Post Ovulation Research Articles

The impact of singing on estrogen and progesterone release among female

Scientific studies have suggested that singing can have positive effect on people’s health. (NAFME, 2012). John Daniel Scott, has cited that people who sing are more likely to be happy because singing elevates the level of neurotransmitters which is associated with pleasure and wellbeing. Most studies on the biological effect of music has resolved around studies involving brain mapping and physiological parameters such as heart rate, blood pressure, through production of messengers such as hormones, cytokines and proteins, music elicit biological responses to stress, emotions and immune functions. Hormone certainly prove necessary for proper physiological growth and development. This study is therefore to know the influence of singing on female sex hormone release which has variety of effects on both sexual organs and diverse target tissues. This research study will be a cross sectional and controlled investigative study. Hormone will be assayed with ELIZA technique and its secretion will be determined by its concentration in the blood. The study will be carried out with a total of thirty female choral singers, grouped into three A, B, C, each group will comprise of ten subjects, using group C as control. Blood sample will collect from each group at pre and post ovulation phase of their menstrual cycle, before and after singing while the control group will not be involved in singing. The mean serum concentration in all group will be compared with group C which serve as control group.

Follicular challenge test to predict suboptimal response to gonadotropin releasing hormone agonist trigger in elective oocyte cryopreservation cycles

This prospective study aimed to test the ability of follicular GnRH agonist challenge test (FACT) to predict suboptimal response to GnRH agonist trigger, assessed by LH levels post ovulation trigger in non-medical oocyte cryopreservation program. The study included 91 women that underwent non-medical fertility preservation. On day two to menstrual cycle, blood tests were drawn (basal Estradiol, basal FSH, basal LH, Progesterone) and ultrasound (US) was performed. On that evening, the women were instructed to inject 0.2 mg GnRH agonist (FACT) and arrive for repeated blood workup 10–12 h later in the next morning, followed by a flexible antagonist protocol. LH levels on the morning after ovulation trigger were compared to FACT LH levels. The results demonstrated that LH levels following agonist ovulation trigger below 15IU/L occurred in 1.09% of cycles and were predicted by FACT, r = 0.57, p < 0.001. ROC analysis demonstrated that FACT LH > 42.70 IU/L would predict LH post trigger of more than 30 IU/L with 75% sensitivity and 70% specificity, AUC = 0.81. LH levels post trigger also displayed significant positive correlation to basal FSH (r = 0.35, p = 0.002) and basal LH (r = 0.54, p < 0.001). LH levels post ovulation trigger were not associated with total oocytes number or maturity rate. The strongest correlation to the number of frozen oocytes was progesterone levels post agonist trigger (r = 0.746, p < 0.001). We concluded that suboptimal response to agonist trigger, as assessed by post trigger LH levels was a rare event. FACT could serve as an adjunct pre-trigger, intracycle tool to predict adequate LH levels elevation after agonist ovulation trigger. Future studies should focus on optimization of agonist trigger efficacy assessment and prediction, especially in high responders.

12 Impact of progesterone signaling on tumor necrosis factor-alpha synthesis by luteal macrophages

Abstract Understanding the mechanism in which luteolysis occurs is an important step in improving the reproduction of livestock in the industry. The corpus luteum (CL) is a transient endocrine organ found on the ovary responsible for progesterone secretion. It is well known that prostaglandin F2-alpha (PGF) is responsible for luteal regression, however, the process by which luteal cells (LC) become sensitive to the luteolytic effects of PGF remain unclear. Therefore, the objective of this study was to determine if suppression of progesterone (P4) synthesis or blocking P4 signaling activates tumor necrosis factor (TNF)-alpha production by the luteal macrophages. Ovaries were collected from an abattoir and transported to the laboratory on ice. Ovary status (i.e. approximate days post ovulation), was determined by phenotypic description of ovarian structures. Only ovaries (n = 5) containing midcycle (d 12/13 post ovulation) CL were used in these experiments. Luteal tissue was isolated and dissociated to collect LC and and luteal macrophages (MAC). Isolated cells were incubated independently at 37°C at 5% CO2 for 24 h in 24-well plates. Luteal cells and MACs were then harvested and placed in the following conditions: 1) LC + MAC + DMSO (control), 2) LC + MAC + trilostane (TRIL 1.0mg/mL), 3) LC + MAC + TRIL 1.5 mg/mL, 4) MACS only, 5) MACS +LPS. Supernatant was harvested at 24 h whereas both cells and supernatant were harvested after 48 h, in vitro. Supernatant was immediately frozen for subsequent P4 analysis, via RIA. Data results were analyzed using a MIXED procedure of SAS and evaluated the effects of cell-type, treatment, and culture time. Statistical significance was determined at P &amp;lt; 0.05 and a tendency at 0.05 &amp;lt; P &amp;lt; 0.10. Overall, TRIL treatment reduced supernatant P4 concentration (8.3 and 6.8 ng/mL to 1.0 and 1.5 mg/mL TRIL, respectively) compared with controls (22.8 ng/mL; P = 0.0012); however there was not a difference when comparing the TRIL treated cultures. Progesterone concentrations tended to decrease over time (P = 0.0661). At 24 h in vitro, P4 concentrations were suppressed in 1.0 mg/mL TRIL and 1.5 mg/mL TRIL treated wells (5.3 and 4.2 ng/mL, respectively), compared with controls (16.4 ng/mL; P = 0.0014). Whereas P4 concentrations were reduced in the 1.5 mg/mL TRIL treated wells (1.6 ng/mL) compared with controls (9.95 ng/mL; P = 0.0419) and tended to differ in the1.0 mg/mL TRIL treated wells (2.4 ng/mL; P = 0.0634), at 48 h in vitro. Progesterone concentrations were undetectable in wells containing MAC only. Collectively, TRIL treatment effectively reduced P4 synthesis in LC cultures. Therefore, the greater concentration of TRIL is needed for P4 suppression in longer term culture systems. Additional analyses will be conducted to determine the effect of P4 suppression on macrophage function.

