Transvaginal Aspiration Research Articles

Transvaginal aspiration, oocyte maturation and production of blastocysts and a pregnancy via ICSI in an endangered breed: Baudet du Poitou donkey

Two Poitou donkey jennies were presented for clinical oocyte recovery and embryo production via intracytoplasmic sperm injection (ICSI). Both jennies underwent transvaginal ultrasound-guided follicle aspiration on two occasions. Recovered oocytes were held overnight then placed into maturation culture, using standard methods for mare oocytes. On the first replicate for both jennies, the oocytes were divided into two groups; one group was denuded and examined at 30 h culture (standard culture duration for mare oocytes) and the second was denuded and examined at 36 h culture. No oocytes with polar bodies were observed at either time. The oocytes were maintained in maturation culture until 46 h, at which time oocytes with polar bodies were observed. Semen was then prepared; oocytes underwent ICSI approximately 48 h after being placed into maturation culture. On the second replicate for both jennies, oocytes were cultured for maturation for 42 h, then denuded and subjected to ICSI at 46 h. Sperm preparation, injection and embryo culture were performed as for mare oocytes. Blastocyst rates per injected oocyte were 8/19 (42 %) overall, being 4/12 and 4/7 for the first and second TVAs, respectively. Blastocysts were vitrified. Three blastocysts were warmed and transferred to Poitou donkey jenny recipients. One embryonic vesicle was visualized on ultrasonography on embryo Day 12, which increased in size on Day 13 but was not present when examined on Day 14. These results demonstrate that oocyte recovery and ICSI are efficient for production of Poitou donkey blastocysts. To the best of our knowledge, this is the first report of production of blastocysts via ICSI in the Poitou donkey, and the first report of transfer of ICSI-produced embryos in the donkey. Further work is needed on factors affecting pregnancy after embryo transfer in the donkey.

CASE REPORT: A SUCCESSFUL TREATMENT STRATEGY FOR CYSTIC OVARIAN DISEASE IN A DAIRY COW

A 5-year-old multiparous lactating Holstein cow was reported to have cystic ovarian disease with a history of whitish vaginal discharge on the 80th day postpartum. On the 83rd day postpartum, transrectal ultrasonography revealed a thick-walled cystic structure with visible cobwebs in the cavity, measuring 4.2[Formula: see text]cm × 3.4[Formula: see text]cm in diameter on the right ovary. The serum progesterone (P4) concentration was 6.82[Formula: see text]ng/mL, leading to a diagnosis of luteinized follicular cyst. Transvaginal aspiration of the cyst was performed to decrease the compression of ovarian stroma by the cyst, followed by intramuscular administrations of cloprostenol and buserelin. A 1.9[Formula: see text]cm diameter corpus luteum was detected on the right ovary nine days after aspiration, with a serum P4 concentration of 1.29[Formula: see text]ng/mL. Continual hormone treatment was followed, consisting of an injection of buserelin and an intravaginal progesterone-releasing device inserted for seven days. After seven days, two corpora lutea were noted on the right ovary with an average diameter of 2.8[Formula: see text]cm and 1.8[Formula: see text]cm, respectively. The device was removed and the cow showed a P4 concentration of 11.22[Formula: see text]ng/mL on that day. Estrus induction was conducted with two low doses of cloprostenol followed by an injection of gonadorelin, to induce a complete luteolysis and the following ovulation to fit a fixed-timed artificial insemination (AI). Pregnancy was confirmed 33 days after AI. Although the number of days open was 102 in this case, it took a total of 19 days from treatment to pregnancy.

Cystic Cervicitis and Infertility, A New Treatment Modality: A Case Report and Review of the Literature

We present a rare case of cystic cervicitis in a 42-year-old woman who came to our unit suffering from primary infertility for one year. Her only complaint was infertility. She was not suffering from chronic pelvic pain or increased vaginal discharge neither before nor after marriage. Diagnosis of cystic cervicitis was made by both transvaginal ultrasound examination and MRI. As her main problem was infertility, she refrained from doing cone biopsy and opted for an ICSI trial and aspiration of the cervical cysts at the same time of ovum pick up (OPU). Ovum pick up resulted in the recovery of two oocytes. On the same setting and using the same needle, aspiration of all of the cervical cysts was performed successfully. Aspirated fluid was sent for cytology as well as culture and sensitivity. To our knowledge, this is the first case, in the english literature, to provide complete diagnosis of, and describe treatment of cystic cervicitis by transvaginal aspiration of the cysts.

