Luteal P4 Research Articles

Correlation Between Luteal Phase Serum Progesterone Levels and Pregnancy Outcome in Frozen Embryo Transfer Cycles: A Prospective Cohort Study

Objectives To study the correlation between luteal phase serum progesterone (P4) levels and pregnancy outcome in frozen embryo transfer (FET) cycles and to find out the cut-off level of luteal phase serum P4 which favours successful pregnancy outcome in FET cycles. Material and Methods This prospective cohort study included 100 women undergoing hormone replacement therapy (HRT)-FET cycle at Akanksha IVF Centre from December 2023 to June 2024, fulfilling the inclusion and exclusion criteria. Serum P4 levels were measured for all the patients in the luteal phase (days 21–23) after FET with intramuscular P4 as luteal phase support. Pregnancy outcome was assessed in terms of implantation rate, biochemical pregnancy rate, clinical pregnancy rate (CPR), first-trimester miscarriage rate and ongoing pregnancy rate. Results The most favourable pregnancy outcomes were observed at serum P4 levels 25.1-35 ng/mL. A statistically significant association was seen between the luteal phase serum P4 levels and implantation rate (36.08%), biochemical pregnancy rate (4%), CPR (52%) and ongoing pregnancy rate (42%). According to the receiver operating characteristic (ROC) curve analysis, the optimal cut-off value for favourable outcomes was determined to be 22.3 ng/mL. However, the analysis also indicated that the luteal phase serum P4 levels did not reliably predict clinical or ongoing pregnancy. Conclusion Larger studies are needed to establish a threshold level of serum P4 in the luteal phase that can differentiate between successful and unsuccessful implantation. However, it is still uncertain whether the issue of unsuccessful implantation can be resolved once it is detected on days 21–23 of the HRT-FET cycle.

Vascular Alterations in Uterine and Ovarian Hemodynamics and Hormonal Analysis throughout Pregnancy Loss in Cows under Heat Stress.

This current study examined the ovarian (OA) and middle uterine arteries (MUA) blood flow under heat stress conditions regarding hormonal status (progesterone (P4), estradiol (E2), and nitric oxide metabolites (NOMs) assays). Eighteen pluriparous cows were examined, with twelve only being subjected to the natural mating as the other six animals were not bred. Pregnancy diagnosis was confirmed at day 30 by embryonic heartbeat and CL graviditatis (n = 6; pregnant), but some animals (n = 6) showed early embryonic death (EED), with the presence of control cows (n = 6). In the pregnant group, luteal diameter (cm) increased after mating, while in the pregnancy loss group it increased (p < 0.05) until reaching day 38 (1.41 ± 0.03), then decreased again. Luteal vascularity was declined in cows with EED after day 36 (p < 0.05) and reached its lowest level at day 50. P4 levels elevated in cows with EED until day 36 (13.64 ± 0.11) then declined. Both ipsilateral OA and MUA Doppler indices were declined in both groups except in cows who suffered from EED; both were elevated from day 38 until day 50 after mating. Ipsilateral peak systolic point (PSV cm/sec) of OA and MUA was elevated in both groups (p < 0.05), but in cows with EED this parameter was declined. E2 and NOMs levels were declined in cows with EED from day 40 and day 38 after mating (p < 0.05), respectively. This study provided novel data on the relations among the luteal diameter, E2, P4, and NOM levels, and luteal hemodynamics that predicts the amount of blood supply, which acts as a sensitive parameter to detect the alterations in luteal function during the first 50 days after mating.

Uterine tone: A neglected criterion for the selection of bovine embryo transfer recipients.

This study evaluated the relationship between CL features assessed by ultrasound (luteal tissue area and blood flow, BF) or rectal palpation (size), uterine tone (UT), plasma progesterone (P4) concentration on Day 7 (D7) and subsequent pregnancy outcomes in bovine embryo recipients. A total of 163 cows and heifers were included in this study. The expected day of ovulation after the synchronization protocol was designated as D0. On D7, ovaries and uterus were examined by ultrasonography and rectal palpation, and subjective scores (1-3 scale) were assigned for CL size, area and BF, and for UT. Blood samples were collected for further P4 analysis. Each embryo recipient then received a grade I frozen-thawed invivo-produced blastocyst, which was transferred to the uterine horn ipsilateral to the CL. Pregnancy diagnosis was performed on D35, and the results were retrospectively compared with the assigned scores for CL and UT. We observed a significant (p < .02) interaction between CL size and UT, with a progressive increase in the likelihood of pregnancy for recipients bearing a large CL among those with turgid UT. Ultrasound scoring of the CL using B-mode and Doppler-mode did not significantly predict pregnancy rates on D35 (p < .6 and p < .5, respectively). However, logistic regression analysis revealed a trend towards a quadratic effect (p < .08 and p < .06) indicating that the probability of pregnancy varied according to the area of luteal tissue and P4 concentrations, respectively. No significant (p > .05) association was found between the probability of pregnancy and the BF area of the CL. In summary, UT before embryo transfer may reflect successful recipient synchronization. Elevated P4 levels, assessed by CL size, may offset uterine contractility, mitigating adverse effects. Additionally, the CL area may be more important than its vascularization area when evaluating recipients D7 after ovulation.