Age and treatment on the day of embryo transfer in recipient mares affect likelihood of pregnancy.

This retrospective evaluation of data from a large commercial embryo transfer facility aimed to determine the extent to which age and treatment on the day of embryo transfer in recipient mares influence the likelihood of pregnancy. Embryo recovery was carried out on days 8-10 post-ovulation using transcervical uterine flushing. Recipient mares grouped according to their age were treated once on the day of embryo transfer (Day 3-8 post ovulation) and were assigned randomly to 1 of 3 groups: Mares in Group A (n=101) received antispasmodic, antimicrobial, and anti-inflammatory drugs. Mares in Group B (n=100) received gentamicin and flunixin meglumine. Group C (control) (n=103) did not receive any treatment. Detomidine (0.008 mg/kg bwt i.v.) was administered to all recipients before transfer of the embryo. The influence of treatment and recipient´s age was calculated using binary logistic regression. Day 16 post-transfer pregnancy rates were highest in Group A (74/101, 73.3a%), when compared to Group B (60/100, 60%), and Group C (57/103, 55.3b%) (a vs b, p<0.05). Pregnancy loss rates at D45 were not different between groups, A (8/74, 10.8%), B (5/60, 8.3%), and C (6/57, 10.5%), respectively (p>0.05). Pregnancy losses were increased in recipient mares 17-22 years (33.3a%) compared to younger recipient mares (2-6 years 7b%, 7-11 years 10%, 12-16 years 8b%) (a:b p<0.05). The regression model showed that the predicted probability for pregnancy after embryo transfer decreased as the age of the recipient mare increased for treated recipients in Group A (p=0.012), there was no effect of treatment and recipient´s age in Group B, and a decreased likelihood of pregnancy in recipients of advanced age (≥12 years of age) in untreated recipients (group C). Likelihood of pregnancy increased following single administration of antispasmodic, antimicrobial, and anti-inflammatory drugs at the time of embryo transfer in recipients 2-12 years of age. Likelihood of pregnancy in recipients decreased in recipients≥12 years of age. These results, obtained under the conditions of a large commercial embryo transfer program, offer an opportunity to improve pregnancy rates in recipient mares≤12 years of age.

Incorporation of L-Carnitine in the OvSynch protocol enhances the morphometrical and hemodynamic parameters of the ovarian structures and uterus in ewes under summer climatic conditions

Heat stress negatively impacts the reproductive performance of sheep including the efficiency of estrous synchronization regimens. This study aimed to investigate the potential effects of L-Carnitine (LC) administration on the efficacy of the OvSynch protocol in ewes under summer climatic conditions. Ewes were synchronized for estrus using the OvSynch protocol and a dose of LC (20 mg/kg body weight) was intravenously (IV) administered on the same day of PGF2α injection to one group (n = 8; LC group), while other ewes (n = 8; control group) received the same protocol without LC. Ultrasonographic evaluation (including B-mode, color, and pulsed Doppler) was used to assess the morphometrical and hemodynamic parameters of ovarian structures [number, size, and blood flow of follicles (GFs) and corpora lutea (CLs)] and uterus during the estrous phase (Day 0), and on Day 8 post ovulation (luteal phase). Uterine artery blood flow (MUA) was assessed by measuring the resistive index (RI) and pulsatility index (PI) at both stages. The serum samples were collected to measure the concentrations of estradiol (E2), progesterone (P4), and total antioxidant capacity (TAC) using commercial kits. Results revealed a significant (P<0.05) increase in the colored pixel area of GFs and uterus in the LC group (392.84 ± 31.86 and 712.50 ± 46.88, respectively) compared to the control one (226.25 ± 17.74 and 322 ± 18.78, respectively) during Day 0. Circulating E2 and TAC levels were significantly (P<0.05) higher in the LC-treated ewes (31.45 ± 1.53 pg/ml and 1.80 ± 0.13 mM/L, respectively) compared to those in the control ewes (21.20 ± 1.30 pg/ml and 0.98 ± 0.09 mM/L, respectively) during Day 0. Moreover, LC improved the colored pixel area of CLs (2038.14 ± 102.94 versus 1098 ± 82.39) and uterus (256.38 ± 39.28 versus 121.75 ± 11.36) and circulating P4 (2.99 ± 0.26 ng/ml versus1.67 ± 0.15 ng/ml) on Day 8. Values of RI of MUA were significantly lower in the LC group compared to the control one on Day 0 and Day 8 (0.48 ± 0.03 versus 0.72 ± 0.03 and 0.58 ± 0.03 versus 0.78 ± 0.02, respectively). In conclusion, LC incorporation in the OvSynch protocol enhanced the morphometrical and hemodynamic parameters of the ovarian structures and the uterus concomitantly with improvements in the TAC, E2, and P4 concentrations in ewes under hot summer conditions.