Effects of cortisol on granulosa cell function during follicle maturation in the horse

Follicle maturation and acquisition of developmental competence by the oocyte require an interplay between granulosa cells and the oocyte, involving Growth Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) mediated modulation of follicular steroidogenesis. In the horse, dynamic intrafollicular concentrations of cortisol were associated with follicle growth and oocyte maturation, suggesting a functional relationship between cortisol and oocyte-derived factors. The aim of this study was to analyze effects of cortisol on steroidogenic activity, apoptosis and proliferative capacity of equine granulosa cells, as well as the relationship with the oocyte-derived factor GDF9. For this purpose, granulosa cells were collected three times in four mares each during the breeding season by transvaginal aspiration in the absence of a dominant follicle. Cells collected on the same day were pooled and then seeded on 12-well plates to grow to 70-80% confluence before being stimulated with 100 ng/ml IGF1 for 24h, in the presence or absence of cortisol in increasing concentrations: 50, 100 and 150 ng/ml. These concentrations were previously observed in antral follicles before deviation (50 ng/ml) and after deviation (120 ng/ml). In blocking experiments, cells were preincubated with 20 uM H89 (protein kinase A inhibitor). All experiments were repeated three times. GraphPad software was used for statistical analysis with one-way analysis of variance (ANOVA), followed by non-parametric multiple comparison post-hoc test.Expression of Star was similar in all groups (P>0.05), independent of cortisol concentration and was decreased (P<0.05) after preincubation with H89. Transcriptional levels of HSD3B2 were higher (P<0.001) after adding IGF1 and cortisol simultaneously, but not separately. The rise in HSD3B2 levels was independent of cortisol dose used (P>0.05). Blocking of Star with H89 increased GDF9 expression (P<0.01), both in the presence and absence of cortisol. Interestingly, addition of cortisol alone in all analyzed dosages raised BCL2 levels (P<0.05) and this effect was inhibited by H89 (P<0.05). However, there was no effect on CASP3 levels (P>0.05), regardless of treatment used. A higher proliferating capability of equine granulosa cells, as assessed by PCNA levels, was observed in response to IGF1 treatment (P<0.05), but only in the presence of 150 ng/ml cortisol. Despite apparently elevated, the expression of Ki67 marker in response to IGF1 in the presence of cortisol proved to be not significant (P>0.05). Taken together, our results demonstrate enhanced steroidogenesis as well as reduced apoptosis in equine granulosa cells in response to cortisol. Moreover, reduced steroidogenic activity (i.e. decreased expression of genes involved in progesterone synthesis) of granulosa cells increased GDF9 expression, which has previously been shown to be detrimental for the acquisition of developmental competence by the oocyte.

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.

Molecular effect of holding equine oocytes: a road to cytoplasmic maturation?