Effect of prostaglandin alone and in combination with trace minerals on the follicular and luteal dynamics, estrus response and pregnancy in sub-estrus buffaloes (Bubalus bubalis).

Sub-estrus is a condition when buffaloes do not display behavioural estrus signs, despite being in estrus and causes a delay in conception and increases the service period. The present study describes the effect of synthetic prostaglandin (PGF2α) alone and in combination with trace minerals on the follicular and corpus luteum (CL) dynamics, serum estradiol (E2) and progesterone (P4) concentration correlating estrus response and pregnancy outcome in sub-estrus buffaloes during the breeding season. A total of 50 sub-estrus buffaloes, identified through ultrasonography (USG) examination, were randomly allocated into three groups, viz. T1 (Synthetic PGF2α, Inj. Cloprostenol 500 μg, i.m, n = 17), T2 (Synthetic PGF2α + Trace mineral supplementation, Inj. Stimvet 1 mL/100 kg body weight, i.m., n = 17) and control (untreated; n = 16). Following treatment, 100% of sub-estrus buffaloes were induced estrus in the T1 and T2 groups, while only 18.75% were induced in the control. The CL diameter and serum P4 concentration were significantly lower at post-treatment, whereas the pre-ovulatory follicle (POF) size and serum E2 concentration were significantly higher in the T1 and T2 groups as compared to the control (p < .05). The buffaloes of the T2 group had a greater proportion of moderate intensities estrus than those of T1. Moreover, the proportion of buffaloes conceived in the T1 and T2 were 41.2% and 52.95%, respectively. The larger POF diameter and higher serum E2 concentration were associated with intense intensity estrus and higher conception rate (66.7%) in sub-estrus buffaloes. Similarly, CL regression rate, POF size and serum E2 concentration were relatively higher in the buffaloes conceived as compared to those not conceived. It is concluded that synthetic PGF2α in combination with trace minerals induces moderate to intense intensities estrus in a greater proportion of sub-estrus buffaloes and increases the conception rate during the breeding season. Moreover, behavioural estrus attributes correlating follicle and luteal morphometry, serum E2 and P4 concentration could be used to optimise the breeding time for augmenting the conception rate in sub-estrus buffaloes.

Initiation and endocrine control of parturition in domestic mammals - Part 1

Endocrine regulation of parturition is based on an intense exchange of signals between the fetus, placenta and mother. Apart from sheep, our knowledge of the endocrine control of parturition is still very incomplete. However, current observations suggest significant differences between the species. For the maintenance of pregnancy, progesterone (P4) is the crucial superordinate regulatory factor, although in some species, such as the horse, functions of P4 are at least partially fulfilled by other progestogens. In general, prepartum P4 withdrawal is considered a prerequisite for the onset of physiological birth. In species with exclusive (dog) or predominant (e. g., cattle, goat, pig) luteal P4 at the end of gestation, luteolysis is the crucial event. In sheep, where P4 is of placental origin prior to parturition, the prepartum P4 decline is due to a switch in placental steroid metabolism. The mechanism of prepartum progestogen withdrawal in the mare is still largely unclear. In sheep, initiation of parturition proceeds from maturation of the fetal hypothalamic-pituitary-adrenal (HPA) axis, which leads to a steep prepartum rise in fetal cortisol concentrations stimulating the collapse of placental P4 production. In cattle, fetal cortisol probably triggers luteolysis via stimulation of placental prostaglandin secretion. In several other domestic mammalian species, there is also evidence that the initiation of parturition proceeds from maturation of the fetal HPA axis. However, the functional relationships between fetal cortisol and prepartum P4 withdrawal are largely unknown in nonruminant species.

P-595 What to expect from “standard vaginal progesterone regimen” in Hormone Replacement Therapy Frozen Embryo Transfer (HRT-FET) – do different products result in different serum levels?