SPIOMET4HEALTH—efficacy, tolerability and safety of lifestyle intervention plus a fixed dose combination of spironolactone, pioglitazone and metformin (SPIOMET) for adolescent girls and young women with polycystic ovary syndrome: study protocol for a multicentre, randomised, double-blind, placebo-controlled, four-arm, parallel-group, phase II clinical trial

BackgroundPolycystic ovary syndrome (PCOS) is the most prevalent, chronic endocrine-metabolic disorder of adolescents and young women (AYAs), affecting 5–10% of AYAs worldwide. There is no approved pharmacological therapy for PCOS. Standard off-label treatment with oral contraceptives (OCs) reverts neither the underlying pathophysiology nor the associated co-morbidities. Pilot studies have generated new insights into the pathogenesis of PCOS, leading to the development of a new treatment consisting of a fixed, low-dose combination of two so-called insulin sensitisers [pioglitazone (PIO), metformin (MET)] and one mixed anti-androgen and anti-mineralocorticoid also acting as an activator of brown adipose tissue [spironolactone (SPI)], within a single tablet (SPIOMET). The present trial will evaluate the efficacy, tolerability and safety of SPIOMET, on top of lifestyle measures, for the treatment of PCOS in AYAs.MethodsIn this multicentre, randomised, double-blind, placebo-controlled, four-arm, parallel-group, phase II clinical trial, AYAs with PCOS will be recruited from 7 clinical centres across Europe. Intention is to randomise a total of 364 eligible patients into four arms (1:1:1:1): Placebo, PIO, SPI + PIO (SPIO) and SPI + PIO + MET (SPIOMET). Active treatment over 12 months will consist of lifestyle guidance plus the ingestion of one tablet daily (at dinner time); post-treatment follow-up will span 6 months. Primary endpoint is on- and post-treatment ovulation rate. Secondary endpoints are clinical features (hirsutism, menstrual regularity); endocrine-metabolic variables (androgens, lipids, insulin, inflammatory markers); epigenetic markers; imaging data (carotid intima-media thickness, body composition, abdominal fat partitioning, hepatic fat); safety profile; adherence, tolerability and acceptability of the medication; and quality of life in the study participants. Superiority (in this order) of SPIOMET, SPIO and PIO will be tested over placebo, and if present, subsequently the superiority of SPIOMET versus PIO, and if still present, finally versus SPIO.DiscussionThe present study will be the first to evaluate—in a randomised, double-blind, placebo-controlled way—the efficacy, tolerability and safety of SPIOMET treatment for early PCOS, on top of a lifestyle intervention.Trial registrationEudraCT 2021–003177-58. Registered on 22 December 2021.https://www.clinicaltrialsregister.eu/ctr-search/search?query=%092021-003177-58.

P-592 Follicular GnRH agonist challenge test (FACT) to predict suboptimal response to GnRH agonist trigger in oocyte cryopreservation cycles

Abstract Study question Can follicular GnRH agonist administration predict suboptimal response to GnRH agonist trigger assessed by LH levels post ovulation trigger in non-medical oocyte cryopreservation program? Summary answer Follicular agonist challenge test (FACT) can serve as an adjunct pre-trigger, intracycle tool to predict adequate response to agonist trigger. What is known already Suboptimal response to GnRH agonist ovulation trigger refers to failure to retrieve the expected number of oocytes. It was demonstrated in up to 5.5% of cycles based on relatively low number of studies with heterogenous suboptimal response definitions and prevalence, no clear cut-offs and mainly post - stimulation tools to predict unsuccessful cycle. The most studied variable associated with suboptimal response was LH levels 10-12 hours after ovulation trigger below 12-15IU. Study design, size, duration We prospectively recruited all women that underwent non-medical fertility preservation in our tertiary university affiliated medical center between October 2020 and February 2022 and agreed to participate in the study. The study included 91 women. Participants/materials, setting, methods On day 2 to menstrual cycle, blood tests were drawn (basal Estradiol/E2, basal FSH/FSH2, basal LH/LH2, Progesterone/P2) and ultrasound (US) was performed. On that evening, the women were instructed to inject 0.2 mg GnRH agonist and arrive for repeated blood workup 10-12 hours later at the next morning, followed by a flexible antagonist protocol. On the morning after ovulation trigger blood tests were again taken and LH levels were compared to FACT LH levels. Main results and the role of chance LH levels following agonist ovulation trigger below 15IU occurred in 1.09 % of cycles and were predicted by FACT, r = 0.57, p &amp;lt; 0.001. ROC analysis demonstrated that FACT LH &amp;gt; 42.70 IU would predict LH post trigger of more than 30 IU with 75% sensitivity and 70% specificity, AUC= 0.81. LH levels post trigger also displayed significant positive correlation to basal FSH (r = 0.35, p = 0.002) and basal LH (r = 0.54, p &amp;lt; 0.001). LH levels post ovulation trigger were not associated with total oocytes number or maturity rate. The strongest correlation to the total number of retrieved oocytes and mature oocytes was progesterone level post ovulation trigger (r = 0.74, p &amp;lt; 0.001). Limitations, reasons for caution Limitation of our study include the relatively small sample size designed to detect 5% suboptimal proportion and might have been insufficient to allow identification of very rare suboptimal responses Wider implications of the findings Suboptimal response to agonist trigger, as assessed with post trigger LH levels is a rare event. FACT could serve as an adjunct pre-trigger, intracycle tool to predict adequate response to agonist ovulation trigger. Future studies should focus on optimization of agonist trigger efficacy assessment and prediction, especially in high responders. Trial registration number 0304-20-ASF

