Early Luteolysis Research Articles

Effects of repeated embryo flushing without PGF2α administration on luteal function, percentage of unwanted pregnancy and subsequent fertility in mares.

PGF2α is commonly given at the end of embryo flushing (EF) to shorten the interval to the next oestrus and ovulation. To determine the effect of repeated EF on plasma progesterone concentration, percentage of mares with endometritis, unwanted pregnancy and subsequent fertility in mares flushed without the use of PGF2α. Controlled experiments. Nine mares were inseminated in seven consecutive cycles (n = 63), to either perform an EF (n = 54) 7-9 days after ovulation or left pregnant (n = 9). PGF2α was not used to induce oestrus. Ultrasound examination and blood sampling were performed just before the EF and 72 h later to determine changes in progesterone concentration and signs of endometritis. The overall percentage of positive EF/pregnancy was 55.5% (30/54) and 66.7% (6/9), respectively. The likelihood of pregnancy/positive EF in the first three cycles was 55.5% (15/29). This was not different (p > 0.1) from the fertility of the last four cycles (69.4%, 25/36). In five EF cycles (9.3%), mares had signs of endometritis and early luteolysis (progesterone <2 ng/mL) 72 h after EF. The reduction in progesterone concentration by 72 h after EF was greater (p < 0.05) for Day 9 (-2.3 ± 0.7 ng/mL) than Day 7 (-1.0 ± 0.8 ng/mL) or Day 8 (-1.3 ± 1.1 ng/mL) cycles. The progesterone concentration in non-flushed mares did not vary significantly during the sampled period (Day 7-12). There were 5 cycles in which the donor mare remained pregnant after the EF, although four were from a single mare. The mare population was limited to barren and maiden mares. The cycle order and operator allocation to each EF were not randomised. EF induces a subtle, but significant reduction in progesterone concentrations compared with non-EF cycles. However, the percentage of mares with EF-induced full luteolysis is low (9.3%). The fertility of mares after repeated EF without administration of PGF2α was unaffected; however, there is a considerable risk of unwanted pregnancy (5/27 = 18.5%) in donors from which an embryo was not recovered.

Effect of embryo flushing technique, number of flushing attempts and previous experience of operator on embryo recovery

The aims of this study were 1) to determine the effect of operator experience and embryo flushing technique on embryo recovery (ER) in each flushing attempt; 2) to determine the percentage of unwanted pregnancies and endometritis following embryo uterine lavage without administration of PGF2α. Seven non-lactating mares were inseminated with the same semen in four cycles (n=28 cycles). Embryo flushing was performed 7-9 days after ovulation by two operators of different experiences: experienced (>500 flushes) and beginner (<5), using two different flushing techniques in a crossover design. Mares were flushed three times with Ringer's Lactate (RL) “in-and-out”: 1L during the first two attempts and 0.5L during the third attempt. The remaining 0.5L was used to flush the tubing system. The two flushing techniques were 1) uterine massage (UM): continuous ballottement and massage of the uterus per rectum during RL recovery in each attempt, 2) control: the RL was allowed to flow back without touching the uterus during the first two attempts, and during the third attempt (0.5L) the uterus was manipulated as in UM. In both groups, 20 IU of oxytocin were administered at the second attempt and the RL was allowed to remain in the uterus for 3 minutes before recovery. No PGF2α was administered at the end of the flushing procedure. The overall ER was13/28 (46.4%). Ten embryos (10/13,77%) were recovered in the first filter, and 3 in the third filter. The experienced operator obtained a higher (P<0.05) ER (10/14) than the beginner (3/14). The ER of controls (6/14) was similar (P>0.1) to the UM (7/14). More embryos (7/7, P=0.06) were recovered in the first filter in the UM compared with the control group (3/6). Two mares entered prolonged dioestrus following a positive ER. Two mares with a negative ER (13%, 2/15) were pregnant 14 days after ovulation: both were from the control group and flushed by the beginner. In these mares, horn expansion was incomplete after RL infusion in every attempt. The last 8 embryos recovered in the study were transferred using the Wilsher´s technique immediately after recovery in the same donors, but none became pregnant. The overall mean inter-ovulatory interval was 22.5d (16-29d). Six mares (21.4%) developed free-intrauterine fluid and early luteolysis, of which 4 mares had been transferred with their own embryos. In conclusion, the previous operator´s experience had the greatest effect on overall ER. The uterine massage influenced the flushing attempt´s number in which the embryo was recovered. Oxytocin had no effect on ER. The risk of unwanted pregnancies in donors flushed by a beginner with no PGF2α treatment, was considerable.

Prostaglandin F2α regulates mitochondrial dynamics and mitophagy in the bovine corpus luteum.