The quality of oocytes has a direct effect on the outcome of in vitro embryo production (IVP). For successful IVP, oocytes need to undergo maturation, which requires coordinated nuclear (NM) and cytoplasmic maturation (CM). The lack of adequate cytoplasmic maturation has been proposed to be associated with a lower developmental competence of the oocyte. The improvement of in vitro techniques relies upon mirroring in vivo conditions. Therefore, acquiring a deeper knowledge of the process involved in CM and pathways leading to germinal vesicle breakdown and maturation can provide the necessary foundation to improve IVP results. Holding oocytes in commercial embryo holding media for 24hrs at room temperature has become a routine procedure for oocyte shipment and laboratory scheduling. However, the effects of this procedure on oocyte CM remain unresolved. In this study, we analyzed the changes induced by the holding period on the transcriptome of equine oocytes (OC) and the surrounding cumulus cells (CC). Cumulus-oocyte-complexes (COCs) were collected from six mares using transvaginal aspiration. Only non-expanded, non-degenerated COCs were randomly allocated to one of two groups: no holding (NH) or 24hrs holding (H). The COCs were denuded, and each single OC and CC were stored individually in RNAlater® at -20°C until further processing. RNA was extracted from 24 samples (NH/CC=6; NH/OC=6; H/CC=6; H/OC=6), followed by librarypreparation and sequencing (150 pb-PE). Reads were trimmed and mapped to the equine reference genome (Ecab 3.0). Mapped reads were quantified using featureCount and analysis of differentially expressed genes (DEGs) was performed using DESeq2 (FDR< 0.1 and log fold change >2). Overall, there were 26,077 genes expressed in CCs and 17,930 in OCs. When compared by holding time, there were 660 DEGs between the CCs (NH vs. H) and 205 between the OCs. In CC, 74 genes had a higher expression and 586 had a lower expression in H than NH groups. In OC, 7 genes were upregulated, and 198 were downregulated in H compared to NH group. Gene ontology analysis showed changes in genes related to glutathione (GSH) synthesis, AP-1 transcription factor complex and mitogen-activated protein kinase (MAPK) pathway in CC. In OC, DEGs were involved in α-tubulin detyrosination and tight junction proteins involved in magnesium transport. Overall, our results suggest modification in processes related to CM, including post-translational changes in tubulin, GSH composition, and activation of MAPK pathways during oocyte holding. Meanwhile, genes considered markers of NM and meiotic resumption remained unchanged, suggesting pre-maturation like changes occur prior to germinal vesicle breakdown. Further studies to understand the implications of this pre-maturation step in the developmental competence of oocytes can help improve the outcome of equine IVP. Funding: Center for Equine Health

Treatment after endometrioma recurrence: a narrative review.

Ovarian endometrioma is a common subtype of endometriosis with a prevalence between 17 and 44%. The reported average recurrence of endometrioma after surgical management is 21.5% after 2 years and 40-50% after 5 years. The aim of this narrative review was to summarize the existing literature focusing on treatment options after endometrioma recurrence in order to provide an evidence-based approach for the clinical practice. Three electronic databases (MEDLINE, EMBASE and Cochrane) were searched until September 2022 for eligible studies. The available studies showed that repeated surgery has a negative impact on ovarian function, without improving the fertility outcomes. Transvaginal aspiration as an alternative option for surgery has a high rate of recurrence, which varies from 8.20 to 43.5% depending on the technique used and on the study population. Pregnancy related outcomes were similar between transvaginal aspiration groups and no intervention groups in patients with endometrioma recurrence. Regarding medical treatments, only four studies were found, showing that progestins reduce the pain and the diameter of the ovarian cyst. Recurrent endometrioma is a challenging condition which could be encountered during the care of women with endometriosis. The decision about the treatment-strategy has to be individualized considering family planning status, age, ovarian reserve and transvaginal ultrasound findings. Well-designed randomized clinical trials are needed to export safer conclusions about the most appropriate treatment in each specific condition after endometrioma recurrence.

Pregnancy rate after trans-vaginal aspiration of ovarian endometrioma in infertile patients

Pregnancy rate after trans-vaginal aspiration of ovarian endometrioma in infertile patients

Preovulatory serum estradiol concentration is positively associated with oocyte ATP and follicular fluid metabolite abundance in lactating beef cattle.

Cattle induced to ovulate a small, physiologically immature preovulatory follicle had reduced oocyte developmental competence that resulted in decreased embryo cleavage and day 7 embryo quality compared with animals induced to ovulate a more advanced follicle. RNA-sequencing was performed on oocytes and their corresponding cumulus cells approximately 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the preovulatory gonadotropin surge suggested reduced capacity for glucose metabolism and oxidative phosphorylation in the cumulus cells and oocytes from follicles ≤11.7 mm, respectively. We hypothesized that induced ovulation of a small, physiologically immature preovulatory follicle results in a suboptimal follicular microenvironment and reduced oocyte metabolic capacity. We performed a study with the objective to determine the impact of preovulatory follicle diameter and serum estradiol concentration at GnRH administration on oocyte metabolic competence and follicular fluid metabolome profiles. We synchronized the development of a preovulatory follicle and collected the follicle contents via transvaginal aspiration approximately 19 h after GnRH administration in lactating beef cows (n = 319). We determined ATP levels and mitochondrial DNA (mtDNA) copy number in 110 oocytes and performed ultra-high-performance liquid chromatography–high resolution mass spectrometry metabolomic studies on 45 follicular fluid samples. Intraoocyte ATP and the amount of ATP produced per mtDNA copy number were associated with serum estradiol concentration at GnRH and time from GnRH administration to follicle aspiration (P < 0.05). mtDNA copy number was not related to follicle diameter at GnRH, serum estradiol concentration at GnRH, or any potential covariates (P > 0.10). We detected 90 metabolites in the aspirated follicular fluid. We identified 22 metabolites associated with serum estradiol concentration at GnRH and 63 metabolites associated with follicular fluid progesterone concentration at the time of follicle aspiration (FDR < 0.10). Pathway enrichment analysis of significant metabolites suggested altered proteinogenesis, citric acid cycle, and pyrimidine metabolism in follicles of reduced estrogenic capacity pre-gonadotropin surge or reduced progesterone production by the time of follicle aspiration.