P-396 Modified Flare/GnRH antagonist protocol versus GnRH antagonist protocol in oocyte cryopreservation cycles

Abstract Study question Is there an advantage for modified flare/ GnRH antagonist protocol in comparison to antagonist protocol in oocyte cryopreservation cycles? Summary answer Modified flare/ GnRH antagonist protocol is at least as good as antagonist protocol in oocyte cryopreservation cycles, potentially improving maturity rate in subgroup of patients. What is known already Ultrashort flare GnRH agonist (GnRHa)/GnRH -antagonist protocol offer benefits of stimulatory effect of repeated microdose flare on endogenous FSH and the advantages of GnRH antagonist suppression, including the option to use GnRHa ovulation trigger to avoid ovarian hyperstimulation syndrome. GnRHa ovulation trigger has been associated with suboptimal response that refers to lesser than expected oocyte yield. Our research group previously demonstrated the value of GnRHa follicular challenge test (FACT) in prediction of suboptimal response assessed by post ovulation trigger LH levels and as a dynamic ovarian reserve test (not published yet). Study design, size, duration A retrospective, age- matched, cohort study that included all non-medical oocyte cryopreservation cycles from October 2020 to December 2021. The study group included 110 women that received modified flare/ GnRH antagonist protocol and were matched with 110 women that received antagonist protocol. Participants/materials, setting, methods Women in the modified flare/ GnRH antagonist protocol were administered GnRHa (Decapeptyl 0.2 mg) on day 2 to menstrual cycle followed by flexible antagonist protocol. They were compared to age matched women that received flexible antagonist protocol. In both study groups GnRHa final ovulation trigger was administered. Main results and the role of chance Among 110 women that underwent non-medical fertility preservation with modified Flare/GnRH antagonist protocol, in comparison to 110 women that received antagonist protocol, a reduced dosage of gonadotropins was consumed with more oocytes retrieved (14.63 vs. 13.86) and more mature oocytes cryopreserved (11.32 vs. 10.14), but differences were not significantly different. In a subgroup with more than 6 oocytes retrieved significantly more mature oocytes were cryopreserved in women that received modified flare/antagonist protocol (14.03± 0.90 vs. 11.63± 0.71, p = 0.03) in comparison to women that received antagonist protocol. Limitations, reasons for caution Limitations of our study are its retrospective nature and lack of information on the oocyte’s fertilization competence and pregnancy outcomes. Wider implications of the findings Future studies will further explore the advantages of this protocol in terms of higher oocytes maturity rate, intra cycle prediction of ovarian response and suboptimal response to GnRHa triggering. Trial registration number not applicable

The main factor of reduced pregnancy rates in post foaling mares is time of embryo transfer after parturition

Post foaling mares can be used as embryo transfer recipients. Higher pregnancy loss and lower pregnancy rates have been reported in recipient mares used at the foal heat versus open recipient mares. The aim of this study was to analyze the influence of post foaling day of transfer, post ovulation day of transfer, month of the year of embryo transfer, mare age and embryo quality over embryo loss and pregnancy rates in postpartum recipients. Retrospective data from three breeding seasons (2020-2022) were analyze at a breeding facility in Montemorelos N.L. Mexico (n=493 embryo transfers). Mares that did not foal the previous breeding season were considered open recipients (n = 383, 7.3 ± 3.4 years old) while post foaling mares were those that foaled in the same transfer season (n=110, 7.2 ± 3.2). Postpartum recipients were divided in two groups considering the post foaling time at transfer (<120 days and>120 days). Embryo donors were flushed on Day 7-9 post ovulation and recovered embryos transferred into recipients that were 3-8 days post ovulation. Ultrasonographic pregnancy diagnosis was undertaken at days 12, 25, 45 and 60. A nonparametric Chi-Square X2 test was performed using SPSS version 16.0 (SPSS, INC., Chicago, IL). Mares hada significant higherpregnancy rate (85.37%) than postpartum mares (67.27%) (P<0.0001). Although mares transferred >120 days post foaling had a pregnancy rate similar to open mares (80%), and higher than those transferred <120 days postpartum (66.6%), no significant differences were found between these two postpartum mares, probably due to the low number of mares on the >120 postpartum days group (25 mares). In contrast with previous reports, we have no differences in embryo losses (p>0.05) between open (9.78%) and postpartum mares (6.75%), and embryo losses were more related to embryo quality than mare status. Age, post ovulation day of transfer and month of the year of embryo transfer, had no influence on pregnancy rate in any of the groups (p>0.05). It is possible that the time from foaling to transfer is one of the main factors explaining reduced pregnancy rates in post-partum mares. This idea is substantiated by our data which show that similar pregnancy rates are obtained when transferring embryos into open or mares >120 days post-foaling. This study reveals an opportunity to optimize the use of post-foaling mares as recipients by considering the appropriate time to transfer embryos after foaling.