Prostaglandins are arachidonic acid-derived lipid mediators involved in numerous physiological and pathological processes. PGF2α analogues are therapeutically used for regulating mammalian reproductive cycles and blood pressure, inducing term labor, and treating ocular disorders. PGF2α exerts effects via activation of calcium and PKC signaling, however, little is known about the cellular events imposed by PGF2α signaling. Here, we explored the early effects of PGF2α on mitochondrial dynamics and mitophagy in the bovine corpus luteum employing relevant and well characterized in vivo and in vitro approaches. We identified PKC/ERK and AMPK as critical protein kinases essential for activation of mitochondrial fission proteins, DRP1 and MFF. Furthermore, we report that PGF2α elicits increased intracellular reactive oxygen species and promotes receptor-mediated activation of PINK-Parkin mitophagy. These findings place the mitochondrium as a novel target in response to luteolytic mediator, PGF2α. Understanding intracellular processes occurring during early luteolysis may serve as a target for improving fertility.

Pregnancy outcomes with frozen-thawed single euploid blastocyst transfer following IVF cycles using HCG versus GNRH agonist trigger for final oocyte maturation

It is now well known that GnRH agonist (GnRHa) when used to trigger final oocyte maturation results in early luteolysis and consequently luteal phase dysfunction in fresh autologous IVF cycles. However, with the use of adjuvant low dose hCG, pregnancy outcomes are favorable and comparable to after hCG trigger(1). This suggests that GnRHa trigger does not impact oocyte or embryo quality. Furthermore, previous studies have shown comparable live birth rates after frozen-thawed embryo transfer (FET) cycles utilizing embryos obtained from cycles triggered previously with GnRHa versus hCG (2). Previous studies have also shown comparable euploid rates after GnRHa and hCG trigger (3). However, there are no studies evaluating pregnancy rates after transfer of euploid frozen embryos derived from cycles triggered with either GnRHa or hCG trigger. To compare pregnancy outcomes for frozen-thawed single euploid embryo transfer cycles that were generated from cycles triggered with either GnRHa or hCG. Retrospective cohort study done at a single academic IVF center evaluating frozen-thawed embryo transfer (FET) cycles in which single euploid blastocysts were transferred over a four year period. All embryos were generated in preceding fresh autologous IVF cycle in which hCG or GnRHa was used for final oocyte maturation. Embryos were subsequently transferred in a medicated or natural frozen cycle (44.3% medicated cycles in hCG group v. 37.0% in GnRHa group, p>0.05.) Patients were excluded if they had >1 embryo transferred, day 3 embryo biopsies, donor oocyte cycles or dual trigger with both hCG and GnRHa. Primary outcome was ongoing pregnancy rate or live birth rate (OPR/LBR) Secondary outcomes were clinical pregnancy rate (CPR), clinical loss rate (CLR) and multiple pregnancy rate (MPR.) Student’s t-test was used to analyze continuous variables and Chi Square test was used for categorical variables. Multivariate logistical regression was performed to control for potential confounding variables. A p-value of <0.05 was considered statistically significant. A total of 215 FET cycles were included for analysis (hCG trigger, n=115 subjects; GnRHa trigger, n=100 subjects.) The GnRHa group was significantly younger (34.8 years old v. 37.2 years old) and had higher baseline AMH values (4.55 ng/ml versus 2.1 ng/ml) than the hCG group, respectively (p<0.05.) There was no significant difference in OPR/LBR (60.9% v. 62.0%), CPR (68.7% v. 69.0%), CLR (8.9% v. 10.1%) or MPR (1.3% v. 2.9%) in the GnRHa v. hCG groups, respectively. Using logistic regression to control for the significant confounding variables of age and AMH value, the adjusted odds ratio for live birth was 1.09 (95% CI 0.60-1.99, p=0.77) when GnRHa trigger was compared to hCG trigger.Table 1Pregnancy Outcomes After Single Euploid Embryo Transfer in FET cycles Triggered by hCG or GnRHa Stratified by AgeAge Group<35 YO>35 YOAll PatientsPregnancy OutcomehCG (n=29)GnRHa (n=55)hCG (n=86)GnRHa (n=45)hCG (n=115)GnRHa (n=100)IR20/29 (69.0%)41/55 (74.5%)60/86 (69.8%)29/45 (64.4%)80/115 (69.6%)70/100 (70.0%)CPR19/29 (65.5%)41/55 (74.5%)60/86 (69.8%)28/45 (62.2%)79/115 (68.7%)69/100 (69.0%)OPR/LBR16/19 (55.2%)36/55 (65.5%)54/86 (62.8%)26/45 (57.8%)70/115 (60.9%)62/100 (62.0%)CLR3/19 (15.8%)5/41 (12.2%)6/60 (10.0%)2/28 (7.1%)9/79 (8.9%)7/69 (10.1%)MPR1/19 (5.3%)1/41 (2.4%)0/60 (0.0%)1/28 (3.6%)1/79 (1.3%)2/69 (2.9%)∗ There were no significant differences in any of the variables listed above (p>0.05) Open table in a new tab ∗ There were no significant differences in any of the variables listed above (p>0.05) Our findings confirm that euploid embryos created after hCG or GnRHa trigger have the same potential for pregnancy.

