retrospective case control study in a private in vitro fertilization center, 95 cases of women undergoing transabdominal follicular aspiration for oocyte retrieval (15 transabdominal only and 80 transabdominal and vaginal combined) were compared with 278 controls of women undergoing the transvaginal aspiration only. Transabdominal oocyte retrieval was performed when one or more ovaries could not be retrieved via the transvaginal approach. Main study outcomes included need for transabdominal retrieval, pregnancy rates, and complications. ResultsA risk assessment scoring system was developed as follows: difficulty seeing ovaries on ultrasound (+4), history of pelvic surgery (+3), and body mass index of 30 kg/m2 or greater (+2). With a cutoff score of 4 or greater, the overall sensitivity is 75%, specificity is 80%, positive predictive value is 57%, and negative predictive value is 90%. No statistically significant differences were found for pregnancy rates or complications. ConclusionThe transabdominal approach is an alternative option that would increase the total number of oocytes retrieved with no statistical difference in complication or pregnancy rates. We also developed a scoring system that can serve as a useful screening tool for identifying women at increased risk of transabdominal oocyte retrieval.

The effect of energy balance on the transcriptome of bovine granulosa cells at 60 days postpartum

Dairy cows expend great amounts of energy during the lactation peak to cope with milk production. A state of negative energy balance (NEB) was suggested as a cause for the suboptimal fertility observed during this period, via an interaction with ovarian function. The objective of this study was to identify the impact of NEB on gene expression in granulosa cells of dairy cows at 60 days postpartum and to suggest a potential treatment to improve ovarian function. Dairy cows at 60 days postpartum from 10 typical medium-sized farms were synchronized using a single injection of prostaglandin. Dominant follicles were collected 42 hours later by transvaginal aspiration. Blood concentrations of beta-hydroxybutyrate (BHB) on the day of aspiration were used to classify animals into two groups: severe NEB (high BHB, n = 12) and mild NEB (low BHB, n = 12). The transcriptomes of granulosa cells from both groups were contrasted using microarrays, and the differentially expressed genes were analyzed using Ingenuity Pathway Analysis to identify affected functions and potential upstream regulators. Genes linked with cellular organization (KRT4 and PPL), proliferation (TACSTD2), and fatty acids metabolism (VNN2) were downregulated in granulosa cells from animals with severe NEB. Several genes linked to decitabine, a hypomethylating agent, and with beta-estradiol, were downregulated in the severe NEB group. Numerous genes linked to vitamins A and D were also downregulated in this group of cows, suggesting a potential deficiency of these vitamins in dairy cows during the postpartum period. This study supports the idea that energy balance has an impact on follicular dynamics which could be detrimental to resumption of fertility after calving.

Eficacia de la utilización de un dispositivo de costo bajo para la aspiración transvaginal de quistes simples anexiales

OBJETIVO: Valorar la eficacia de la utilización de un dispositivo de costo bajo para la aspiración transvaginal de quistes simples anexiales. DISEÑO: Estudio tipo observacional y prospectivo. LUGAR: Consulta privada del distrito de Socabaya, Arequipa, Perú. Participantes: Pacientes portadoras de quistes simples anexiales no complicados. INTERVENCIONES: Previa firma del consentimiento informado, a 15 pacientes portadoras de quistes simples anexiales no complicados se les realizó una aspiración transvaginal del quiste, mediante la utilización de un dispositivo (guía) artesanal de costo bajo, acoplado al transductor endovaginal de un ecógrafo portátil. Las muestras tomadas fueron enviadas para estudio patológico. Se realizó seguimiento clínico y ecográfico de las pacientes hasta por 12 meses. PRINCIPALES MEDIDAS DE RESULTADOS: Dificultad y complicaciones del procedimiento, remisión del quiste. RESULTADOS: El procedimiento realizado se logró sin dificultades y sin complicaciones en las 15 pacientes. Hubo remisión completa del quiste en 6 de 15 pacientes (40%) y parcial en las restantes 9 (60%). El volumen promedio del quiste aspirado fue 85 mL. Los casos de recidiva, luego de un mes presentaron 29,2% del volumen inicial aspirado, a los 6 meses, 28,7%, y a los 12 meses, 30,1%. El CA 125 y el estudio patológico de los contenidos del quiste fueron negativos para malignidad. Todas las pacientes se mantuvieron asintomáticas en los 12 meses de seguimiento. CONCLUSIONES: La utilización del dispositivo de costo bajo es eficaz y bien tolerado, para la aspiración transvaginal de quistes simples anexiales, permitiendo controlar esta patología de una manera mínimamente invasiva.