Successful vitrification of embryos ≤500µm without puncture or aspiration of the blastocoele

To achieve good pregnancy rates following vitrification, embryos >300µm require micromanipulator-assisted puncture and blastocoele collapse. Without using a micromanipulator, Wilsher et al (Equine Veterinary Journal.2021;53:1227-1233) reported high pregnancy rates following vitrification of embryos 300-560µm using manual puncture but failed to achieve any pregnancies with non-punctured embryos. These manually punctured embryos were exposed to an equilibration medium (EM; HEPES culture medium + 7.5% DMSO + 7.5% ethylene glycol [EG]) for 6min. Extending the time to 8min in the non-puncture group did not improve results. The aim of this study was to determine if non-punctured embryos, exposed to considerably longer in EM (i.e.,15min) would, upon warming and transfer to recipients, result in acceptable pregnancy rates. Thirty, Grade 1 embryos were recovered on Day 7-8 post ovulation and divided into three groups based on size, G1 (≤300 µm; n = 8), G2 (>300 to ≤500µm; n=16) and G3 (>500µm; n=6). Median (range) embryo diameters for Groups 1 to 3 were 237.5 (180–280), 410 (310–500) and 575 (510–660) µm, respectively. Embryos were vitrified with a commercial human vitrification kit (Kitazato) utilising 2-step concentrations of DMSO and EG (7.5-15% v:v) in a HEPES culture medium containing gentamycin, trelahose and hydroxypropyl cellulose. At room temperature (RT) embryos were placed in EM for 15min prior toa vitrification solution for ≤90 sec, during which time they were loaded onto a Cryolock device, capped and plunged into LN2. Embryos were warmed in 1M sucrose solution of holding media (HM; H199 + 20% FBS + antibiotics) at 42°C for 1min before being transferred to HM + 0.5M sucrose at RT for 4min, then into Vigro Hold for a further 4min. Within 15min, embryos were transferred to recipient mares 6 days post ovulation. Mares were checked thrice for pregnancy and to ensure continuing embryonic development before administration of prostaglandin. Two mares were left pregnant until the heartbeat and expanding allantois stage in groups achieving pregnancies. G1 and G2 embryos achieved a higher pregnancy rate than G3 in which no embryos survived (87.5% [7/8] vs. 75% [12/16] vs. 0% [0/6], respectively; P<0.001). One early embryonic death (EED) occurred in G2 (350µm) between Days 12-14. The four embryos in G2 that did not result in a pregnancy were 330, 380, 425 and 500µm. This study shows that non-punctured embryos <500µm can survive vitrification and result in excellent pregnancy rates (19/24 [79%]) following a 15min exposure to equilibration medium containing 7.5%EG and 7.5%DMSO. Larger numbers of pregnancies are required to ensure that the longer exposure time to cryoprotectants does not increase the risk of EED. Nevertheless, this protocol negates the need to puncture embryos <500µm providing an easy and successful way to vitrify them.

The mystery of the capsular vesicles: preliminary studies

An unexpected experimental finding was the presence of capsular vesicles (CVs) in the uterine flushings of mares 48h after transfer of a punctured Day 8 embryo. These CVs were thought to originate from fragments of capsule, in the same way that trophoblast fragments readily form vesicles in vitro. However, recovery of the capsule with the puncture hole alongside numerous CVs showed this was not the case. A preliminary study was undertaken to see if CVs formed in recipient mares following transfer of trophoblast vesicles (TVs) for 24h and if CV formation was uterine stage specific. Day 11 embryos were recovered from donor mares and the capsule removed before fragmenting the trophoblast. Small fragments of trophoblast were incubated in DMEMF12 (+ 5% FBS and pen/strep) for 48h in a Tri-gas incubator at 38°C. After 48h any degenerate TVs were discarded, and the remainder were washed in H199 (+ 5% FBS + pen/strep). Twenty to 50 TVs were transferred to the uteri of 25 recipient mares divided into 4 groups; G1, Days 3–5 post ovulation (n=6); G2, Days 6–8 (n=7); G3, Days 9–11 (n=6); Days 12–14 (n=6). The recipient mares’ uteri were flushed 24h after TV transfer and the recovered fluid searched. In all groups TVs were recovered with varying degrees of degeneration. Data were analyzed using aOne Way ANOVA on ranks with a post-hoc Dunn's test. The median number of CVs varied between G1–4 (0 vs. 68 vs. 17.5 vs. 6.5, respectively), with G1 versus G2 varying significantly (P<0.05). When adjusted for CVs per transferred TV, median values for G1–4 were (0 vs. 1.6 vs. 0.51 vs. 0.225), with G1 versus G2 remaining significantly different (P<0.05). The highest number of CVs recovered was 148 (2.96 per TV) from a Day 8 recipient, with a trend for a reduction in CV production after Day 8. These results suggest that CVs can form in the uterus following TV transfer providing the uterus is at a stage when an embryo would normally be forming a capsule, since transfers before Day 6 failed to produce CVs. Immunohistochemistry needs to be undertaken to confirm the CVs are identical in composition to in vivo produced capsules. Capsule formation in the mare is believed to involve interaction of the embryo with the endometrium, as in vitro produced embryos fail to produce a proper capsule. In vivo, developmentally regulated changes in composition of the capsule occur as its thickness increases up until at least Day 11. The mechanism of the formation of capsular vesicles remains a mystery as they are free in the uterus and do not appear to form around the trophoblastic vesicles.

