Transvaginal Ultrasound-guided Aspiration Research Articles

Effect of equine transvaginal follicle aspiration on serum concentrations of acute phase proteins and their utility in diagnosis of ovarian abscess.

Transvaginal ultrasound-guided follicle aspiration (TVA) is being performed frequently in equine practice, to recover oocytes for in vitro embryo production via intracytoplasmic sperm injection. While complications from TVA are rare, one of the most prevalent major complications is formation of an ovarian abscess, which can result in the necessity for euthanasia. The acute-phase serum proteins, fibrinogen and serum amyloid A (SAA) are markers of inflammation that might be of use in diagnosis of ovarian abscess. However, no information is available on the effect of standard TVA on these acute phase proteins. We evaluated serum fibrinogen (n=8) and SAA (n=6) concentrations in mares immediately before and on Days 3, 7 and 14 after performance of TVA. Serum amyloid A levels were at baseline on all days. There was no significant difference in mean serum fibrinogen concentrations between D0 and any day after TVA. There was no significant difference in fibrinogen levels between mares that had undergone repeated TVA and a separate group of mares (n=14) that had never been subjected to TVA. We then describe four cases of mares that had undergone TVA and subsequently presented with signs associated with possible ovarian abscess, in which serum fibrinogen aided in interpretation. We conclude that fibrinogen and SAA were not elevated in mares undergoing TVA, and thus, elevations in mares following TVA should be investigated further and may support early diagnosis of an ovarian abscess.

Complications associated with equine transvaginal ultrasound-guided follicle aspiration: Practitioner survey and field results

Complications associated with equine transvaginal ultrasound-guided follicle aspiration: Practitioner survey and field results

Complications in equine transvaginal ultrasound‒guided follicular aspiration

Transvaginal ultrasound-guided follicular aspiration has revolutionised equine assisted reproduction, particularly in in vitro embryo production. This literature review examines the complications associated with transvaginal ultrasound-guided follicular aspiration in mares and its potential impact on future fertility. While transvaginal ultrasound-guided follicular aspiration is generally safe, it has inherent risks, ranging from mild rectal bleeding to severe complications such as peritonitis and acute haemorrhage. Factors influencing complication rates include veterinarian experience, mare anatomy and procedural technique. Despite concerns, studies suggest that repeated transvaginal ultrasound-guided follicular aspiration does not significantly impair future fertility, with normal pregnancy rates observed in mares undergoing artificial insemination or embryo transfer after transvaginal ultrasound-guided follicular aspiration. However, further research is needed to fully understand the long-term effects on ovarian function and oocyte quality. Veterinary practitioners must prioritise appropriate training and procedural precautions to minimise the risk of complications and safeguard mare welfare and fertility in the expanding field of equine assisted reproduction.

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.

Success rates and factors influencing pregnancy outcome after 464 transvaginal ultrasound-guided twin reductions in the mare.

Transvaginal ultrasound-guided aspiration (TUA) is used for post-fixation twin reduction in mares. However, there is limited information regarding factors that influence pregnancy outcome after TUA. To evaluate the effect of day of gestation on which TUA is performed, aspiration volume, puncture of the conceptus, medication administered before and after TUA, embryo location, mare age and parity and operator experience on pregnancy and foaling rates after TUA. Retrospective case series. Data were collected from case records of 464 TUAs performed by 14 operators in 422 mares diagnosed pregnant with dizygotic twins in two different facilities between 2010 and 2019. Pregnancy status was determined by ultrasonography at 5-7 days and 3-4 weeks after the TUA was performed. Subsequent pregnancy and foaling results were obtained by follow-up communication. The effects of mare, gestation- and TUA-related variables on pregnancy and foaling rates were analysed by the chi-square-test for homogeneity and Fisher's exact test and logistic regression. TUA was performed between 21 and 82 days of gestation in unilaterally (267/359 [74.4%]) and bilaterally fixed (92/359 [25.6%]) twin pregnancies. A singleton pregnancy (218/381 [57.2%]), persistent twin pregnancy (60/381 [15.8%]), or the loss of both conceptuses (103/381 [27%]) was confirmed 5-7 days after TUA was performed. At 3-4 weeks post TUA 50.3% (163/324) of mares were diagnosed with a single viable pregnancy and 40.1% (127/317) went on to deliver a live single foal. TUA performed early in gestation (D 25-35) resulted in the birth of a live singleton foal in 49.3% (74/150) of mares. Missing retrospective data despite extensive follow-up. This is the first large scale study to demonstrate that acceptable pregnancy and foaling rates can be achieved in mares diagnosed with twins when TUA is performed early in gestation (<40 days).