Abstract Study question Do luteal serum progesterone levels (P4) vary in relation to time of blood sampling, the type and dosing of vaginal progesterone products used for HRT-FET? Summary answer Significant differences in luteal serum P4 levels exist between time of blood sampling as well as between different vaginal progesterone products and the administration regimen. What is known already Mid-luteal P4 levels below 9-11 ng/ml have been shown to negatively affect reproductive outcomes in HRT-FET cycles. Formulations of vaginal progesterone products differ as progesterone can be suspended in vegetable hard fat, in a vegetable lipophile liquor, be prepared in an oil-in-water emulsion carrier, or mixed in a tablet. Moreover, dosing regimens vary from one to three times daily, and BMI, age, and parity affect serum P4 levels; importantly, no consensus exists in terms of optimal time point to measure P4 in relation to administration, the specific serum P4 cut-off level to use or the most optimal vaginal progesterone product. Study design, size, duration Cohort study including 488 HRT-FET blastocyst transfers performed from January 2020 to November 2022 in patients undergoing a “standard vaginal progesterone regimen”. Each patient participated only once in the study. To compare the vaginal progesterone product used in the present study, we reviewed the most recent literature, including data from six studies, using three different products in six different regimens; all studies were cohort studies, including between 227 -1150 patients undergoing a “standard” HRT-FET protocol. Participants/materials, setting, methods A total of 488 patients in a public Fertility Clinic underwent HRT-FET, including endometrial preparation with oral oestradiol (6mg/24hours), followed by vaginal micronized progesterone, 400mg/12hours (Cyclogest®). Blood sampling on the blastocyst transfer day was standardised to two to four hours after progesterone administration. In contrast, P4 was measured randomly on the day of pregnancy testing. For comparison a review of the literature from six HRT-FET cohort studies using either Crinone®, Utrogestan® or “PIVET”-pessaries was performed. Main results and the role of chance The mean serum P4 (2-4 hours after administration) on the day of blastocyst transfer in our cohort was 15.4 ±6.6ng/ml. On the day of pregnancy testing (random time points) serum P4 levels were divided into four groups, depending on the time from progesterone administration to blood sampling: &amp;lt;2hours, &amp;gt;2&amp;lt;4hours &amp;gt;4&amp;lt;6hours and &amp;gt;6hours.The mean P4 levels were 14.7 ±6.3ng/ml, 15.6 ±5.8ng/ml, 14.3 ±4.9ng/ml and 12.9 ±6.4, respectively, and a significant difference was seen, between P4 levels measured 2-4 hours and &amp;gt;6 hours after vaginal administration (p = 0.03). As regards the product, the mean P4 levels in this study (Cyclogest®) was significantly different from the reviewed P4 levels of other “standard” HRT-FET cohorts, using three different products (Uterogestan®, Crinone® and the “PIVET-pessary”) 12.1 ±7.0ng/ml (p &amp;lt; 0.001), 7.6 ±3.2ng/ml (p &amp;lt; 0.001) and 29.3 ±20.3ng/ml (p &amp;lt; 0.001), respectively. As regards the dose, a significant difference in P4 levels was seen between dosing regimens in two cohorts when using the same product (Crinone®) 90mg x 3 vs. 90 mg x 2; 11.3 ±4.7ng/ml and 7.6 ±3.2ng/ml, respectively, p &amp;lt; 0.001. Interestingly, when comparing another product (Utrogestan®) used in different dosing regimens, 400mg x 2 vs. 200mg x 3, no difference in P4 levels was seen, 12.1 ±7.0ng/ml and 11.3 ±5.1ng/ml, P = 0.09. Limitations, reasons for caution In HRT-FET serum P4 levels reflect the exogenous administration, only, and P4 fluctuations depend on the route and number of administrations, as well as absorption and metabolization. The reviewed data used for this comparison between different progesterone products and regimens derive from cohorts with different characteristics and blood sampling timings. Wider implications of the findings Luteal serum P4 measurement is important, however, standardization of blood sampling in relation to the latest vaginal progesterone administration is mandatory to obtain correct levels. Clinicians should be aware of the significant differences in P4 levels related to dose and vaginal absorption between different types of vaginal progesterone products. Trial registration number EudraCT no.: 2019-001539-29

Regulatory T cells are paramount effectors in progesterone regulation of embryo implantation and fetal growth.

Progesterone (P4) is essential for embryo implantation, but the extent to which the pro-gestational effects of P4 depend on the maternal immune compartment is unknown. Here, we investigate whether regulatory T cells (Treg cells) act to mediate luteal phase P4 effects on uterine receptivity in mice. P4 antagonist RU486 administered to mice on days 0.5 and 2.5 postcoitum to model luteal phase P4 deficiency caused fewer CD4+Foxp3+ Treg cells and impaired Treg functional competence, along with dysfunctional uterine vascular remodeling and perturbed placental development in midgestation. These effects were linked with fetal loss and fetal growth restriction, accompanied by a Th1/CD8-skewed T cell profile. Adoptive transfer at implantation of Treg cells - but not conventional T cells - alleviated fetal loss and fetal growth restriction by mitigating adverse effects of reduced P4 signaling on uterine blood vessel remodeling and placental structure and by restoring maternal T cell imbalance. These findings demonstrate an essential role for Treg cells in mediating P4 effects at implantation and indicate that Treg cells are a sensitive and critical effector mechanism through which P4 drives uterine receptivity to support robust placental development and fetal growth.