Supplemental progesterone induces temporal changes in luteal development and endometrial transcription in beef cattle

Long-acting injectable progesterone (iP4) supplementation during early diestrus is a strategy to increase conception rates in cow-calf beef operations. However, iP4 treatment causes early functional and structural regression of the corpus luteum (CL) in a proportion of iP4-treated animals, resulting in pregnancy loss. The hypothesis evaluated was that iP4 accelerates downregulation of sex-steroid receptors (PGR, ESR1, ESR2) during early to mid-diestrus and the upregulation of genes controlling PGF2α secretion (OXTR, PTGS2, AKR1B1) during late diestrus in the endometrium. Ovulations of cyclic, multiparous Nelore (Bos indicus) cows were synchronized, and cows were divided to receive placebo or 300 mg iP4 3 d postovulation (D3). Growth and vascularization of luteal tissue were evaluated by ultrasonography. Blood samples were collected from 3 d postovulation to 3 d after luteolysis, and P4 plasma concentrations were measured by radioimmunoassay. On days 3, 5, 7, 9, 11, 13, and 16 luminal endometrial samples were taken using a cytologic brush. Transcript abundance was measured by qPCR. Structural luteolysis occurred 3 d earlier in cows receiving iP4 compared to the control group. Analyzing only cows that received iP4, those that presented early luteolysis (ie, ≤ D16) showed a decrease in CL area and P4 concentration after D5, compared to the control group. Cows that presented early luteolysis showed a reduced abundance of transcripts on D5 for the ESR2 gene and a greater abundance of transcripts for OXTR and ESR1 on D16, compared to cows that did not present early luteolysis. The iP4-induced early luteolysis can be explained by two nonexclusive possibilities: the activation of uterine mechanisms that trigger early secretion of endometrial PGF pulses and the formation of a subfunctional CL that is prone to early regression.

Supplementation with long-acting progesterone in early diestrus in beef cattle: II. Relationships between follicle growth dynamics and luteolysis

The aims were to characterize follicular dynamics in response to supplemental progesterone (P4) and to investigate the relationships between follicular growth and onset of luteolysis in P4-treated cows, submitted or not to artificial insemination (AI). Nonsuckled beef cows detected in estrus were assigned to receive AI or to remain non-AI. Three days after ovulation (ie, D3), AI cows were injected with 150 mg of long-acting P4 (AI + injectable P4 formulation [iP4]; n = 22), and the non-AI cows were assigned to receive 150 mg iP4 (n = 19) or saline (control, n = 19). Between D3 and D21, growth dynamics of the dominant follicles (DFs) was monitored by ultrasonography. Plasma P4 concentrations were measured every other day from D9 to D19. Pregnancy status (ie, P: pregnant and NP: nonpregnant) was examined by ultrasound on D28 to D32. Injectable P4 formulation supplementation decreased average maximum diameter of first-wave DF (DF1). Neither day of emergence of DF2 or DF3 nor the proportion of two- or three-wave cycles were altered by supplemental P4. Daily mean diameter of DF2 and DF3 was also similar between control and iP4 groups. Consistently, daily mean diameter of DF1 in iP4-treated cows was smaller for cows that underwent luteolysis by D15 compared to a later onset. Progesterone concentrations between D9 and D19 decreased earliest in the iP4 group, latest in the control group and was intermediate for the NP-AI + iP4 group. In addition, three-wave cycles presented a delayed decrease on plasma P4 concentrations than two-wave cycles. Further analysis revealed that on two-wave cycles, P4 concentrations on D15 were lowest in the iP4 and NP-AI + iP4 animals compared to the control and P-AI + iP4 groups. Conversely, for three-wave cycles, on D15, P-AI + iP4, NP-AI + iP4, and controls had greater P4 concentrations than the iP4 group. In summary, our data indicate that impairment of first follicular growth was associated with P4-induced shortened luteal lifespan (D14–D15) and that three-wave cycles after AI can be more supportive for pregnancy maintenance in P4-treated cows. We speculate that such conditions play a critical role in the embryonic ability to inhibit iP4-induced early luteolysis reported in part I of this series.