Embryo production by Holstein heifers superovulated with a recombinant long-acting follicle-stimulating hormone analog.

This study aimed to evaluate embryo yield in Holstein heifers superovulated with a single injection of recombinant, long-acting human follicle-stimulating hormone (rFSH) vs. multiple injections of pituitary-derived follicle-stimulating hormone (FSH). In experiment 1, heifers were assigned randomly to one of four experimental groups: Control (280mg of pituitary-derived FSH; six injections of 40mg and two injections of 20mg, each ~12h apart, n = 16); rFSH1 (50 µg of FSH analog protein, n = 16); rFSH2 (75 µg of FSH analog protein, n = 16); or rFSH3 (100 µg of FSH analog protein, n = 16). The rFSH was administered as a single injection. Estrous cycles were presynchronized with gonadorelin acetate (GnRH) and an intravaginal progesterone insert (CIDR) on day 8, followed by cloprostenol sodium (PGF2α) on day 3 and day 2 with removal of the CIDR, and GnRH on day 0. On day 5, ovarian follicles ≥ 5mm in diameter were ablated by transvaginal ultrasound-guided aspiration. On day 6.5, the heifers received a CIDR and the first injection of either rFSH or pituitary-derived FSH. On day 9, the heifers received two injections of PGF2α, 12h apart. On day 10.5, the CIDR was removed, and on day 11, the heifers received a GnRH injection. Heifers were AI-inseminated 12- and 24-h post-GnRH injection, and uterine contents were flushed transcervically on day 18 (7 d after the GnRH injection). Ovarian follicles ≥ 5mm and corpora lutea (CL) were counted via ultrasound on days 5, 9, and 18. In experiment 1, group did not affect (P = 0.52) the number of follicles ≥ 5mm (Control = 15.9 ± 1.2; rFSH1 = 17.5 ± 1.3; rFSH2 = 17.1 ± 1.3; rFHS 3 = 18.6 ± 1.4 follicles) or the number of CL (P = 0.96) on day 9 (Control = 1.1 ± 0.3; rFSH1 = 1.1 ± 0.3; rFSH2 = 1.1 ± 0.3; rFSH3 = 0.9 ± 0.2). Furthermore, there was no effect (P = 0.28) of rFSH dose on freezable embryos (grade 1 and 2 embryos) collected on day 18 (Control = 4.7 ± 1.1; rFSH1 = 4.7 ± 1.2; rFSH2 = 4.4 ± 1.1; rFSH3 = 2.6 ± 0.7 embryos). In experiment 2, Control (n = 8) and rFSH1 (n = 16) groups were repeated in three replicates using the same protocols as experiment 1. Consequently, Results showed that rFSH produced fewer total number of ova/embryos (Control = 9.9 ± 1.5 vs. rFHS1 = 5.9 ± 0.9, P = 0.04) and fewer freezable embryos (Control = 5.3 ± 1.0 vs. rFSH1 = 1.4 ± 0.3, P < 0.01). In conclusion, the single rFSH injection effectively induced superovulation; however, its repeated use reduced embryo production.

Evaluation of the effects of the recommended oral dose of diflubenzuron on bovine sperm and oocyte quality using CASA and OPU-IVEP.