Establishing an in vitro production program for buffalo embryos (Bubalus bubalis) in Colombia

Objective. Evaluate the results of the standardization of the in vitro production program of buffalo embryos, using oocytes obtained by ultrasound guided oocyte puncture during the 2012 breeding season in Colombia. Materials and methods. Fifty seven buffalo females were selected for ultrasound guided transvaginal aspiration of follicles, oocytes were identified within follicular fluid, classified and transported to the laboratory and matured in vitro for 18 to 20 hours. Frozen semen of seven Mediterranean bulls were used, motile sperm was obtained using the Percoll technique and oocytes were inseminated with 2 million sperm/ml. Presumptive zygotes were cultured for 6 days, grade 1 embryos obtained were frozen using ethylene glycol. Embryos were transferred to females on day 5 during natural cycle. Results. 97 aspirations were performed on the 57 animals, in 8.2% of the aspirations no oocytes were found. 8 oocytes/aspiration were obtained. Of the 783 oocytes, 92% were classified as viable (721/783) and were fertilized. The cleavage and blastocyst rate were 23% and 19% respectively. 37 embryos were transferred and 11 pregnancies were obtained, confirmed by rectal palpation 60 days after transfer, with a pregnancy rate of 29.7%. Conclusions. The results presented here are comparable with others in literature and shows the feasibility of producing in vitro embryos and pregnancies after the standardization of current protocols, with normal and sexed semen and transfer during natural cycle in buffalo.Key words: Buffaloes, embryo, fertilization in vitro, reproduction (Source: MeSH).

Heterotopic cervical pregnancy: Conservative treatment with transvaginal embryo aspiration

To discuss two cases of heterotopic cervical pregnancy (HCP) managed with transvaginal embryo reduction. Two patients diagnosed with HCP after assisted reproductive treatment were treated with ultrasonographically guided transvaginal embryo aspiration of ectopic fetus. In one case, the pregnancy resulted in healthy delivery while the second case was terminated due to chorioamnionitis followed by preterm premature rupture of membranes at 23 weeks of gestation. This minimally invasive approach in an hemodynamically stable patient can be considered in the management of a first-trimester HCP. However, the patient must be informed for the potential risks related to the procedure.

Transvaginal Aspiration of Ectopic Gestational Sac: A Novel Treatment Option in Tubal Ectopic Pregnancies

ABSTRACTObjectiveTo present five cases of unruptured tubal ectopic pregnancies treated with ultrasound-guided aspiration with local potassium chloride (KCl) or systemic methotrexate (MTX) administration.DesignCase series.SettingA hospital-based prospective study.PatientsFive women with unruptured tubal ectopic pregnancy.InterventionsTransvaginal ultrasound-guided aspiration of tubal ectopic pregnancy followed by local KCl or intramuscular injection of MTX (50 mg/m2).Main outcome measuresSuccessful conservative treatment of tubal ectopic pregnancies with preservation of fallopian tubes.ResultsFour (80%) out of five women weres uccessfully treated, without the need for operative intervention. One patient required salpingectomy for ruptured ectopic pregnancy during the course of treatment.ConclusionTransvaginal ultrasound-guided aspiration of ectopic gestational sac along with either local KCl or systemic methotrexate administration can be safely used to treat unruptured tubal ectopic pregnancies in selected patient population.How to cite this articleMishra VV, Choudhary S, Sharma U, Agarwal R. Transvaginal Aspiration of Ectopic Gestational Sac: A Novel Treatment Option in Tubal Ectopic Pregnancies. J South Asian Feder Obst Gynae 2015;7(1):1-4.