Supplementation with long-acting progesterone in early diestrus in beef cattle: I. effect of artificial insemination on onset of luteolysis

Progesterone (P4) supplementation in early diestrus advances changes in the endometrial transcriptome, stimulating embryonic development. However, it also induces early onset of luteolysis. Occurrence of luteolysis before D16 postmating can be detrimental to fertility. A potential counteracting role of the elongating conceptus on early luteolysis is understood poorly. The aim of the study was to evaluate the effect of artificial insemination (AI; ie, pregnancy) on the temporal dynamics of luteolysis of cows supplemented with P4. Nonsuckled beef cows were inseminated at 12 h after estrus (D0: ovulation) or were not inseminated (no-AI). On D3, the AI cows were assigned to receive a single dose of 150 mg of injectable long-acting P4 via intramuscular injection (AI + iP4; n = 23), and the no-AI cows were assigned to receive iP4 (iP4; n = 21) or saline (control, n = 22). Corpus luteum (CL) development and regression were determined by ultrasonography (US) between D3 and D21. Plasma P4 concentrations were measured on D3 and every other day from D9 to D21. Pregnancy status was determined by US (D28‒D32). iP4 supplementation reduced luteal development (D5–D10) compared to the control group and increased incidence of luteolysis between D14 and D15. On D15, the proportion of cows that underwent luteolysis and plasma P4 concentrations differed between the iP4 group (47.6; 2.10 ± 0.47) and the control group (13.6; 4.40 ± 0.46) and was intermediate in the AI + iP4 group, respectively (26.1%; 3.70 ± 0.45 ng/mL; P < 0.05). The AI effects were due to the pregnant cows (n = 7). Considering nonpregnant cows only, the proportion of early luteolysis in the AI + iP4 group (37.5%) was similar to the iP4 group. Pregnancy was not established in cows having a shortened luteal lifespan. Indeed, interval to luteolysis in the AI + iP4 group (15.50 ± 0.66 d) was similar to the iP4 group (16.38 ± 0.46 d), but less than the control group (17.38 ± 0.40 d; P = 0.05). In conclusion, the effect of AI on extending luteal lifespan occurred exclusively in cows that maintained pregnancy.

Gonadotropin Releasing Hormone Agonist Final Oocyte Maturation and Human Chorionic Gonadotropin as Exclusive Luteal Support in Normal Responders

Background/Aims: Gonadotropin releasing hormone (GnRH) agonist triggering results in an endogenous gonadotropin flare. Although it effectively stimulates ovulation, GnRH agonist triggers results in an early luteolysis and requires modification of the luteal support. The current study aims to evaluate GnRH agonist triggering with exclusive human chorionic gonadotropin (hCG) luteal support. Methods: In this prospective observational study, 56 normogonadotropic-assisted reproductive technology patients, stimulated using a GnRH-antagonist protocol, were studied. Final oocyte maturation was achieved with 0.2 mg triptorelin acetate followed by progesterone free luteal support with human choriogonadotropin (1,500 IU * 2). A control group was selected from a pool of 1,023 normogonadotropic patients who received Choriogonadotropin alfa for final oocyte maturation and progesterone suppositories for luteal support. Results: No significant difference was found for the number of oocytes, oocyte maturation rate, fertilization and implantation rate, clinical pregnancy rate (25 vs. 26.7%) and live birth rate (25 vs. 21.4%). Progesterone levels in conception cycles were significantly higher in the study group than corresponding levels in the control group. Conclusion: GnRH agonist triggering with exclusive hCG support may be a valid alternative to hCG triggering with progesterone support. This protocol combines the potential advantages of a physiological trigger with a simple, patient-friendly, luteal support.

Impact of estradiol cypionate prior to TAI and progesterone supplementation at initial diestrus on ovarian and fertility responses in beef cows

In cattle, early diestrus progesterone (P4) supplementation modulates endometrial function to exert pro- and anti-pregnancy establishment effects; specifically, P4 stimulates conceptus growth, but also induces early onset of luteolysis. This paradoxical effect is frequently related to the inconsistent fertility outcomes that result from P4 supplementation experiments. Aim was to investigate the impact of exogenous estradiol (E2) treatment at the end of timed fixed AI (TAI) on frequency of early luteolysis and pregnancy of beef cows supplemented with P4. Ovulations (D0 of study) of suckled multiparous (n = 643) and primiparous (n = 193) Nelore cows (Bos indicus) were synchronized with an E2/P4-based protocol for TAI and assigned to receive 1.0 mg of estradiol cypionate (CP) or nothing (NoCP) on D–2 and 150 mg of injectable long-acting P4 (iP4) or Placebo (NoiP4) on D4 on a 2 × 2 factorial arrangement. On D15, the iP4 supplementation increased (P < 0.05) the frequency of early luteolysis (NoCP + iP4: 26.0%; [13/50] vs. NoCP: 8.0% [4/50]), but CP prevented this effect (CP + iP4: 8.3% [4/48] and CP: 6.4% [3/47]). The CP improved pregnancy/AI (P/AI) of multiparous (CP: 51.6% [165/320] and NoCP: 35.0% [113/323]; P < 0.001) and primiparous cows (CP: 40.4% [40/99] and NoCP: 24.5% [23/94], P < 0.05), regardless of iP4 treatment. The iP4 supplementation affected P/AI of CP and NoCP treated cows according to follicle size at TAI. For the CP treated cows, the iP4 supplementation improved P/AI of sub-populations of cows with follicles <12.35 mm (42.0% [34/81] vs. 53.1% [34/64]), while for NoCP treated cows, the improvements occurred in subpopulations of cows with follicles ≥12.35 mm (46.1% [35/76] vs. 58.7% [37/63]). In conclusion, strategies associating E2 and P4 supplementation decrease the incidence of early onset of luteolysis and improve P/AI of suckled beef cows with smaller follicles.