The aim of this study was to evaluate potential effects of diflubenzuron on the production and quality of gametes, and on in vitro embryo production (IVEP) outcomes, in cattle. Two experiments were performed, the first to evaluate effects on semen, and the second on cumulus-oocyte complexes (COC) and on IVEP. Nelore (Bos taurus indicus) bulls (n = 14) or heifers (n = 16) were allocated into control (CG) or treatment (DIF) groups. All groups received a mineral mix supplement added (DIF) or not (CG) with diflubenzuron (30 mg/head/day), during 8 weeks. Animals were weighed and blood samples were collected throughout the experimental period. Every other week, bulls were subjected to semen collection and heifers to transvaginal ultrasound-guided follicle aspiration sessions. Semen underwent physical and morphological evaluation, and samples were stored for further computer-assisted sperm analysis. The COC recovered were evaluated according to morphology and those classified as viable were sent to an IVEP laboratory. Diflubenzuron had no effect (P > 0.05) on average body weight or in any blood hematological or biochemical endpoints, regardless of gender. In experiment 1, there was no difference (P > 0.05) between DIF and CG groups for sperm concentration, morphology, or kinetics. In experiment 2, there was also no effect of diflubenzuron on the number of total, viable, or grade I oocytes, as well as on cleavage or blastocyst rates (P > 0.05). In summary, the oral administration of diflubenzuron, within the recommended dose, has no short-term negative effects on sperm production and quality or on oocyte yield and developmental potential in vitro, in cattle.

Successful use of sexed stallion semen for in vitro embryo production; a preliminary study

The use of sex-sorted semen to obtain offspring of a desired sex in commercial livestock breeding programs has been in a state of rapid growth as sorting technology has evolved (Neculai-Valeanu and Ariton, Animals 2021, 11, 1182-1194, 2021). In horses, sex-sorted semen has resulted in successful pregnancies using low-dose or hysteroscopic artificial insemination, albeit with decreased pregnancy rates (Clulow et al, Animal Reproduction Science 108: 287–297, 2008; Gibb et al, Eq Vet J 49:160–166, 2017). Even though sex-sorted semen has been used to produce viable in vitro produced (IVP) embryos, the cleavage and blastocyst rates remain low (Colleoni et al, Reproduction, Fertility and Development 21(1): 228-229, 2008). Using current sperm sorting technology (ST Inc, Navasota, TX), we investigated fertilization, cleavage, and embryo development rates after ICSI using a time-lapse imaging system. To sex sort the semen, the ejaculate was diluted in 1:1 in a casein/cholesterol-based semen extender (BotuSemen Gold) supplemented with 60ug/ml of Hoechst 33342. The diluted semen was centrifuged through a colloid suspension (EquiPure) for 25 min at 300g using 3mL of EquiPure per billion sperm. After centrifugation, the sperm pellet was split into two aliquots: NON-SEXED, resuspended in BotuCrio, cooled and frozen in 0.5 ml straws at ∼ 100 million/ml, and SEXED, resuspended in a proprietarychemically-defined medium (EquiShip, ST Genetics), and transported to the sorting facility at ∼20°C. At the laboratory, the sperm were passed through the SexedULTRA™ Genesis III flow cytometer. After approximately 40 million total events X- and Y-bearing sperm were recovered, diluted in BotuCrio at a concentration of 2 million/ml, loaded into 0.5 ml straws and frozen. Oocytes were obtained from mares using transvaginal ultrasound-guided aspiration, shipped/held for 24h, followed by in vitro maturation. The matured MII oocytes from each aspiration were randomly split and injected with SEXED and NONSEXED sperm. A total of 56 oocytes were fertilized. We observed 24/29 and 23/27 cleaved oocytes, resulting in 11 and 12 blastocysts in SEXED and NONSEXED groups, respectively. No significant difference was detected in the cleavage and blastocyst rates among the groups. Embryo kinetic analysis revealed a slightly shorter duration from injection to blastocyst formation in the SEXED versus NONSEXED groups (183h and 188h, respectively). Higher number of embryos are required to compare the morpho-kinetics of male and female embryos at each developmental stage. This is a preliminary report, showing the successful use of frozen sexed semen in a commercial in vitro embryo production setting and further studies are ongoing.