38 EFFECT OF OOCYTE MATURATION DURATION ON BLASTOCYST RATES AFTER EQUINE SOMATIC CELL NUCLEAR TRANSFER

In equine cloning, the scarcity of equine oocytes places emphasis on development of the most efficient nuclear transfer (NT) methods possible. In other species, using oocytes matured for the shortest duration needed to reach metaphase II has increased NT efficiency. In the present study, we examined the effect of duration of oocyte maturation at the time of enucleation on equine cloned blastocyst production. Oocytes were collected from live mares by transvaginal ultrasound-guided aspiration of all visible follicles ≥5 mm in diameter. The oocytes were held overnight (16–22 h) at room temperature, matured in vitro, and reconstructed with donor cells as described in our previous study (Choi et al. 2013 Theriogenology 79, 791–796). In Experiment 1, oocytes were divided into 2 groups and matured for 20 or 24 h. After enucleation, oocytes were reconstructed by direct injection of donor cells. Reconstructed oocytes were held for 5 h and then activated by treatment with 5 μM ionomycin for 4 min, then injection with sperm extract, followed by incubation in 2 mM 6-DMAP for 4 h. The activated reconstructed oocytes were cultured in global human embryo culture medium under 5% CO2, 6% O2, and 89% N2 at 38.2°C for 7 to 11 days (20 mM glucose was added at Day 5) and blastocyst rate was recorded. Because a low maturation rate was found at 20 h in Experiment 1, in Experiment 2 oocytes were denuded at 20 h and those that were mature were enucleated and used for NT; those that had not cast out a polar body at 20 h were cultured for an additional 3 h (20 + 3h) and then evaluated for polar body formation and used for NT, which was conducted as in Experiment 1. Data were analysed by Fisher's exact test. In Experiment 1, 203 oocytes were collected in 46 aspiration sessions. The rate of oocyte maturation to metaphase II was significantly lower for oocytes cultured for 20 h (35/116, 30%), than for those cultured for 24 h (47/80, 59%). However, the rate of blastocyst development was significantly higher for oocytes cultured for 20 h (11/27, 41%) than for 24 h (2/38, 5%). In Experiment 2, 89 oocytes were collected in 18 aspiration sessions. After 20 h of maturation culture, 22 oocytes were mature (25%). After an additional 3 h of culture, 21 additional oocytes had matured. There were no significant differences between the two treatments (20 and 20 + 3h) in reconstruction rates (77%, 17/22, and 90%, 19/21, respectively) or blastocyst rates (24%, 4/17, and 32%, 6/19, respectively). These results indicate that duration of in vitro maturation, or the duration of presence of cumulus cells, influences blastocyst development after somatic cell NT in the horse. This appears to be due to a benefit of using oocytes immediately after they reach metaphase II; if this is ensured as in Experiment 2, the duration of maturation itself had no effect.This work was supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts.

Transvaginal ultrasound-guided aspiration of benign ovarian cysts

In our study, we evaluate the diagnostic and therapeutic efficacy of transvaginal ultrasound-guided aspiration of benign ovarian cysts in selected patients. A total of 46 women with benign ovarian cysts were referred to our outpatient clinic. The aspirated fluid was collected and sent for cytological analysis. All women were re-evaluated at 1, 3 and 6 months after the procedure. The cytological analysis was negative for malignancy in all cases. Our study showed an overall recurrence rate for ovarian cysts of 39.1%. Women with endometriotic ovarian cysts have an increased incidence of recurrence, 62.5% (n = 5), in comparison with serous cysts, 35.2% (n = 12) and serous-haemorrhagic cysts, 15% (n = 1), χ2 = 9.913, df = 2, p = 0.007. The results of our study reveal that transvaginal ultrasound-guided aspiration of benign ovarian cysts is a simple, safe and effective procedure.

Letter to the Editor: Re: Comparison of IVF-ET outcomes in patients with hydrosalpinx pretreated with either sclerotherapy or laparoscopic salpingectomy

Dear Sir, We have read the article published by Na et al. [1] with great interest. The conclusions reported by the authors was that treating hydrosalpinx prior to the IVF procedure is thought to improve the likelihood of successful IVF outcome. None of the cases experienced persisting pain, need for surgery or hospitalization. Recent studies have argued for sclerotherapy after aspirating a hydrosalpinx under ultrasound in hope of better success rate in IVF/ICSI cycles than that obtained without aspiration [2,3]. To overcome the drawback of high recurrence rate associated with ultrasound-guided transvaginal aspiration of hydrosalpinges, ethanol sclerotherapy has been introduced as a new therapeutic option. Ethanol rapidly coagulates the active endothelial cells lining the cyst wall but penetrates the fibrous capsule slowly causing minimal local or systemic complications [4]. However, in an experimental controlled animal study, Atilgan et al. [5] shed lights on the possible damage level of adjacent ovarian reserve in rats after local ethanol application through inducing fibrosis and apoptosis. As yet, no data are available in the literature concerning its effect on the ovarian reserve in human as well as its long-term sequelae especially in women suffering from infertility. Here we report the laparoscopic findings of five patients who had undergone ethanol sclerotherapy for hydrosalpinges before attempting IVF/ICSI. All patients were planned for laparoscopy before repeat IVF/ICSI cycles because of recurrence with persistant pelvic pain not resolved by analgesics. All women had a medical record of a prior diagnostic laparoscopy before first IVF/ICSI trial reporting no adnexal adhesions. The most distressing finding was that all patients had severe and unusual pelvic adhesions surround the ovaries. The adhesions were thick, string-like, and the majority of which were adhered to the Douglas pouch and the lateral pelvic wall. Interestingly, in two cases, there were thick adhesions between the rectum and the intestine, which might have been caused by the leakage of injected ethanol. It has been suggested that adhesion formation could occur after transvaginal aspiration alone [3]. However, the findings described here show that all the cases undergoing ethanol injection for hydrosalpinx had severe and unfamiliar thick ovarian adhesions. Although the exact mechanism of extensive adhesions after leaked ethanol sclerotherapy are unknown, one may infer that chemical irritation of the visceral peritoneum together with the fact that the ovary is sensitive to noxious chemicals, potential damage might remain to be excluded. Furthermore, the possible toxicity of injected ethanol to ova located in the surrounding ovarian cortex together with the actively dividing oocytes has not been completely excluded. Based on these findings, ethanol sclerotherapy for hydrosalpinx before IVF/ICSI cycles, despite being less invasive when compared with surgery, seems to be a traumatic and harmful therapy and should be used cautiously. Further randomized trials are required to confirm these data.