GnRH-agonist triggering for final oocyte maturation in GnRH-antagonist IVF cycles induces decreased LH pulse rate and amplitude in early luteal phase: a possible luteolysis mechanism

The use of GnRH agonist to trigger final oocyte maturation in GnRH-antagonist in vitro fertilization (IVF) cycles has been shown to significantly reduce or even eliminate the risk of ovarian hyperstimulation syndrome (OHSS) by inducing rapid luteolysis early in the luteal phase. The exact mechanism of this early luteolysis is still widely unknown. Since luteinizing hormone (LH) has a major role in corpus luteum support, we sought to explore the pattern of LH secretion early in the luteal phase. Ten high risk patients for developing OHSS and triggered with GnRH agonist were included. Frequent blood sampling (every 20 min for 6 h) to measure LH, estradiol and progesterone was done on the day of oocyte collection (n = 5, Group 1) and on the day of embryo transfer, 48 h after oocyte collection (n = 5, Group 2). We found that the mean LH concentration and its secretion rate decreased significantly in Group 2 compared to Group 1. Both groups had similar number of LH pulses characterized by very small amplitude. In Group 2, there was a steady significant decrease in estradiol and progesterone over time. The results of this study show that LH secretion deviates significantly from normal physiologic pattern, which can explain, at least in part, the post-GnRH-agonist trigger early luteolysis mechanism.

Proteomic Profile of Mabuya sp. (Squamata: Scincidae) Ovary and Placenta During Gestation.

Reptiles are one of the most diverse groups of vertebrates, providing an integrated system for comparative studies on metabolic, animal physiology, and developmental biology. However, the molecular data available are limited and only recently have started to call attention in the "omics" sciences. Mabuya sp. is a viviparous placentrotrophic skink with particular reproductive features, including microlecithal eggs, early luteolysis, prolonged gestation, and development of a highly specialized placenta. This placenta is responsible for respiratory exchange and the transference of all nutrients necessary for embryonic development. Our aim was to identify differentially expressed proteins in the ovary and placenta of Mabuya sp. during early, mid, and late gestation; their possible metabolic pathways; and biological processes. We carried out a comparative proteomic analysis during gestation in both tissues by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, two-dimensional gel electrophoresis, and matrix-assisted laser desorption/ionization. Differential protein expression in both tissues (Student's t-test P < 0.05) was related to several processes such as cell structure, cell movement, and energy. Proteins found in ovary are mainly associated with follicular development and its regulation. In the placenta, particularly during mid and late gestation, protein expression is involved in nutrient metabolism, transport, protein synthesis, and embryonic development. This work provides new insights about the proteins expressed and their physiological mechanisms in Mabuya sp. placenta and ovary during gestation.

Current Medical Strategies in the Prevention of Ovarian Hyperstimulation Syndrome

The purpose of this review is to analyze current medical strategies in the prevention of ovarian hyperstimulation syndrome (OHSS) during ovarian stimulation for in vitro fertilization. Owing to contemporary preventive measures of OHSS, the incidence of moderate and severe forms of the syndrome varies between 0.18% and 1.40%. Although none of medical strategies is completely effective, there is high-quality evidence that replacing human chorionic gonadotropin (hCG) by gonadotropin-releasing hormone (GnRH) agonists after GnRH antagonists and moderate- quality evidence that GnRH antagonist protocols, dopamine agonists and mild protocols reduce the occurrence of OHSS. Among various GnRH agonists, buserelin 0.5 mg, triptorelin 0.2 mg and leuprolide acetate (0.5-4 mg) have been mostly utilized. Although GnRH trigger is currently regarded as the best tool for OHSS prevention, intensive luteal support with exogenous administration of estradiol and progesterone or low-dose hCG on the day of oocyte retrieval or on the day of GnRH agonist trigger are required to achieve optimal conception rates due to early luteolysis. Among currently available dopamine agonists, cabergoline, quinagolide and bromocriptine are the most common drugs that should be used for prevention of both early and late OHSS. Mild stimulation protocols offer attractive option in OHSS prevention with satisfactory pregnancy rates.