Effects of feeding exogenous vitamins and microelements on follicular development and molecular function of granulosa cells in donkeys

In mammalian ovarian physiology, vitamins and microelements are essential for follicular development, ovulation, and the synthesis and secretion of hormones and growth factors. However. the molecular mechanisms by which they influence maturation and growth of follicles in donkeys are still unknown. Here, we investigated how vitamins and microelements affected the development of follicles and the proliferation of ovarian granulosa cells (GCs). For this experiment, 40 jennies (average body weight: 292.946±33.543 kg, age: 3∼4 years) were randomly assigned into 4 groups (n=10) and fed 50% (A), 75% (B), 100% (C), and 150% (D) of a mixture of vitamins A, D and E and microelements once a day (dose 5 ml) for two consecutive months. After that, ultrasonography was applied to record the change in follicular dynamics in three estrous cycles. Using a transvaginal ultrasound-guided aspiration device to obtain follicular fluid (containing GCs) from the follicles (Ф=30mm-35mm), multi-omics analysis revealed its molecular mechanisms and physiological functions. The results showed that the time required for growing follicles to reach ovulation (Ф>40mm) was shortened in the vitamin D group compared to the three other groups(P<0.05). Of note, the follicles (Ф=30mm-35mm) in the vitamin C group had less discrete diameters and developed more evenly and had significantly more twin ovulations than the other three groups (P<0.05). The omics analysis showed that945 metabolites, including organic acids and derivatives (24.44%), lipids and lipid-like molecules (24.34%), organic heterocyclic compounds (13.44%), and benzenoids (12.06%) were identified in follicular fluid. The results also revealed 232, 207, 210 and 202 differential cumulative metabolites (DAMs) in group A vs. group B, group B vs. group C, group C vs. group D and group A vs. group C, respectively. The DAMs were most significantly enriched in protein digestion and absorption, ABC transporters, amino acid biosynthesis, mineral absorption, aspartate-arginine biosynthesis, and ovarian steroid synthesis process. Meanwhile, nine DEGs (INSR, CYP19A1, ALOX5, PLA2G4A, ADCY6, CYP1B1, PRKACB, CYP17A1, and PLA2G4F) associated with the secretory function of GCs were involved in the ovarian steroidogenesis pathway, and they were significantly correlated with estradiol (E2), cholesterol, progesterone (P4), and testosterone (T)(P<0.05). Furthermore, STEAP2, CYBRD1, ATP1B3, ATP1A1, SLC26A6, SLC5A1, SLC6A19, SLC40A1, and TRPV6 were closely associated with the mineral absorption of GCs, and they had a positive correlation with glycine and L-methionine (P<0.05). In conclusion, exogenous supplementation of jennies with vitamins A, D and E and micronutrients can contribute to follicular growth and ovulation and this was associated with the interaction and expression of key genes related to -metabolites in ovarian steroidogenesis and mineral absorption pathways.

Effect of the interval from GnRH administration after ovarian super-stimulation on the recovered oocytes, and effect of the transferred cloned blastocysts on the pregnancy rate and pregnancy loss in dromedary camel