Heat-induced increases in body temperature in lactating dairy cows: impact on the cumulus and granulosa cell transcriptome of the periovulatory follicle.

Cows acutely heat stressed after a pharmacologically induced luteinizing hormone (LH) surge had periovulatory changes in the follicular fluid proteome that may potentiate ovulation and impact oocyte developmental competence. Because the cellular origins of differentially abundant proteins were not known, we have examined the cumulus and granulosa cell transcriptomes from the periovulatory follicle in cows exhibiting varying levels of hyperthermia when occurring after the LH surge. After pharmacological induction of a dominant follicle, lactating dairy cows were administered gonadotropin releasing hormone (GnRH) and maintained in thermoneutral conditions (~67 temperature-humidity index [THI]) or heat stress conditions where THI was steadily increased for ~12 h (71 to 86 THI) and was sufficient to steadily elevate rectal temperatures. Cumulus-oocyte complexes and mural granulosa cells were recovered by transvaginal aspiration of dominant follicle content ~16 h after GnRH. Rectal temperature was used as a continuous, independent variable to identify differentially expressed genes (DEGs) increased or decreased per each 1 °C change in temperature. Cumulus (n = 9 samples) and granulosa (n = 8 samples) cells differentially expressed (false discovery rate [FDR] < 0.05) 25 and 87 genes, respectively. The majority of DEGs were upregulated by hyperthermia. Steady increases in THI are more like the "turning of a dial" than the "flipping of a switch." The moderate but impactful increases in rectal temperature induced modest fold changes in gene expression (<2-fold per 1 °C change in rectal temperature). Identification of cumulus DEGs involved in cell junctions, plasma membrane rafts, and cell-cycle regulation are consistent with marked changes in the interconnectedness and function of cumulus after the LH surge. Depending on the extent to which impacts may be occurring at the junctional level, cumulus changes may have indirect but impactful consequences on the oocyte as it undergoes meiotic maturation. Two granulosa cell DEGs have been reported by others to promote ovulation. Based on what is known, several other DEGs are suggestive of impacts on collagen formation or angiogenesis. Collectively these and other findings provide important insight regarding the extent to which the transcriptomes of the components of the periovulatory follicle (cumulus and mural granulosa cells) are affected by varying degrees of hyperthermia.

Developmental Competence of equine MI oocytes following in vitro maturation culture