Effects of embryo flushing on serum progesterone levels and the inter-ovulatory period in Arabian donor mares

To obtain the maximum number of cycles throughout the breeding season, avoid unwanted pregnancies and possible endometritis, the luteal phase of donor mares is shortened after embryo flushing. This is achieved by treating the donor with prostaglandin F2α (PGF2α) to destroy the corpus luteum and hence shorten the inter-ovulatory interval (IOI). It has been suggested that manipulation of the cervix and/or uterus during embryo flushing may result in release of endogenous of PGF2α and/or oxytocin, resulting in altered luteal function with a consequent decrease in progesterone and shortening of the cycle. Conversely, other authors have proposed no change in or extension of cycle length following embryo flushing. The purpose of this study was to evaluate the effects of embryo flushing with or without administration of PGF2α on serum progesterone levels and inter-ovulatory interval (IOI). Over two breeding seasons, 26 cycles of 6 Arabian donor mares, aged 4-15 years were studied. Inseminated mares were flushed on Day 8 or 9 post ovulation using Ringer's Lactate and a standard closed embryo flushing technique. After flushing the mares were divided into 2 groups; Group 1 (n=9) were treated with dl-Cloprostenol and Group 2 (n=17) were untreated. Blood wascollected on the day of embryo flushing and 48h later and the serum frozen until analysis using a progesterone ELISA kit (DRG Instruments, Marburg, Germany). For statistical analysis, a Shapiro-Wilks normality test was performed before 2-way ANOVA, followed by Tukey's post hoc and unpaired T tests. A significant decrease in plasma progesterone concentrations was observed in Group 1 mares (12.08 ± 3.91 ng/ml at flushing vs 0.7 ± 0.28 ng/ml at 48h, p<0.001) but not in Group 2 (12.62 ± 4.76 ng/ml at flushing vs 10.89 ± 3.57 ng/ml at 48h, p=0.549). The inter-ovulatory interval was shorter in Group 1 compared to Group 2 mares (18.56 ± 1.94 vs. 20.94 ± 1.84 days, respectively; p=0.006). Only 2 cycles had follicles ≥28mm at the time of embryo flushing and PGF2α was not administered in those cycles. Although the IOI was shortened by a mean of 2.38 days in mares given PGF2α, this was less than the 5-6 days which the dioestrous phase of the cycle had been shortened by. Based on the results obtained in this study, manipulation of the reproductive tract during embryo flushing does not result in endogenous release of PGF2α and/or oxytocin that is sufficient to cause luteolysis of the CL or cause a significant decrease in serum progesterone levels in embryo donor mares.

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

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

Interleukin-1 beta treatment alters prostaglandin-related mRNA expression and cytokine abundance in equine endometrial culture

Interleukin-1β (IL-1β) is a proinflammatory cytokine that modulates the immunoinflammatory response and induces prostaglandin synthesis. IL-1β has a variety of roles in the endometrium during inflammation, decidualization, pregnancy, and post-partum. It can cause changes in endometrial gene expression and may affect cytokine, chemokine, and prostaglandin-related pathways. The relationship between IL-1β, prostaglandins, and downstream inflammatory responses in the equine uterine environment is not fully known. Our objective was to determine the effect that IL-1β has on prostaglandin-related gene expression, as well as cytokine and chemokine abundance, in endometrial cell culture. Light breed mares (n=8) were examined daily by transrectal ultrasound until ovulation. An endometrial biopsy was collected at day 11 post ovulation, and used for primary endometrial cell culture. Cells were cultured in DMEM with 10% FBS. At 80% confluence, cells were either not supplemented or supplemented with: 100 nM Progesterone (Prog); 1 nM Estradiol-17β (Est); or 100 nM Prog and 1 nM Est (P+E). After 24 hours, cells were treated with recombinant equine IL-1β at either 10 ng/mL (Low) or 1000 ng/mL (High), or 0.2% DMSO as an untreated control. Twenty-four hours post-treatment, supernatant was collected, and a bead-based multiplex assay was used to quantify concentration of twelve cytokines and chemokines. Total RNA was extracted from the cells and mRNA expression was evaluatedfor 10 prostaglandin-related genes, using the ΔCT method with ACTB as the reference gene. Data analysis was performed using JMP Pro 16, using a restricted maximum likelihood model with treatment and supplementation as factors, and mare as a random factor. Treatment with IL-1β High resulted in increased levels of CCL2, CCL5, and CCL11 (p<0.001) in cell culture supernatant, as well as increased mRNA expression of PTGS2 (p<0.02), regardless of supplementation. Cells supplemented with Prog or P+E and treated with IL-1β High had increased expression of IL1R1, IL1RAP, PTGS1, and PTGES compared to non-supplemented cells (p<0.05). IL-1β High treatment increased LiF expression in cells supplemented with Prog or P+E compared to untreated cells (p<0.05). IL-1β likely acts to modulate inflammatory pathways in the endometrium, altering chemokine levels and activating expression of PTGS2, leading to changes in downstream prostaglandin synthesis and other related pathways. Progesterone appears to increase expression of several target genes when compared to non-supplemented cells. Progesterone has been shown to be involved in the suppression of several inflammatory pathways, and investigation into the ovarian steroid regulation of genes and proteins in the equine endometrium is warranted. Further studies on the effect of IL-1β on molecular pathways and prostaglandin regulation could lead to a greater understanding of the uterine environment in the pregnant and non-pregnant mare.