The present study was conducted to evaluate the number and maturity of the recovered oocytes after two intervals of in-vivo maturation. In addition to evaluating the effect of the developmental stage, as well as the number of cloned transferred blastocysts on the pregnancy rate and early pregnancy loss (EPL) in dromedary camel. The donor animals (n = 52) were super-stimulated using a single injection of 3000 IU of eCG followed by GnRH administration for oocyte maturation. Cumulus oocyte complexes (COCs) were collected by transvaginal ultrasound-guided aspiration (OPU) either 24–26 h or 18–20 h after GnRH administration. A fewer number of COCs with a lower percentage of oocyte maturity was observed at 24–26 h in comparison to 18–20 h. The effect of the cloned blastocysts' transferred number and developmental stage on the pregnancy rate and EPL was investigated. The total pregnancy rates at 10 days post-ET, 1 and 2 months were 21.9, 12.4, and 8.6%, respectively. Transfer of two or 3–4 embryos per surrogate was accompanied with a higher pregnancy rate at 1 and 2 months than a single embryo transfer. Rates of EPL were 43.5 and 60.1% at 1 and 2 months of pregnancy, respectively. The transfer of two embryos per surrogate was associated with a lower rate of EPL than ET of a single embryo at 1 and 2 months of pregnancy. Also, the ET of 3–4 embryos per surrogate showed a higher rate of EPL than the ET of two embryos at 2 months of pregnancy. ET of hatching (HG) blastocysts showed higher pregnancy rates and fewer EPL than ET of unhatched (UH) or fully hatched (HD) cloned blastocysts at 1 and 2 months of pregnancy. In conclusion, a high number of in-vivo matured oocytes can be recovered by ultrasound-guided transvaginal OPU from super-stimulated females using 3000 IU eCG and an interval of 18–20 h after GnRH administration. The transfer of two hatching cloned blastocytes per surrogate increases the pregnancy rate and decreases EPL in dromedary camels.

203 Effect of aspiration pressure at the time of transvaginal ultrasound-guided follicle aspiration on cumulus-oocyte complex quality and embryo development in alpacas (

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Effect of day of estrus cycle at time of transvaginal follicle aspiration for oocyte recovery on rates of in vitro maturation and blastocyst production after intracytoplasmic sperm injection.

To determine the effect of stage of estrus cycle (day after ovulation) at the time of transvaginal ultrasound-guided follicle aspiration (TVA) on parameters related to the success of in vitro equine embryo production. 14 healthy mares were used; 11 completed the study and were included for analysis. Mares underwent TVA of all follicles ≥ 5 mm diameter at each of 3 timepoints: 7 days after ovulation, 14 days after ovulation, and S-DSF (subordinate to a dominant stimulated follicle), during estrus at 24 hours after gonadotropin administration. The 3 treatments were assigned to each mare in random order; mares underwent follicle growth and ovulation between treatments. Recovered oocytes were matured in vitro, subjected to intracytoplasmic sperm injection (ICSI), and cultured to the blastocyst stage in vitro. Total follicle numbers differed significantly between individual mares but did not differ between treatments. The number of follicles of different sizes significantly (P < 0.05) differed between treatments, with mares in the Day 7 treatment having more follicles 5 to 9 mm in diameter and fewer follicles 20 to 24 mm in diameter than mares in the other 2 treatments. After in vitro maturation culture, there were significantly more mature oocytes in the S-DSF treatment than in the other 2 treatments. There were no differences in blastocyst rate after ICSI among treatment groups. Timing of TVA for aspiration of S-DSFs may increase the number of mature oocytes available for ICSI. Understanding of the effects of timing of TVA will help veterinarians to maximize the efficiency of this procedure.

Supplemental Nicotinic Acid Elevates NAD+ Precursors in the Follicular Fluid of Mares.

Simple SummaryMiscarriage and embryonic death have been associated with a deficiency in NAD+ resulting from a lack of dietary niacin in women and mice. Mares often suffer from high rates of early embryonic loss, but the dietary requirement of niacin in pregnant mares remains unknown. The aim of this study was to determine the effect of supplementing nicotinic acid, a form of niacin often supplemented in horse diets, on the elevation of other NAD+ precursors in the blood and follicular fluid of mares. We orally administered nicotinic acid to mares over consecutive days and collected blood and follicular fluid at the end of the feeding period. The results show that multiple examined NAD+ precursors were elevated in the follicular fluid of mares at the end of the feeding period, which we propose will aid in reducing early embryonic loss in the mare by promoting good quality oocytes.A deficiency in NAD+ has previously been linked with increased occurrences of congenital abnormalities and embryonic death in humans and mice. Early embryonic death is a major factor involved in pregnancy loss in mares, and very little is known regarding the NAD+ requirements for optimum reproductive function in horses. The aim of this study was to determine the effect of supplementing the diet of mares with nicotinic acid (NA) on the composition of NAD+ metabolites in the blood and follicular fluid. Vehicle alone or NA (3 g per os) were administered to seven mares over a minimum of 3 consecutive days during the follicular phase of the oestrous cycle. Blood samples were collected immediately prior to supplemental feeding and follicular fluid aspiration. Follicular fluid was collected from the dominant follicle through transvaginal ultrasound-guided aspiration. Blood and follicular fluid samples were processed and analysed by mass spectrometry. The concentration of nicotinamide mononucleotide (NMN) in the follicular fluid of NA-fed mares was 4-fold greater than that in the corresponding plasma and 10-fold greater than that in the follicular fluid of vehicle-fed mares. The concentrations of NA, nicotinamide (NAM) and nicotinuric acid (NUR) tended to be greater in the follicular fluid of NA-supplemented mares than in the corresponding plasma. The results show that NA supplementation increased the bioavailability of NAD+ precursors in the follicular fluid of the dominant follicle, which is proposed to better promote the maturation of good quality oocytes, especially in older mares.