With an increasing demand for the in vitro production (IVP) of equine embryos over the past 10 years, the value of each oocyte recovered has also increased. Intracytoplasmic sperm injection (ICSI) is performed to fertilize the oocytes reaching MII stage of nuclear maturation, which is determined by microscopic evidence of an extruded polar body (PB). Cytoplasmic maturation, during which replication of organelles, proteins and RNA's necessary for postfertilization development occurs, is more difficult to evaluate. However, equine oocytes contain a large amount of cytoplasmic lipids and the migration and polarization of lipid granules can be microscopically visualized in mature oocytes. Furthermore, the distribution of lipids may reflect a level of cytoplasmic capacitation and developmental competence, although the cleavage rate of equine oocytes fertilized following maturation culture does not appear to be significantly different based on lipid polarization (Ambruosi et al. Therio 2009; 71:1093-1104). The present study evaluated the migration and polarization of cytoplasmic lipids (granularity) following in vitro maturation to determine if blastocyst development can occur in the absence of PB extrusion. Cumulus-oocyte-complexes (COC; n=411) obtained from ovarian follicles by transvaginal aspiration were stored in a holding medium overnight at 20°C before undergoing incubation at 38.5°C in 50ul drops of equilibrated maturation media as described by Foss. et al (EVJ Supp 2013;45:39-43). After 28.5 hours, oocytes were denuded followingexposure to 80IU hyaluronidase and evaluated for the presence of an extruded PB as well as lipid reorganization. The cytoplasm of MI oocytes that did not exhibit PB extrusion was graded according to the percentage with a dark granular appearance. All oocytes were fertilized by conventional ICSI with thawed spermatozoa from ICSI-fertile stallions. Fertilized oocytes were cultured as described by Foss et al (EVJ Suppl 2013;45:39-43). When examined under an inverted 1000X light microscope, 66% (N=273/411) of the denuded oocytes exhibited an intact membrane with an extruded polar body and were considered mature (MII), while 26% (N=106/411) of the oocytes without a visible polar body were classified as immature (MI). Of the MI oocytes injected, 23% (N=24/106) were deemed to have attained some level of cytoplasmic capacitation (Cc=40-70% granularity). A cleavage rate of 25.5% was recorded for all injected MI oocytes regardless of Cc status. Of the 24 MI oocytes with some Cc, nine (37%) cleaved and continued to develop, and seven of those developed to the blastocyst stage. Of the 82 MI oocytes that did not demonstrate Cc, 18 (22%) cleaved but no blastocysts (0%) were obtained. This study found a blastocyst developmental potential of 6.6% per total MI's injected and 29% per MI oocytes with at least a 40-70% Cc. Therefore, injecting oocytes that do not show nuclear maturation, yet have reached a level of cytoplasmic capacitation, may still result in blastocyst development.

Effect of Transvaginal Aspiration of Oocytes on Blood And Peritoneal Fluid Parameters in Mares

Transvaginal aspiration of oocytes (TVA) is a commonly used clinical procedure to obtain oocytes for in vitro production of embryos in horses. The goal of this study was to evaluate the effect of the TVA procedure on blood and peritoneal parameters, and to investigate the association of these findings with variables such as use of antibiotics, number of ovarian punctures, and length of the procedure. Physical examination was performed and blood and peritoneal fluid were obtained from 14 mares before they underwent TVA and the same parameters were assessed 24 hours after the procedure. On examination, 13/14 mares remained clinically healthy after the procedure. One mare developed low-grade fever, transient anorexia and mild colic that resolved within 12 hours post-TVA. The use of antibiotics, length of procedure and number of ovarian punctures did not have an effect on the measured parameters. All the mares presented significant changes in the leukogram, but these mostly remained within normal reference range values. The peritoneal parameters were also consistently affected after TVA. A significant post-TVA increase in lactate, total protein, and peritoneal neutrophil count was observed in peritoneal fluid. Peritoneal lactate level was elevated above baseline physiological levels in more than 50% of the mares. Results from this study indicate that there is an expected degree of inflammation after TVA procedures and peritoneal fluid parameters could be successfully used to monitor inflammation in the early stages.

376 Ovarian remnant syndrome and recurrent ovarian serous cystadenoma

Introduction and aims: Ovarian remnant syndrome (ORS) is a rare gynecological complication, most often induced by difficult salpingoophorectomy that leave residual ovarian tissue on the pelvic wall. Recurrent serous cystadenoma may arise from ORS. Our objective was to enhance the importance of diagnosis and surgical management in patients with ORS. Methods: We describe the case of a patient with continuous interventions before radical surgery for ORS. She had a right nephrectomy at age 43, a total abdominal hysterectomy at 45, and bilateral adnexectomy for serous cystadenoma at age 47. After thirteen years of continuous transvaginal aspirations for recurrent cystadenoma, and two laparotomies for cystectomies she was referred to our center. She presented with an abdomino-pelvic mass of 22cm, pain, and compression symptoms. Basic surgical principles were followed: high religation and resection of gonadal vessels; bilateral stripping and excision of the pelvic side wall peritoneum; wide excision of tissue surrounding the remnant ovary. Results and discussion: She needed partial bladder and sigma resection, and comprehensive ureterolysis for complete surgical resection, without significant postoperative complications. Histollogically, remnant ovarian tissue was associated with serous cystadenoma. After being heavily pretreated the modest risk of bowel, bladder or ureteral trauma caused definitive sidewall stripping and vaginal excision. Nearly two years later it occurred a low-rate growing cystic colonic perianastomosis recurrence of 3cm, which has reached 6 cm to date, and remains completely asymptomatic. Conclusions: Meticulous surgery is advised to assure the best outcome to patients. Even a benign non-threatening lesion such as ovarian serous cystadenoma may become a locorregional problem, with aggressive behavior similar to low-grade malignant lesions.