Effect of cooled vs frozen stallion semen on the efficiency of equine in vitro embryo production

Since 2008, our practice has been optimizing the equine in vitro embryo production system. Oocyte collection, in vitro maturation and culture media, sperm processing techniques and the Piezo driven sperm injection have remained relatively unchanged since then. However, our population of patients is now younger donor mares (< 17 years old) and in some instances, the use of extended-cooled semen has been prioritized. The purpose of this clinical report is to provide evidence that utilization of cooled semen 24-36 h post collection for ICSI increases the overall embryo production efficiency. Mares (n=350, 90% <17 years old and 8 different breeds) were presented for transvaginal follicular aspiration (TVA) and ICSI. Antral follicle count was determined prior to TVA and oocyte retrievals (n=680) were performed between day 12-20 post ovulation, or 19-25 days after the last TVA. In vitro embryo production was performed as previously described (LF Campos-Chillon, CM Owen, JL Altermatt, J Equine Vet Sci. 2019; 73:51-55). Briefly, sperm injections were performed with a Piezo driven system with the client's stallion of choice (n=262), and in vitro embryo culture was done for up to 9 days. Data (frozen vs cooled semen) was analyzed by One-way ANOVA using Minitab. The average follicle count was 15 (2-35) with an oocyte recovery rate of 82 ± 8%. Oocyte maturation rate expressed as oocytes witha polar body was 70.6 ± 1.1%; for frozen (n=476) vs cooled (n=204) semen injections, cleavage (57.6 ± 1.8 and 70.1 ± 2.9%) and blastocyst (13.4 ± 1.2 and 25.8 ± 1.5%) rates were different (p<0.001) respectively. In addition, number of embryos produced per cycle were 1.16 ± 0.08 for frozen and 2.24 ± 0.13 for cooled semen (p<0.001). Pregnancy rates of fresh embryo transfers at day 35 were higher (p<0.004) for embryos obtained with cooled (n=90, 91.5 ± 0.04%) than with frozen (n=250, 72 ± 0.05%) semen. Furthermore, of the 23% of cycles (n=156) that did not produce an embryo in the first TVA attempt, 18 and 82 % were performed with cooled and frozen semen, respectively. Overall, when cooled semen was used, there was a one-fold increase in embryos obtained per TVA, 20 percentile points higher pregnancy rate, and only 4 % of cycles using cooled semen yielded no embryo vs 19% with frozen semen. These results suggest that cooled semen used for ICSI increases the overall efficiency of the in vitro embryo production system. However, further prospective studies controlling stallion and mare effect of cooled and frozen semen in the model are warranted. In addition, studies involving sperm motility/viability, decondensation parameters and PLCz content and its effect in semen samples as recently described by Gonzales-Castro (Repro Fert Dev. 2022, 35:150-150).

Equine endometrial Th1 and Th2 evaluation during early pregnancy

As in all mammals, continuous feto-maternal dialogue is essential for successful pregnancy in mares. Nevertheless, several features of pregnancy in the mare are unusual and differ markedly from equivalent events in other species. The nature of the signal by which the equine embryo biochemically indicates its presence in the mare´s uterus for pregnancy maintenance remains a mystery to the present day, but the equine conceptus must begin to release this factor resulting in maternal recognition of pregnancy around day 10 to day 13 after ovulation. In humans and some animal species, the Th1/Th2 balance is essential for immunological tolerance in pregnancy. This immunohistochemical study aimed to assess the T-bet (Th1) and GATA3 (Th2) lymphocyte populations in the endometrium on days 7, 10, and 13 after ovulation in cyclic and pregnant mares. Mares were randomly assigned to the day of sample collection in both groups. In the first cycle, endometrial biopsies from 30 cyclic mares were collected on days 7 (n = 10), 10 (n = 10), and 13 (n = 10) post-ovulation (Cyclic group). In the second cycle, the same mares were bred to a fertile stallion, and endometrial samples were collected on days 7, 10, and 13 post ovulation. Immediately after biopsies, uteri were flushed, and those with an embryo recovered were allotted to the Pregnant group. Of the 30 mares flushed, embryos were recovered from6 mares on day 7, 6 on day 10, and 6 on day 13. Samples from mares without embryo recovery were excluded from both groups. Immunohistochemistry was used to detect Th1 (T-bet PA5-28881, Invitrogen) or Th2 (GATA3 L50-823, Thermo Fisher) proteins in the endometrium. Only stained lymphocytes from the stratum compactum (SC) were counted. Five fields were analyzed at 400x magnification. A two-way ANOVA was performed to evaluate the absolute number of T-bet/GATA3 cells and their ratio as a dependent factor and experimental groups, day, and their interactions as independent variables. T-bet-stained cells decreased from days 7 to 13 in both the Cyclic (day 7=73 vs 13=48) and Pregnant groups (day 7=83 vs 13=39) (P=0.0179); no difference was observed between groups. GATA3 stained cells remained constant over the days, with more stained cells in the Pregnant (day 7=37; 10=35; 13=34) than in the Cyclic group (day 7=23; 10=12; 13=12) (P=0.0001). The Th1/Th2 ratio did not show a difference (P>0.2222). Th2 cells are recruited into the SC of the endometrium of the pregnant mare. These cells could be secreting interleukins and establishing an anti-inflammatory microenvironment in early pregnancy. In conclusion, the presence of Th2 cells in pregnant mares could be important for embryonic development and maternal recognition of pregnancy regarding immunological tolerance.

Influence of Intrauterine Fluid Detection, Number of Transfers and Age of the Recipient on Pregnancy Rate and Early Embryonic Loss in a Commercial Embryo Transfer Program.