Effects of intrauterine infusion of Trueperella pyogenes on endometrial mRNA expression of proinflammatory cytokines and luteolytic cascade genes and their association with luteal life span in dairy cows

Objectives were to determine the effects of intrauterine infusion (IUI) of Trueperella pyogenes on endometrial expression of proinflammatory cytokines and luteal life span. Holstein cows (n = 32) were allocated randomly, in two replicates (15 then 17 cows), to receive one of three treatments on Day 5 of the estrous cycle: TP (n = 13), IUI containing 109 colony-forming units/mL of T pyogenes; tumor necrosis factor (TNF; n = 9), IUI containing 1 μg of TNFα; and control (n = 10), IUI of saline solution. Five cows per treatment had uterine biopsies collected at 6, 12, and 24 hours after treatment to evaluate the endometrial messenger RNA expression of TNFα (TNF), interleukin-1β (IL1B), IL6, IL8, prostaglandin E synthase (PGES), prostaglandin F synthase (PGFS), and oxytocin receptor (OXR), and histologic evidence of inflammation. Messenger RNA expression was measured using quantitative reverse transcription polymerase chain reaction. The remaining cows had ovaries scanned and blood collected for progesterone evaluation; however, only seven, four, and three cows in the TP, TNF, and control groups were used for comparison in replicate 2. The GLIMMIX procedure of SAS was used for statistical analysis. All TP and TNF cows had moderate to severe endometrial inflammation, whereas only one control had mild inflammation. Premature luteolysis occurred in three, one, and zero cows in the TP, TNF and control groups, respectively. Delayed luteolysis occurred in one TP and one TNF cow. Interleukin-1β expression was greater in the TP cows than in the TNF cows at 24 hours after IUI. Moreover, IL6 expression tended to be greater for the TP cows than for the control cows at 12 hours after IUI. Interleukin 8 expression was greater in the TP cows than in the control and TNF cows at 24 hours after IUI. Oxytocin receptor expression tended to be greater for the TP cows and was greater for the TNF cows than for the control cows at 12 hours. The messenger RNA expressions of TNF, PGES, and PGFS were not affected by treatment, time, or their interaction. In conclusion, IUI of T pyogenes or TNFα led to histologic evidence of inflammation and early luteolysis in some cows, which may have been caused by increased endometrial expression of proinflammatory cytokines (i.e., IL1B, IL6), chemokines (i.e., IL8), and luteolytic cascade factors (i.e., OXR).

Relationships among nitric oxide metabolites and pulses of a PGF2α metabolite during and after luteolysis in mares

Hourly circulating concentrations of a PGF2α metabolite (PGFM), progesterone (P4), and LH were obtained from a reported project, and concentrations of nitric oxide (NO) metabolites (NOMs; nitrates and nitrites) were determined in eight mares. Unlike the reported project, hormone concentrations were normalized to the peak of the first PGFM pulse of luteolysis (early luteolysis), second PGFM pulse (late luteolysis), and a pulse after luteolysis. The duration of luteolysis was 23.1 ± 1.0 hours, and the peak of the first and second PGFM pulses occurred 6.5 ± 0.9 and 14.8 ± 0.8 hours after the beginning of luteolysis. Concentration of P4 decreased progressively within and between the PGFM pulses Changes were not detected in LH concentration in association with the PGFM pulses. Concentration of NOMs was greater (P < 0.05) at the peak of the PGFM pulse during early luteolysis (88.8 ± 15.0 μg/mL) than during late luteolysis (58.8 ± 9.0 μg/mL). Concentration of NOMs began to decrease (P < 0.05) 4 hours before the peak of the PGFM pulse of early luteolysis. Concentration began to increase (P < 0.05) an hour after the peak of the PGFM pulse of late luteolysis. An NOM decrease and increase was not detected during the PGFM pulse after luteolysis. On a temporal basis, results indicated that NO either is not required for luteolysis in mares or has a role in or responds only during late luteolysis. A caveat is that the relative contribution of the CL versus other body tissues to circulating concentrations of NOMs in mares has not been determined.

Introduction: Gonadotropin-releasing hormone agonist triggering of final follicular maturation for in vitro fertilization

These Views and Reviews articles examine the use of GnRH agonist for triggering the final stage of follicular maturation in IVF, resulting in excellent follicle maturation. An advantage of the GnRH agonist trigger is the ability to retrieve oocytes in high responders, yet markedly reducing the risk of ovarian hyperstimulation syndrome (OHSS). However, the induction of early luteolysis after the GnRH agonist trigger requires the use of aggressive steroidal luteal support or low-dose hCG to allow successful fresh ET and live birth. With the enhanced effectiveness of vitrification, segmentation in GnRH agonist-triggered cycles with freezing all embryos for transfer in subsequent cycles may be the optimal strategy for eliminating OHSS.

Physiopathologic mechanisms involved in mare endometrosis.