​Effect of Season on Quantity and Competence of Oocytes Recovered Transvaginally from Holstein Cows for In vitro Fertilization

Background: Season of the year can affect the reproductive behavior in Holstein cows, altering the competition of the oocytes, reflecting a reduced production of embryos. The objective of this study was to evaluate the average of total oocytes, competition of oocytes and embryos in the in vitro production process at different season of the year in Holstein cows. Methods: During the four seasons of the year, was performed on each of the oocyte donor cows (winter, n = 957; spring, n = 1571; summer, n = 1776; autumn, n = 1128), by in vivo transvaginal follicular aspiration technique after the collection were subjected to the embryos production in vitro. Result: The highest number of total embryos were produced in winter and autumn, compared to spring and summer (3.76±0.16 and 3.54±0.18 vs. 2.73±0.11 and 2.45±0.10; respectively, P less than 0.05). During winter, a higher percentage of oocyte competition was observed, followed by autumn and spring and less competition shown in summer (26.03±0.39, 19.08±0.29, respectively, P less than 0.05). The quantity and competence of the oocytes collected and in vitro embryo production were drastically reduced during the hottest months of the year in this area of intense heat.

Transvaginal Aspiration for Management of Huge Ovarian Endometrioma in Pregnancy

Study Objective To demonstrate a minimally invasive approach of a huge ovarian cyst during pregnancy. Design A step-by-step explanation of the patient's condition, diagnosis, surgical technique, and post-operative results. Setting The patient underwent transvaginal procedure in the lithotomy position and under intravenous sedation and ventilation by face mask. Patients or Participants A 34-year-old woman, gravida 1 at 12 weeks 5 days’ gestation, presented with huge ovarian cyst. A transvaginal ultrasound demonstrated ovarian cyst measuring 10.4 × 8.2 cm with no evidence of malignancy, no free fluid, and a normal-appearing contralateral ovary. The crown-rump length measured 5.92 cm. On non-enhanced magnetic resonance imaging (MRI), 11 × 7 cm sized cystic lesion with high signal intensity in T1-weighted image (WI) and T1-weighted fat suppressed image, low signal intensity in T2 WI suggesting endometrioma, located in cul-de-sac was noted. Because the cyst was huge and deeply located in cul-de-sac, and the uterus was enlarged to 13 weeks size, approach through laparoscopy or even laparotomy was thought to be difficult and the risk of manipulation for uterus was high. So, we decided to aspirate the ovarian cyst through the vaginal route. Interventions Ultrasound-guided transvaginal aspiration was performed using an 18-G needle. Measurements and Main Results The operation time was 4 hours and 35 minutes with 305 mL dark brownish fluid drained. The patient recovered well and was discharged 2days after the procedure. There was no intraoperative or postoperative complication. Postoperative ultrasound revealed neither recurrence nor fetal abnormality. Until delivery, the patient was free of symptom and ultrasound revealed neither recurrence nor fetal abnormality. Conclusion In select pregnant woman with endometrioma requiringintervention, the risks of surgery may be minimized with aspiration. Transvaginal ultrasound-guided aspiration is a safe, feasible, and effective minimally invasive procedure for huge ovarian endometrioma in pregnancy. To demonstrate a minimally invasive approach of a huge ovarian cyst during pregnancy. A step-by-step explanation of the patient's condition, diagnosis, surgical technique, and post-operative results. The patient underwent transvaginal procedure in the lithotomy position and under intravenous sedation and ventilation by face mask. A 34-year-old woman, gravida 1 at 12 weeks 5 days’ gestation, presented with huge ovarian cyst. A transvaginal ultrasound demonstrated ovarian cyst measuring 10.4 × 8.2 cm with no evidence of malignancy, no free fluid, and a normal-appearing contralateral ovary. The crown-rump length measured 5.92 cm. On non-enhanced magnetic resonance imaging (MRI), 11 × 7 cm sized cystic lesion with high signal intensity in T1-weighted image (WI) and T1-weighted fat suppressed image, low signal intensity in T2 WI suggesting endometrioma, located in cul-de-sac was noted. Because the cyst was huge and deeply located in cul-de-sac, and the uterus was enlarged to 13 weeks size, approach through laparoscopy or even laparotomy was thought to be difficult and the risk of manipulation for uterus was high. So, we decided to aspirate the ovarian cyst through the vaginal route. Ultrasound-guided transvaginal aspiration was performed using an 18-G needle. The operation time was 4 hours and 35 minutes with 305 mL dark brownish fluid drained. The patient recovered well and was discharged 2days after the procedure. There was no intraoperative or postoperative complication. Postoperative ultrasound revealed neither recurrence nor fetal abnormality. Until delivery, the patient was free of symptom and ultrasound revealed neither recurrence nor fetal abnormality. In select pregnant woman with endometrioma requiringintervention, the risks of surgery may be minimized with aspiration. Transvaginal ultrasound-guided aspiration is a safe, feasible, and effective minimally invasive procedure for huge ovarian endometrioma in pregnancy.