Management of monozygotic twins in the mare

In the horse, only a small number of monozygotic twins and triplets have been reported and commonly, the outcome is poor. As in man, an association between the occurrence of monozygotic twins and the use of assisted reproductive technologies (ART) has been suggested. In the mare, transfer of in vitro produced (IVP) embryos appears to increase the incidence of monozygotic twinning (1.6%; 4/254) compared to transfer of in vivo embryos (0%; 0/413) (Dijkstra et al. Equine Vet J. 2020; 52: 258-261). Due to the type of placental arrangement, these monozygotic twin pregnancies are difficult to reduce to a singleton using reduction techniques commonly applied to dizygotic twins. In this report, 8 cases of monozygotic twins are presented, all of which resulted from transfer of a single IVP embryo. Seven were presumed to be monochorionic diamniotic/diallantoic and one dichorionic diamniotic/diallantoic, based on membrane classification by ultrasound and/or post-expulsion examination. Similar to previously described cases, twin pregnancy only became apparent at 25 days of gestation, when two embryos with heartbeat were visualized. Depending on gestational stage, different twin reduction techniques were applied. In one case, only referred at day 40 of gestation andclassified as dichorionic diamniotic/diallantoic, a transvaginal ultrasound guided aspiration (TUGA) was performed. Reduction of two other twins was performed by per rectum compression of the fetal thorax at day 65 of gestation. In two other cases, cranio-cervical dislocation was performed at day 75-80 of gestation. Finally, transcutaneous ultrasound guided twin reduction was attempted at day 120 of gestation in one case. However, only one of the 6 treated monozygotic twins was successfully reduced, resulting in a live foal born following thorax compression at 65 days of gestation; all other cases resulted in death of both fetuses. In addition, two remaining cases (2/8) lost their pregnancy spontaneously at around 42 days and 8 months of gestation, whereby in the latter the presence of twins was not established at previous ultrasound examinations. Based on these limited cases, only one monozygotic twin was successfully reduced, using the thoracic compression method at day 65 of gestation. These results highlight the need for further investigation into the best reduction method, depending on the gestational stage and on how much the demise of one fetus affects the survival of its co- twin, which may depend on placental arrangement.