The selection of the recipient mare is one of the most important factors involved in the success of equine embryo transfer. The aim of this study was to determine whether the age of the recipient, the number of transfers and the detection of intrauterine fluid during the follicular phase or after ovulation can affect pregnancy rate at 14 and 45 days (PR 14 and PR 45) or early embryonic loss (EEL). A total of 1222 ETs were included in the study. Mares receiving the first embryo of the year had a higher PR 14 and 45 days compared to mares at the third transfer (78.8% and 70.1% vs. 65.6% and 54.1%, respectively). The detection of intrauterine fluid post ovulation negatively affected PR 14 (60.5% vs. 77.6%) and should therefore be considered an abnormal finding, probably being a sign of uterine inflammation or delayed uterine clearance. On the contrary, the age of the recipient mare and detection of fluid during follicular phase did not affect PR 14. Only the age of the recipient mare influenced the EEL, since mares aged 10-13 years had a higher EEL compared to mares aged 3-5 years (15.6% vs. 6.4%). Embryo size and grade affected PR 14 and 45.

Synchronization of the ovulation and copulation timings increased the number of in vivo fertilized oocytes in superovulated female mice.

The number of sperm that reaches the oocytes in mammalian species is limited. In mice, 8-10 oocytes are ovulated, a similar number of sperm reaches the oocytes, and nearly all oocytes are fertilized via natural mating. Meanwhile, our improved superovulation technique (ultrasuperovulation: administration of inhibin antiserum and equine chorionic gonadotropin [IASe]) produced 100 oocytes from a single female C57BL/6 mouse but resulted in only approximately 20 fertilized oocytes via mating. We hypothesized that sperm shortage in the ampulla might cause this low fertilization rate. Mice were mated in the proestrus stage or after hormone injection, but ovulation timing was not considered. In clinical application, the rhythm method supports fertilization by testing the ovulation period and synchronizing the ovulation and copulation timings. Therefore, this study examined the effects of ovulation and copulation timings on in vivo fertilization in female mice with IASe. Synchronization of the ovulation and copulation timings increased fertilization efficiency in female mice with ultrasuperovulation. The number of embryos obtained post ovulation was three times higher than that obtained pre ovulation. This study suggests that synchronized ovulation and copulation timings improve the efficiency of in vivo fertilization in IASe-treated female mice. This technique can be used to produce genetically modified mice and develop technologies for infertility treatment.

The Correlation of Endogenous Progesterone Concentration in Diestrus on Early Pregnancy Rate in Thoroughbred Mares

The aim of this project was to test the hypothesis that progesterone concentration 5 days after ovulation did not differ between pregnant and nonpregnant Thoroughbred mares on stud farms located in the Waikato region of New Zealand. A prospective cohort study was performed involving five stud farms in the Waikato region of New Zealand during the 2018 breeding season. A total of 275 mares were enrolled in the study. Mares were served by 34 individual stallions. Blood samples were taken from each mare 5 days after ovulation (D0) and measured for progesterone concentration. Early pregnancy was confirmed at D14 by transrectal palpation and ultrasonography of the mares reproductive tract. Progesterone concentration at Day 5 post-ovulation was higher in mares determined to pregnant at Day 14 of gestation than in mares determined to be non-pregnant at Day 14 (6.4 ± 3.0 ng/ml vs. 5.5 ± 3.3 ng/ml respectively; P = .02). A negative association between increasing mare age and pregnancy rate was found but mare age had no effect on progesterone concentration at D5. In this study we found that although higher serum progesterone concentration at Day 5 post ovulation was associated with a higher pregnancy rate at Day 14, no predictive or definitive minimum required progesterone concentration could be identified. Additional studies are required to determine if a synthetic progestogens can serve to supplant natural progesterone to increase pregnancy rate in naturally bred mares.

An approach to uncover the relationship between 17b-estradiol and ESR1/ESR2 ratio in the regulation of canine corpus luteum.

The canine corpus luteum (CL) is able to synthetise, activate and deactivate 17b-estradiol (E2) and also expresses nuclear estrogen receptors in a time-dependent manner during diestrus. Nevertheless, we are still missing a better comprehension of E2 functions in the canine CL, especially regarding the specific roles of estrogen receptor alpha (ERa) and ERb, encoded by ESR1 and 2, respectively. For that purpose, we analyzed transcriptomic data of canine non-pregnant CL collected on days 10, 20, 30, 40, 50 and 60 of diestrus and searched for differentially expressed genes (DEG) containing predicted transcription factor binding sites (TFBS) for ESR1 or ESR2. Based on biological functions of DEG presenting TFBS, expression of select transcripts and corresponding proteins was assessed. Additionally, luteal cells were collected across specific time points during diestrus and specificity of E2 responses was tested using ERa and/or ERb inhibitors. Bioinformatic analyses revealed 517 DEGs containing TFBS, from which 67 for both receptors. In general, abundance of predicted ESR1 targets was greater in the beginning, while abundance of ESR2 targets was greater in the end of diestrus. ESR1/ESR2 ratio shifted from an increasing to a decreasing pattern from day 30 to 40 post ovulation. Specific receptor inhibition suggested an ERa-mediated positive regulation of CL function at the beginning of diestrus and an ERb-mediated effect contributing to luteal regression. In conclusion, our data points toward a broad spectrum of action of E2 and its nuclear receptors, which can also act as transcription factors for other genes regulating canine CL function.