PSXVI-16 The quality of cows’ oocyte derived at different timing regimens of transvaginal follicular puncture

Abstract Recovery of oocytes from live animals through ultrasound-guided transvaginal follicular aspiration (Ovum Pick-Up, OPU) is an essential element of modern livestock development. The aim of our work was to study the effect of two different timing regimens on the quality of oocytes, recovered from cycled Simmental heifers (n = 4). Follicle aspirations were performed once a week (1/w) or twice a week (2/w). The same donor animals were used for each timing regimens. 228 and 224 follicles were aspirated at 2/w and 1/w regimen, respectively, and 280 cumulus-oocyte complexes (COCs) were recovered (140 COCs at each of timing regimen), which corresponds to the recovery rate of 61.4±5.9% and 62.1±6.3%. On average, 4.4 oocytes were received from each donor per session. We observed a significant increase by 1.2 times (P &amp;lt; 0.05) of the proportion of OPU-oocytes of good quality, which were characterized by normal morphology, when performing the procedure in the 2/w compared to 1/w regimen (65.7±4.0% vs. 53.6±3.0% of the total number of recovered oocytes, respectively). The proportion of oocytes at the MII stage of meiosis after 24 hours of maturation was similar in both groups: 73.3±5.6% (2/w regimen) and 74.7±2.4% (1/w regimen). Proportion of oocytes with signs of apoptotic changes was also not significantly differed between the groups: 8.0±3.1 and 9.8±3.3%, respectively. We did not observe the effect of the frequency of OPU sessions on the embryo cleavage rate (on average 63.5%) and the blastocysts development rate (on average 16.7%). Our data will be used to optimize the timing regimen of oocyte retrieval from living Simmental donor heifers. The studies were performed under financial support of the RFBR (project No. 20-316-90003) and the Russian Ministry of Science and Higher Education.

OP07.05: Recurrence rate after ultrasound‐guided transvaginal aspiration of benign adnexal masses with and without sclerotherapy: a systematic review and meta‐analysis

OP07.05: Recurrence rate after ultrasound‐guided transvaginal aspiration of benign adnexal masses with and without sclerotherapy: a systematic review and meta‐analysis

Culture protocols for horse embryos after ICSI: Effect of myo-inositol and time of media change

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.