223 CRYOPRESERVATION OF IMMATURE BOVINE CUMULUS–OOCYTE COMPLEXES BY SLOW RATE FREEZING AND VITRIFICATION

Cryopreservation of mature oocytes can result in damage to the metaphase spindle due to the temperature sensitivity of microfilaments and microtubules. Cryopreservation of immature oocytes may circumvent this problem because these structures have not formed yet and the genetic material is enclosed within a nuclear envelope. Because intact oocyte cumulus-oocyte complexes (COC) are essential for normal maturation, we chose to cryopreserve immature intact COC. The aim of this study was to determine if immature COC cryopreserved by slow rate freezing or vitrification would resume meiosis upon thawing. In 2 separate experiments, immature COC (n = 102 and 79) were collected from cross-bred cattle by transvaginal ultrasound-guided aspiration and divided into 2 groups. For both experiments, the first group (n = 64 and 40) was placed directly into maturation medium (TCM 199 supplemented with 10% fetal bovine serum, 0.2 mM sodium pyruvate, 2 mM glutamine, and 5 μg mL–1 of FSH) and cultured for 22 h under 5% CO2 in air atmosphere at 39°C. In experiment 1, COC (n = 38) in the second group were cryopreserved by a slow rate freezing protocol. The COC were equilibrated for 5 min in 1.5 M ethylene glycol (EG), and 5 COC were loaded into 0.25-mL straws and placed into the cooling chamber of a Freeze Control unit at –6°C. After 5 min, straws were seeded, then cooled at 0.5°C per min to –35°C before plunging into LN. After storage in LN for 5 days, straws were removed from LN, thawed by placing straws in a 35°C water bath, and COC put into maturation as described above. In experiment 2, COC (n = 39) in the second group were cryopreserved by vitrification using a 3-step procedure. The COC were equilibrated in solutions consisting of 10% glycerol then 10% glycerol and 20% EG in PBS for 5 min each. The COC were then placed in a solution of 24% glycerol and 26% EG, and 1 to 3 COC were placed onto a cryotop device in minimal medium and plunged into LN within 45 s. After storage in LN for 2 days, COC were thawed by placing the cryotop device directly into a warmed dilution solution consisting of 0.5 M galactose in PBS. After 5 min in the dilution solution, COC were put into maturation as described above. For all groups, after 22 h of maturation cumulus cells were stripped from the oocytes by vortexing and oocytes were placed on slides for fixation in methanol acetic acid and stained with 1% orcein to determine the nuclear stage. In experiment 1, oocytes cryopreserved by slow rate freezing matured to MII at the same rate as control oocytes (45 v. 55%, P = 0.44). In experiment 2, oocytes cryopreserved by vitrification matured to MII at a lower rate than controls (49 v. 79%, P = 0.01). These results show that cryopreservation of immature intact COC is a viable alternative to cryopreservation of mature oocytes. Further studies are needed to optimize either slow rate freezing or vitrification of intact COC and determine the developmental competence of cryopreserved oocytes following fertilization.