In vitro production of ICSI derived horse blastocysts

In vitro production of horse embryos via intracytoplasmic sperm injection (ICSI) is a useful clinical and research technique. However, current rates of blastocyst production are still sub-optimal. Here, we compared results from our commercial ICSI program to evaluate whether slight modifications in protocols can result in improved blastocyst rates. Firstly, we evaluated the time cumulus oocyte complexes (COCs) can be maintained in holding medium before starting in vitro maturation (IVM) (Group A: 4-10 hours and Group B: overnight). Secondly, we evaluated the effects of using a microfluidic device (Zymöt) for the preparation of sperm samples for ICSI (Group C). This device comprises a microporous membrane that allows to select spermatozoa in base of their motility, avoiding samples centrifugation. This could eventually reduce DNA fragmentation and oxidative stress, which have been associated with conventional sperm preparation protocols. Frozen-thawed cut straws with semen from different stallions were used and prepared either with a gradient and swim-up protocol or with the Zymöt microfluidic device. COCs were recovered from immature follicles using transvaginal ultrasound-guided follicle aspiration (TVA) procedures. After collection, COCs were transferred to holding medium for 4 to 10 hours or maintained in holding medium overnight at room temperature (22–24°C) before starting IVM. In vitro maturation was performed in T-199 Earle's medium supplemented with 5 mU/ml FSH, 50 ng/ml EGF and 10% FBS. After IVM, oocytes were denuded from cumulus and those showing a polar body were subjected to piezo-ICSI (PiezoXpert, Eppendorf). Spermatozoa were first immobilized with strong piezo pulses and then injected individually in the matured oocytes after penetrating the zona pellucida and oolemma with low intensity pulses.The injected oocytes were then cultured in a mix of KSOM-DMEM/F12 medium supplemented with 6% FBS, which was refreshed on Day 5 of development. Embryo culture was performed in humidified benchtop incubators at 6.5% CO2 and 5%O2 at 38.2°C. A Chi-square test was used to compare results.

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.

Transvaginal Ultrasound-Guided Fine-Needle Aspiration Biopsy of Pelvic Lesions.

To assess adequacy of transvaginal ultrasound-guided fine-needle aspiration biopsy (TVUS-FNAB) for pathologic diagnosis of pelvic masses performed using onsite cytopathology consultation. In this Institutional Review Board approved, Health Insurance Portability and Accountability Act (HIPAA) compliant study, radiology records were retrospectively queried to identify patients who underwent TVUS-FNAB of a pelvic mass over a 11-year duration. TVUS-FNAB adequacy was determined by correlating cytopathology results with transvaginal ultrasound-guided core-needle biopsy (TVUS-CNB) or surgical pathology results when available, and with clinical diagnostic confidence when additional pathology confirmation was not available. Secondary analysis included patient age, history of hysterectomy, or pelvic malignancy. Target-specific features analyzed included mass size, depth, location, and final pathologic diagnosis. Sixty patients underwent TVUS-FNAB of pelvic masses, 43 of which underwent FNAB only and 17 underwent both TVUS-FNAB and TVUS-CNB during the same procedure. TVUS-FNAB alone was adequate for diagnosis in 51 (85%) cases and addition of core-needle biopsy (CNB) achieved a diagnosis in additional 4 patients, increasing overall diagnostic accuracy to 92% (55/60). FNAB inadequacy had statistically significant association with increasing mass depth, occurrence of a minor intraprocedural complication, and decision to perform a CNB (P <.05). Number of FNAB passes, mass size, history of hysterectomy, and final diagnosis were not statistically significant predictors of FNAB adequacy. TVUS-FNAB has a high specimen adequacy rate when performed with an onsite cytopathologist and can be considered first-line approach for image-guided sampling of pelvic lesions with option to add CNB if preliminary cytopathologic review does not confirm sample adequacy.

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.

β-NGF Stimulates Steroidogenic Enzyme and VEGFA Gene Expression, and Progesterone Secretion via ERK 1/2 Pathway in Primary Culture of Llama Granulosa Cells.