Identification and treatment of a cervical sinus tract in a patient with 10 years of infertility

To introduce a special case of endometrial cavity fluid (ECF), highlighting the application of hysteroscopy and laparoscopic surgical techniques in the treatment of cervical sinus tract. Narrated video featuring the diagnosis and surgical management of a case of recurrent ECF. Informed consent was obtained from the patient, and approval was granted by the ethics committee of the First Affiliated Hospital of the Wenzhou Medical University. Academic tertiary hospital. A 36-year-old woman, gravida 0, had menstrual spotting for 13 years after abdominal myomectomy of a 104 × 86 × 111-mm myoma on the posterior uterine wall near the cervix. She failed to conceive after her marriage for 10 years, and 5 operations, including hysteroscopy and laparoscopy, were performed to increase pregnancy opportunities. She also underwent invitro fertilization and embryo transfer procedures many times, but failed. Transvaginal sonography preoperatively suggested that ECF sometimes appeared and sometimes disappeared. The local echo of the posterior wall of the cervix was enhanced. A 40-mm cystic dark area was found beside the right ovary, which seemed to connect with the cervical hyperechoic part. Additionally, a solid mass of the right adnexa with abundant blood supply was detected. First, hysteroscopy was performed to explore the ECF. A deep and narrow cervical sinus with a steady stream of accumulated blood overflowed in the lower part of the cervix, and a normal uterine cavity was found. Laparoscopic adhesiolysis and enucleation of the cystic structure that connected to the sinus tract then were performed. Hysteroscopy was repeated to determine the thinnest cervical region by the light transmission test. A horizontal incision was made on the thinnest layer. Scar tissues were removed. The incision was sutured in full layer intermittently and continuously under laparoscopy. The postoperative thickness of the muscular layer in the sinus was confirmed by light transmission test of hysteroscopy. The patient was discharged on the third day after operation, uneventfully. Histopathologic examination showed that the cystic structure and scar tissue contained smooth muscle tissue and were covered by both mucinous columnar epithelium of the cervical canal and endometrial glandular epithelium. Restoration of normal anatomy, removal of uterine effusion, and symptomatic relief. At the 6-month follow-up, the patient's menstrual cycles returned to normal without the recurrence of menstrual spotting. The ultrasound scan also showed a symmetrical uterus without ECF. Patients with ECF who underwent assisted reproductive surgeries were related to the poor prognosis. However, the treatment should be different according to the causes, appearance time, and accumulation amount, including expectant treatment, postponement of embryo transfer, transvaginal aspiration, laparoscopic salpingectomy, or proximal tubal occlusion. For patients with recurrent ECF and/or special appearance on ultrasound, endoscopic examination is necessary. In addition, patients with large myomas at difficult locations required a uniform strategy to reduce the intraoperative and postoperative complications, especially for the nulligravida women.

Changes in anti-müllerian hormone after ultrasound guided aspiration and ethanol sclerotic therapy of ovarian cyst

ObjectiveTo evaluate the effect of transvaginal ultrasound-guided aspiration and ethanol sclerotherapy on anti-müllerian hormone (AMH) in patients with ovarian endometriomas. Setting: Teaching hospital affiliated with Chang Gung University, Taipei. Material and methodsWe retrospectively reviewed 124 patients, with ovarian endometriomas who underwent transvaginal aspiration and sclerotherapy of endometrioma(s) at a tertiary medical center, Chang Gung Memorial Hospital, Taipei, Taiwan. Preoperative evaluation included AMH, midcycle serum CA-125 level, and ultrasonography to exclude possibility of malignancies. Patients underwent ultrasonographic guided transvaginal aspiration and sclerotherapy with 95% ethanol irrigation of the cystic cavity. Patients were grouped into group 1, n = 44, retention of ethanol, and group 2, n = 80, no retention. Serum AMH level was checked at 6 months after aspiration. Those who were infertile prior to therapy were followed up for subsequent pregnancies (either by assisted reproductive technologies, or by natural conception). ResultsThe mean pre-operative AMH levels for the group without retention of ethanol and with ethanol retention were 3.80 and 3.06 respectively (p > 0.05). The change in AMH at 6-month follow up for retained group patients was significantly more than for non-retained group patients, with mean decrease of 0.72 (23.6%) and 0.10 (2.7%) respectively (p < 0.05). 54.5% (retained) and 47.2% (non-retained) of patients failed to achieve pregnancy during the observation period. ConclusionsTransvaginal aspiration of endometriomas followed by sclerotherapy with ethanol can be effective in preserving ovarian reserve, provided that no ethanol is left in situ.