160 EFFECT OF cAMP MODULATORS ON IN VITRO MATURATION OF BOVINE OOCYTES

The efficiency of in vitro production (IVP) has remained low due to an inadequate in vitro maturation (IVM) system. Thus far, the most promising methods of improving IVM utilise cAMP modulators to slow the maturation process by keeping cAMP high. This experiment compares standard IVM to an extended IVM as previously described by Albuz (2010 Hum. Reprod. 25, 2999–3011). The extended IVM consists of a 2-h pre-IVM phase with FSK (an adenylate cyclase activator) and 3-isobutyl-1-methylxanthine [IBMX, a nonspecific phosphodiesterase (PDE) inhibitor], followed by a 31-h IVM phase with cilostamide (a PDE3 inhibitor). 3-Isobutyl-1-methylxanthine inhibits all PDE in both the oocyte and CC, whereas cilostamide inhibits PDE3 only in the oocyte, allowing a gradual reversal of inhibition. Bovine oocytes (n = 363) were obtained by transvaginal ultrasound-guided aspiration of both Brahman and Angus cattle over 4 collection days. Oocytes from each cow were divided into 2 groups. The first group was placed in standard 23-h IVM medium composed of TCM-199 supplemented with 10% fetal bovine serum, sodium pyruvate, penicillin and streptomycin, glutamine, and FSH, and cultured in 5% CO2 at 39°C. A subset of oocytes was removed from maturation at 8, 13, 18, and 23 h. The second group was placed into a pre-IVM medium of HEPES-TALP supplemented with 100 μM FSK and 500 μM IBMX for 2 h at 39°C, and then moved into standard maturation medium supplemented with 20 μM cilostamide for 31 h (5% CO2, 39°C). Oocytes were sampled at 8, 13, 18, 23, 28, and 33 h. Oocytes were stained with 1% orcein and nuclear status was examined for each sample time (Table 1). Data were analysed using a chi-squared test. At 8 h, there was a significant difference (P < 0.001) between GV stage of IVM and extended IVM (7.1 v. 73.2%), and between metaphase I (MI) stage IVM and extended IVM (76.2 v. 4.9%). At the 23-h time sample, there was a significant difference between metaphase II (MII) IVM and MII extended IVM (78.9 v. 30.6%; P < 0.001). There was also a significant difference between MII oocytes at 23 h IVM and MII oocytes at 33 h extended IVM (78.9 v. 33.3%; P < 0.001). These results are consistent with the hypothesis that cAMP modulators slow the nuclear maturation process. However, results also suggest that the inhibitory effect of the cAMP modulators is not completely reversible. Oocytes appear to arrest at MI by 23-h extended IVM and do not progress to MII at the same rate as standard IVM. Table 1.Nuclear status of bovine oocytes after standard IVM and extended IVM with cAMP modulators

33 PRODUCTION OF A CLONED FOAL USING MITOCHONDRIAL DNA-IDENTICAL OOCYTES

Recently we reported the birth of a viable foal produced by nuclear transfer (NT) using oocytes recovered from immature follicles of live mares by transvaginal ultrasound-guided aspiration (TVA; 2013 Theriogenology 79, 791–796). This procedure opens the door for production of mitochondrial DNA-identical cloned foals; typically, use of heteroplastic oocytes results in cloned offspring that have different mitochondrial DNA from that of the donor. We selected 2 mares (BL and SM) from the maternal line of the donor, a 23-year old stallion. Genetic analysis confirmed that the mares’ mitochondrial genotype was identical to that of the donor. Oocytes were obtained from the mares by TVA of all follicles ≥5 mm diameter, and were matured in vitro for 20 to 26 h. Donor fibroblasts were treated with 15 μM roscovitine for 24 h, then were directly injected into enucleated oocytes using a Piezo drill. Reconstructed oocytes were activated with 5 μM ionomycin for 4 min followed by injection with sperm extract, then incubation in 2 mM 6-dimethylaminopurine for 4 h. Oocytes from mare SM were assigned to treatment with either Scriptaid (500 nM) or Scriptaid plus vitamin C (50 μg mL–1) for 14 to 16 h, starting at the onset of 6-dimethylaminopurine exposure; mare BL did not provide sufficient oocytes for treatment grouping. Presumptive zygotes were cultured in vitro for 7 to 11 days and blastocysts were shipped for transfer to recipient mares, 1 embryo per mare. In mare BL, 10 aspiration sessions were conducted, 78 follicles were aspirated and 45 oocytes were collected, of which 4 were degenerating. After in vitro maturation, 12/40 (30%) oocytes were mature. Five of 12 oocytes lysed during manipulation; the remaining 7 were cultured and 1 blastocyst (14%) was obtained, which did not yield a pregnancy. In mare SM, 3 aspiration sessions were conducted and 53 oocytes were recovered from 81 follicles. After in vitro maturation, 31/53 (58%) were mature. Four oocytes were lysed during manipulation, 27 were cultured, and 4 blastocysts (15%) were produced, 2 from scriptaid treatment and 2 from scriptaid plus vitamin C. Transfer of these blastocysts yielded one pregnancy (scriptaid treatment); the mare delivered a healthy foal at 328 days of gestation. These results indicate that NT can be successful using low numbers of immature oocytes from selected mares. However, the individual mare may greatly affect the outcome in terms of oocyte number and quality; in this case, mare BL not only yielded fewer oocytes per aspiration session (4.5 v. 17.7 for mare SM; P < 0.001, t-test), but also fewer reconstructed oocytes per oocyte recovered (7/45 v. 27/53, respectively; P < 0.001, Fisher's exact test). Efficiency (14 to 15% blastocysts per reconstructed oocyte cultured; 1 foal from 5 embryos transferred) was similar to that achieved previously in our laboratory using heteroplastic oocytes. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, by Kit Knotts, and by Jack Waggoner.