Endometrosis is a degenerative chronic process, characterized by paramount fibrosis development in mare endometrium. This condition is one of the major causes of subfertility/infertility in mares. As in other organs, fibrosis might be a pathologic sequel of many chronic inflammatory diseases. However, aetiology and physiopathologic mechanisms involved in endometrial fibrosis are still controversial. This review presents new hypotheses based on our newest data. As the first line of innate immune defence, systemic neutrophils arrive in the uterus at mating or in the presence of pathogens. A novel paradigm is that neutrophils cast out their DNA in response to infectious stimuli and form neutrophil extracellular traps (NETs). We have shown that bacterial strains of Streptococcus zooepidemicus, Escherichia coli or Staphylococcus capitis, known to cause endometritis in mares were able to induce NETs release in vitro by equine PMN to different extents. An intriguing dilemma is the dual action of NETs. While NETs play a desirable role fighting micro-organisms in mare uterus, they may also contribute to endometrial fibrosis. A long-term in vitro exposure of mare endometrium explants to NETs components (myeloperoxidase, elastase and cathepsin G) up-regulated fibrosis markers TGFβ and Tissue inhibitor of metalloproteinase (TIMP-1). Also, pro-fibrotic cytokines regulated collagen deposition and fibrosis. Changes in expression of connective tissue growth factor (CTGF), interleukins (IL)1-α, IL-1β, IL-6 and receptors in endometrium with different degrees of fibrosis and/or inflammation were observed. A putative role of CTGF, IL and NETs components in endometrosis development should be considered. Additionally, we speculate that in sustained endometritis in mares, prostaglandins may not only cause early luteolysis or early pregnancy loss, but may also be related to endometrial fibrosis pathogenesis by stimulating collagen deposition.

Efficiency of oestrous synchronization by GnRH, prostaglandins and socio-sexual cues in the North African Maure goats.

This study aims to develop at different seasons, for local North African Maure goats, synchronizing protocols simultaneously to the standard 'S' protocol using progestagens in association with prostaglandins and gonadotropin. In late May, 40 goats were assigned to either the 'S' protocol or to a protocol where oestrus and ovulation were induced by the buck effect in single-injection progesterone-treated goats and provoking early luteolysis using prostaglandin 9days after exposure to bucks 'B'. During the 72h after the treatments ended, 15 and 5 goats expressed oestrus in the 'S' and 'B' protocols (p<0.01). Mean time to oestrus was shorter for 'S' than for 'B' goats. Ovulation rate averaged 2.1±0.22 and 1.60±0.35 for, respectively, 'S' and 'B' goats (p>0.05). During mid-September, 60 goats were assigned to either 'S' treatment, 'PGF' treatment where oestrus and ovulation were synchronized using two injections of prostaglandin 11days apart or to 'GnRH' treatment where the goats had their oestrus and ovulation synchronized with a GnRH (day 0)-prostaglandin (day 6)-GnRH (day 9) sequence. More 'S' goats were detected in oestrus over the 96-h period after the end of the treatments (88.8, 73.7 and 55% in 'S', 'PGF' and 'GnRH' treatments, respectively; p<0.05). Mean ovulation rates were 2.3±0.27, 1.33±0.27 and 1.33±0.27 for, respectively, 'S', 'PGF' and 'GnRH' goats (p<0.001). Despite a similar ovulatory response to 'S' protocol, efficiency of prostaglandin and GnRH-based treatments should be tested in mid-breeding season.

11 STRATEGIES TO MODULATE THE PERI-OVULATORY ENDOCRINE MILIEU BY CONTROLLING PROGESTERONE CONCENTRATIONS PRE- AND POST-INSEMINATION IN BEEF COWS