The beta-nerve growth factor (β-NGF) from llama seminal plasma exerts ovulatory and luteotrophic effects following intramuscular or intrauterine infusion in llamas and alpacas. In this study, we investigate the in vitro effect of llama β-NGF on the expression of genes involved in angiogenesis and progesterone synthesis as well as progesterone release in preovulatory llama granulosa cells; we also determine whether these changes are mediated via the ERK1/2 signaling pathway. From adult female llamas, we collected granulosa cells from preovulatory follicles by transvaginal ultrasound-guided follicle aspiration; these cells were pooled and incubated. After 80% confluence, the cultured granulosa cells were treated with β-NGF, β-NGF plus the MAPK inhibitor U0126, or luteinizing hormone, and the abundance of angiogenic and steroidogenic enzyme mRNA transcripts were quantified after 10 and 20 h by RT-qPCR. We also quantified the progesterone concentration in the media after 48 h by radioimmunoassay. We found that application of β-NGF increases the abundance of mRNA transcripts of the vascular endothelial growth factor (VEGFA) and the steroidogenic enzymes cytochrome P450 side-chain cleavage (P450scc/CYP11A1), steroidogenic acute regulatory protein (STAR), and 3β-hydroxysteroid dehydrogenase (HSD3B1) at 10 and 20 h of treatment. Application of the MAPK inhibitor U0126 resulted in downregulation of the genes encoding these enzymes. β-NGF also enhanced progesterone synthesis, which was prevented by the prior application of the MAPK inhibitor U0126. Finally, western blot analysis confirmed that β-NGF activates the ERK1/2 signaling pathway. In conclusion, our results indicate that β-NGF exerts direct luteotropic effects on llama ovarian tissue via the ERK 1/2 pathway.

Two waves of transcriptomic changes in periovulatory human granulosa cells.

How does the human granulosa cell (GC) transcriptome change during ovulation? Two transcriptional peaks were observed at 12h and at 36h after induction of ovulation, both dominated by genes and pathways known from the inflammatory system. The crosstalk between GCs and the oocyte, which is essential for ovulation and oocyte maturation, can be assessed through transcriptomic profiling of GCs. Detailed transcriptional changes during ovulation have not previously been assessed in humans. This prospective cohort study comprised 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital-affiliated fertility clinic in 2016-2018. From each woman, one sample of GCs was collected by transvaginal ultrasound-guided follicle aspiration either before or 12h, 17h or 32h after ovulation induction (OI). A second sample was collected at oocyte retrieval, 36h after OI. Total RNA was isolated from GCs and analyzed by microarray. Gene expression differences between the five time points were assessed by ANOVA with a random factor accounting for the pairing of samples, and seven clusters of protein-coding genes representing distinct expression profiles were identified. These were used as input for subsequent bioinformatic analyses to identify enriched pathways and suggest upstream regulators. Subsets of genes were assessed to explore specific ovulatory functions. We identified 13 345 differentially expressed transcripts across the five time points (false discovery rate, <0.01) of which 58% were protein-coding genes. Two clusters of mainly downregulated genes represented cell cycle pathways and DNA repair. Upregulated genes showed one peak at 12h that resembled the initiation of an inflammatory response, and one peak at 36h that resembled the effector functions of inflammation such as vasodilation, angiogenesis, coagulation, chemotaxis and tissue remodelling. Genes involved in cell-matrix interactions as a part of cytoskeletal rearrangement and cell motility were also upregulated at 36h. Predicted activated upstream regulators of ovulation included FSH, LH, transforming growth factor B1, tumour necrosis factor, nuclear factor kappa-light-chain-enhancer of activated B cells, coagulation factor 2, fibroblast growth factor 2, interleukin 1 and cortisol, among others. The results confirmed early regulation of several previously described factors in a cascade inducing meiotic resumption and suggested new factors involved in cumulus expansion and follicle rupture through co-regulation with previously described factors. The microarray data were deposited to the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/gds/, accession number: GSE133868). The study included women undergoing ovarian stimulation and the findings may therefore differ from a natural cycle. However, the results confirm significant regulation of many well-established ovulatory genes from a series of previous studies such as amphiregulin, epiregulin, tumour necrosis factor alfa induced protein 6, tissue inhibitor of metallopeptidases 1 and plasminogen activator inhibitor 1, which support the relevance of the results. The study increases our understanding of human ovarian function during ovulation, and the publicly available dataset is a valuable resource for future investigations. Suggested upstream regulators and highly differentially expressed genes may be potential pharmaceutical targets in fertility treatment and gynaecology. The study was funded by EU Interreg ÔKS V through ReproUnion (www.reprounion.eu) and by a grant from the Region Zealand Research Foundation. None of the authors have any conflicts of interest to declare.