171 IN VIVO AND IN VITRO MATURATION OF WOOD BISON (BISON BISON ATHABASCAE) CUMULUS–OOCYTE COMPLEXES DURING THE OVULATORY SEASON

Technologies are being developed to conserve the genetic diversity of wood bison. Knowledge of the characteristics of in vivo and in vitro maturation of the cumulus–oocyte complex (COC) are needed in wood bison to design efficient in vitro embryo production protocols. The objectives were to (1) determine the optimal interval after hCG treatment for in vivo maturation of COC in superstimulated wood bison, and (2) compare the characteristics of COC after in vitro and in vivo maturation. Ovarian synchronization was induced in 25 bison during October and November by giving a luteolytic dose of prostaglandin followed 8 days later by follicular ablation (Day –1). Ovarian superstimulation was induced with FSH (Folltropin-V) given i.m. on Day 0 (300 mg) and Day 2 (100 mg). A second luteolytic dose of prostaglandin was given on Day 3. Bison were assigned randomly to 5 groups (n = 5/group). The COC were collected by transvaginal follicle aspiration on Day 4 and were either assessed immediately (0 h, control), or matured in vitro for 24 or 30 h (in vitro maturation), or collected on Day 5 (in vivo maturation), 24 or 30 h after bison were given 2000 IU of hCG i.m. on Day 4. In vitro maturation was done in TCM-199 with 5% calf serum, 5 μg mL–1 LH, 0.5 μg mL–1 FSH, and 0.05 μg mL–1 gentamicin, at 38.5°C and in a 5% CO2 humidified atmosphere. Nuclear maturation was classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), or metaphase II (MII) with anti-lamin AC/DAPI staining. Groups were compared by analysis of variance and Fisher's exact test (Table 1). A mean (±s.e.m.) of 7.3 ± 1.7 COC were collected per bison, with no difference among groups. The COC in the control (0 h) group were at the nonexpanded GV stage. Cumulus cells were more expanded after in vivo than in vitro maturation, and the percentage of fully expanded COC was the highest in the 30-h in vivo maturation group (87%; P < 0.05). The greatest number of oocytes reached MII stage after 24 h of in vitro maturation, and 30 h of in vivo maturation. In conclusion, nuclear maturation occurred more quickly in vitro compared with in vivo, but the degree and incidence of cumulus expansion was greater after in vivo maturation. The competence of oocytes to undergo fertilization and develop into embryos remains to be investigated. Table 1.Cumulus expansion and nuclear maturation of wood bison oocytes

A new device for the aspiration of follicular fluid for acid-base balance analysis in cattle