Recent evidence indicates that progesterone (P4) secretion from corpus luteum (CL) is affected by preovulatory follicle (POF) size and that increased circulating P4 during early diestrus has a positive effect on embryo development and pregnancy rates in cattle. Despite the embryotrophic effects, early administration of P4 may impair CL growth and anticipates luteolysis. Owing to develop favourable peri-ovulatory endocrine milieus to pregnancy success, we evaluated in beef cows (1) the effect of CL presence during dominant follicle growth on follicular vascularization, size and ovulation rate and (2) the effects of POF size and supplementation of long-acting P4 post-AI on CL development and regression. Sixty-six Nelore cows received an intravaginal P4 device and an injection of oestradiol benzoate on Day 10 (5–10 days post-oestrus), and were split to receive sodium cloprostenol [prostaglandin F2α (PGF2); large follicle (LF); n = 31) or not [small follicle (SF); n = 35]. Devices were removed and PGF2 was injected on Day 1.75. Ovulation was induced with a gonadotropin-releasing hormone (GnRH; buserelin acetate) on Day 0. Cows were inseminated twice on Days 0.5 and 1. Cows that ovulated were assigned to receive placebo (LF/control group, n = 14; and SF/control group, n = 9) or 150 mg of long-acting P4 on D4.5 (LF/P4 group, n = 13; and SF/P4 group, n = 12). Colour-Doppler ultrasound scanning was done on Days 10, 6, 2, 0.5, 1, 1.5, 4.5, 7.5, and 15.5. Structural luteolysis was detected by 25% decrease in CL area and 50% in luteal blood flow of subsequent scanning. Data were analysed for the main effects of POF size and supplementation and their interaction using the PROC MIXED procedure of SAS (SAS Institue Inc., Cary, NC, USA). Follicle diameter (mm) on Day 2 (11.4 ± 0.4 v. 8.4 ± 0.3) and POF (14.1 ± 0.3 v. 12.5 ± 0.3) were greater (P &lt; 0.05) in LF group. Percentage of luteal blood flow in follicle wall was greater (P &lt; 0.05) on Day 0.5 (33.9 ± 3.4 v. 22.8 ± 3.6) and 1 (48.9 ± 4.8 v. 28.3 ± 4.3) in the LF group. Ovulation rate was lower (P &lt; 0.05) in the SF group (60%, 21/35) than in the LF group (90%, 28/31), suggesting that a functional CL during dominant follicle growth is less favourable to pregnancy success due to 30% reduction in fertilizing potential of non-ovulated follicles. For luteal area (cm2), an effect (P &lt; 0.05) of POF size was detected on Day 4.5 (1.7 ± 0.1 v. 1.3 ± 0.1), 7.5 (2.8 ± 0.2 v. 2.2 ± 0.1), and 15.5 (2.5 ± 0.1 v. 2.2 ± 0.2) in the LF and SF groups, respectively. Luteal blood flow was greater (P &lt; 0.05) in the LF groups (40.4 ± 1.7 and 47.6 ± 3.4) than in the SF groups (35.7 ± 1.5 and 37.9 ± 4.1) on Day 4.5 and 15.5, respectively. This indicates that larger and more vascularized POF may be associated with greater CL vascularization in addition to greater CL size. The frequency of cows with early luteolysis did not differ (P &gt; 0.1) between P4-treated (5/25) and placebo-treated (1/23) cows. We suggest that the P4 supplementation did not severely induce early luteolysis in inseminated cows and that the P4-embryotrophic effects may down-regulate the mechanisms involved in the earlier luteolytic process caused by greater exposure of uterus to P4. Support for this study was provided by the São Paulo Research Foundation (FAPESP, Sao Paulo, Brazil) and Ourofino (Cravinhos, SP, Brazil).

Pregnancy-associated genes contribute to antiluteolytic mechanisms in ovine corpus luteum

The hypothesis that ovine luteal gene expression differs due to pregnancy status and day of estrous cycle was tested. RNA was isolated from corpora lutea (CL) on days 12 and 14 of the estrous cycle (NP) or pregnancy (P) and analyzed with the Affymetrix bovine microarray. RNA also was isolated from luteal cells on day 10 of estrous cycle that were cultured for 24 h with luteolytic hormones (OXT and PGF) and secretory products of the conceptus (IFNT and PGE2). Differential gene expression (>1.5-fold, P < 0.05) was confirmed using semiquantitative real-time PCR. Serum progesterone concentrations decreased from day 12 to day 15 in NP ewes (P < 0.05) reflecting luteolysis and remained >1.7 ng/ml in P ewes reflecting rescue of the CL. Early luteolysis (days 12-14) was associated with differential expression of 683 genes in the CL, including upregulation of SERPINE1 and THBS1. Pregnancy on day 12 (55 genes) and 14 (734 genes) also was associated with differential expression of genes in the CL, many of which were ISGs (i.e., ISG15, MX1) that were induced when culturing luteal cells with IFNT, but not PGE2. Finally, many genes, such as PTX3, IL6, VEGF, and LHR, were stabilized during pregnancy and downregulated during the estrous cycle and in response to culture of luteal cells with luteolytic hormones. In conclusion, pregnancy circumvents luteolytic pathways and activates or stabilizes genes associated with interferon, chemokine, cell adhesion, cytoskeletal, and angiogenic pathways in the CL.

Use Of Combined Oral And Vaginal Estradiol With IM And Vaginal Progesterone In Luteal Phase Of Antagonist Cycles Triggered With GnRH Agonist Results In Good Clinical Pregnancy Rates

BACKGROUND: High responder patients have a reduced risk of OHSS when treated with a GnRH antagonist (GnRH-ant) to prevent premature ovulation, followed by a leuprolide acetate GnRH agonist (GnRH-ag) ovulation triggering protocol. In contrast to cycles triggered with HCG, the shorter half-life of the endogenous LH surge with the GnRH-ag trigger and subsequent pituitary suppression, likely contribute to the decrease in OHSS but also lead to early luteolysis in patients without luteal support (e.g.