The aim of this study was to evaluate a new device for the ultrasound-guided transvaginal aspiration of follicular fluid for acid-base balance analysis (ABB set) in comparison with the original modified commercial OPU set. In the ABB set, an aspiration syringe was placed in the front part of the new tool’s handle, next to the transducer, so as to enable direct collection of the sample into the syringe. To obtain a sufficient amount of testable fluid, reservoirs of urine (rubber balloons) were used for later aspiration under laboratory conditions in Experiment 1. Fifteen triads of samples (each triad with two punctures) were collected. While the first sample of each triad was taken using the ABB set (ABB sample), two samples were taken by one puncture using the original modified commercial OPU set: aerobic phase of sampling (AE sample) with air present in the tubing at the start of sampling and the subsequent anaerobic phase of sampling (AN sample). Values determined in the second sample from the triad (AE) varied from the values in both ABB and AN samples (pH 7.685 vs. 7.704 vs. 7.692, pCO<sub>2</sub> 11.13 vs. 10.3 vs. 10.85, pO<sub>2</sub> 6.87 vs. 8.67 vs. 7.02). In Experiment 2, ultrasound-guided transvaginal aspirations were carried out in 13 cows bearing ovarian cysts with diameters of at least 3 cm, using plastic aspiration syringes (Experiment 2P) and in 12 cows using glass aspiration syringes (Experiment 2G). The sequence of samples was the same as in Experiment 1. We found a significantly higher pH in AE in comparison to AN (7.357 vs. 7.348), lower pCO<sub>2</sub> (6.85) and higher O<sub>2</sub> (14.12) in samples of AE in comparison to samples of ABB and AN (pCO<sub>2</sub> 7.36, 7.30; O<sub>2</sub> 9.95, 10.63 respectively) in cystic fluid in Experiment 2P. We found a significantly higher pH (7.4), lower pCO<sub>2</sub> (5.98) and a higher pO<sub>2</sub> (12.35) in AE samples in comparison to ABB and AN samples of cystic fluid (pH 7.386, 7.385; pCO<sub>2</sub> 6.39, 6.35 and O<sub>2</sub> 10.56, 10.65, respectively) from Experiment 2G. We conclude that the acid-base balance assay was affected by air, present in the tubing during aerobic sampling in comparison to anaerobic and ABB set sampling. These pre-analytical changes can be prevented by the use of the ABB set because the results obtained with the ABB set were not different from that of the AN samples. We also confirmed pre-analytical changes in acid-base balance parameters in the cystic fluid after it had been stored in plastic aspiration syringes. Our new ABB set equipped with a glass aspiration syringe is suitable for sampling follicular fluid for both acid-base balance and gas analysis.

Ultrasound-Guided Aspiration and Ethanol Sclerotherapy (EST) for Treatment of Cyst Recurrence in Patients after Previous Endometriosis Surgery: Analysis of Influencing Factors Using a Decision Tree

Study ObjectiveTo evaluate the efficiency of transvaginal aspiration accompanied by ethanol sclerotherapy for treating cyst recurrence in patients who have previously undergone surgery to treat endometriosis and to analyze various factors that influence success rates using a data mining system. DesignRetrospective cohort study (Canadian Task Force classification II-3). SettingTeaching hospital affiliated with Chang Gung University, Taoyuan, Taiwan. PatientsOne hundred ninety-six patients with endometrioma recurrence. InterventionA total of 274 transvaginal aspirations followed by sclerotherapy with 95% ethanol. Treatment times varied from immediate removal (0–10 minutes) to in situ retention. Patients were followed up at 3, 6, and 12 months to detect complications, determine the size and persistence of cysts, obtain the pelvic pain score, and assess for pregnancy or the need for repeat surgical intervention. A decision tree was used to determine factors from the collected data that most influenced the success of treatment. Measurements and Main ResultsCyst size was consistently reduced until 6 months after ethanol sclerotherapy. The mean (SD) cyst reduction rate was 37.2% (42.2%), and the pain score reduction rate was 20.5% (71.5%). The antral follicle count was simultaneously increased by 36.4%. Sixty-three patients (23%) required repeated surgery during the observation period and were treated with either repeat aspiration (13.5%) or major laparoscopic or open laparotomic interventions (8.4%). Eighteen of 101 infertile patients (17.8%) achieved pregnancy. The total recovery rate (pregnancy or no persistence of symptoms or cyst) was significantly higher in patients in the groups that received longer treatment (7–10 minutes and retention) than in the groups with shorter treatment (0–6 minute) (47.0% vs 28.7%; p < .005). The highest recovery rate was observed in patients with longer treatment time, smaller cysts (≤5.05 cm), lower CA 125 level (≤62.03 IU/mL), and fewer cysts (≤3 cm) (35 of 49 [71.4%]). In patients with larger cysts and cysts with clear contents, better success can be achieved with longer treatment. The use of postoperative ovarian suppression, traditional Chinese medicine, or no therapy for 6 months before the study was not significant among groups. ConclusionUltrasound-guided sclerotherapy with 95% ethanol retention is an effective alternative therapy for recurrent ovarian endometrioma, in particular in selected patient groups.

Deleterious impact of propofol on in vitro fertilization. a prospective randomized trial

Propofol is widely used for anesthesia in oocyte transvaginal aspiration. In vitro studies on animal models have shown that propofol could be deleterious for fertilization and embryo development in a dose- and time-dependent manner. The purpose of this study was to analyse the potential negative effects of propofol on human in vitro fertilization outcomes. Prospective-double blind, randomized trial. 144 patients with more than four metaphase II oocytes retrieved were enrolled in this study and randomly separated. Two protocols of anesthesia were used. Group A: (GA, n=74) received intravenous (IV) propofol 2 mg/kg, midazolam 0.015 mg/kg and fentanyl 1 μg/kg. Group B: (GB, n=74) IV propofol 1.5 mg/kg, midazolam 0.05 mg/kg and fentanyl 1.5 μg/kg. Maintenance of anesthesia was achieved by intermittent IV bolus when required. Quantity of propofol, duration and quality of anesthesia, number of oocytes, embryo quality and pregnancy rate were evaluated. Mean doses of propofol were GA: 0.277 ± 0.080 mg/kg.min and GB: 0.195 ± 0.055 mg/kg.min (p< .01). No significant differences were found among anesthetics parameters, patient age, number of oocytes retrieved, fertilization rate and embryo quality in both groups. A significative difference was found in clinical pregnancy rate between groups (GA: 27.0% vs GB: 43.3%, p < .05). Our findings suggest that the administration of high doses of propofol produces a negative late effect on human embryo development. The use of lower doses of propofol and the consistent lower exposure of the oocytes to the drug, show an improvement in clinical pregnancy rate, maintaining quality of anesthesia and optimal postoperative recovery. According to our results, we postulate that the reduction of propofol could improve the results of in vitro fertilization treatments.

Transvaginal sono-guided aspiration of gestational sac concurrent with a local methotrexate injection for the treatment of unruptured cesarean scar pregnancy

Cesarean scar pregnancy (CSP) is one of the rarest forms of ectopic pregnancy. A delay in treatment can lead to massive bleeding, uterine rupture, and life-threatening maternal morbidity. We present a conservative method for the management of CSP at a single tertiary centre over a 6-year period. Eleven patients with unruptured CSPs who were treated by transvaginal aspiration of the gestational sac followed by a local methotrexate injection were evaluated. Gestational age at diagnosis ranged from 5 + 2 weeks to 7 + 4 weeks. Seven of the patients had undergone two prior Caesarean sections (63.6 %). The levels of β-hCG at the time of diagnosis ranged from 1,290 to 81,586 mIu/ml. The mean time of the procedure was 8.2 ± 1.6 min. During follow up, 54.5 % of the patients may need an additional systemic MTX injection due to an elevation of β-hCG. Estimated blood loss of the procedure was <50 ml and no blood transfusion is needed. This method has a shorter operative time, less blood loss and no hospitalization is needed for CSPs. All patients had their uterus successfully preserved without maternal morbidity or mortality. Transvaginal sono-guided sac aspiration concurrent with a local MTX injection is an effective management option for preserving the fertility of women with an unruptured CSP. However, additional systemic MTX injection may be needed if β-hCG levels >20,000 mIU/ml at diagnosis.

A viable foal obtained by equine somatic cell nuclear transfer using oocytes recovered from immature follicles of live mares

The presence of heterogenous mitochondria from the host ooplast affects the acceptance of offspring obtained by somatic cell nuclear transfer. This might be avoided by obtaining oocytes from selected females, but is then complicated by low numbers of available oocytes. We examined the efficiency of equine somatic cell nuclear transfer using oocytes recovered by transvaginal aspiration of immature follicles from 11 mares. Use of metaphase I oocytes as cytoplasts and of scriptaid (a histone deacetylase inhibitor) treatment during oocyte activation were evaluated to determine if these approaches would increase blastocyst production. In experiment 1, blastocyst development was 0/14 for metaphase I oocytes and 4/103 (4%) for metaphase II oocytes. Three blastocysts were transferred to recipient mares, resulting in two pregnancies and one live foal, which died shortly after birth. In experiment 2, blastocyst development was 2/47 (4%) for control oocytes and 1/83 (1%) for scriptaid-treated oocytes. No foals were born from two blastocysts transferred in the control group. The blastocyst from the scriptaid treatment resulted in birth of a live foal. In conclusion, this is apparently the first report of production of a viable cloned foal from oocytes collected from immature follicles of live mares, supporting the possibility of cloning using oocytes from selected mares.