Doppler ultrasonography: an improvement in real-time recipient selection in equine ET programs

In this study, we test the hypothesis that a CL with greater blood perfusion has a more significant impact than luteal size and P4 concentrations on pregnancy per embryo transfer (P/ET). The study was carried out from November/2021 to May/2022 on a stud farm of the Mangalarga Marchador breed in Itaperuna-RJ, Brazil. Recipient mares (n=110), aged between 3-16 years, weighing 370-460 kg, with adequate BCS and on days 3 to 9 after spontaneous ovulation, received a single fresh grade I or II blastocyst, retrieved by uterine flushing of donor mares. Immediately before the ET, the CL characteristics were evaluated by B-mode and Color-Doppler ultrasonography (US) and a blood sample was collected for plasma P4 assay. Recipients were retrospectively classified according to CL area [small (≤6 cm2) or large (>6 cm2)], CL blood perfusion [low ≤55 % or high >55%] and plasma P4 concentration [low ≤9.98 ng/mL or high >9.98 ng/mL]. Pregnancy diagnosis was performed 30 days after ET and P/ET analysis was performed using the GLIMMIX of SAS procedure. The overall P/ET was 73% (83/110). The P/ET was not influenced by the luteal area, but effects of CL blood perfusion class (P=0.007) and the P4 plasma concentration class (P=0.02) on P/ET were observed. The P/ET was greater in the high luteal perfusion group [89.1%, 41/46 vs.65.6%, 42/64] and in the high P4 group [85.4%, 41/48 vs. 67.7%, 42/62]. When area, luteal blood perfusion and P4 concentration were compared with the probability of pregnancy through ROC curve analysis, luteal blood perfusion (AUC=0.66) was the most significant (P=0.01) predictor of pregnancy compared to area (AUC=0.53) and plasma P4 (AUC=0.60). Finally, recipients were grouped into four different groups by analyzing CL blood perfusion classes and P4 (high CLBP and high P4 vs. High CLBP and low P4 vs. low CLBP and high P4 vs. low CLBP and low P4) and compared with the P/ET using the Fisher test. P/ET differed between groups (P=0002), as indicated by the higher P/ET in the high CLBP and high P4 95% (19/20) and high CLBP and low P4 84.6% (22/26) groups, compared to animals with low CLBP and low P4 55.5% (20/36). In conclusion, although the P4 concentration is a potential indicator of pregnancy success, it cannot be used in real-time when selecting the most appropriate recipient in a commercial ET program. Also, luteal blood perfusion determined by Doppler-US is an innovative and easily accessible tool that can be used in real-time to select the recipients with the greatest chances of maintaining pregnancy in ET programs in horses.

Impaired luteal progesterone synthesis observed in gilts with PMSG/hCG-induced estrus affects the expression of steroid, prostaglandin, and cytokine receptors in the endometrium and myometrium during the peri-implantation period

Abstract The present study aimed to examine the effect of impaired progesterone (P4) synthesis, observed in gilts with gonadotropin-induced estrus, on the uterine expression of receptors important for pregnancy establishment. Twenty prepubertal gilts received 750 IU PMSG and 500 IU hCG 72 h later, while 18 prepubertal gilts in the control group were observed daily for estrus behavior. Gilts were inseminated in their first estrus and slaughtered on days 10, 12, and 15 of pregnancy to collect endometrial and myometrial tissues for mRNA analysis using real-time PCR. As we previously described, gilts with PMSG/hCG-induced estrus showed decreased luteal P4 synthesis on days 10 and 12 of pregnancy. PMSG/hCG treatment did not affect P4 receptor mRNA expression in either uterine tissue. In the endometrium, a greater mRNA transcript abundance of estrogen receptors (ESR1 and ESR2), androgen receptor (AR), prostaglandin (PG) E2 receptors (PTGER2 and PTGER4), PGF2α receptor (PTGFR), interleukin 6 receptor (IL6R), and tumor necrosis factor α receptors (TNFRSF1A and TNFRSF1B) was detected in gilts with natural than with PMSG/hCG-induced estrus (P&lt;0.05). In the myometrium, the mRNA expression of AR, PTGER2, and PTGFR was lower, while PGI2 receptor (PTGIR) transcript abundance was elevated in the gilts treated with PMSG/hCG as compared with the control animals (P&lt;0.05). In summary, a decreased luteal P4 level during the peri-implantation period in gonadotropin-stimulated pigs affects endometrial and myometrial receptor expression, with the endometrium being more sensitive to impaired P4 synthesis. Whether the observed changes alter uterine receptivity to local and systemic factors remains to be elucidated.

Injectable progesterone for estrus and ovulation induction in seasonal anestrous ewes

We evaluated, for the first time, the effects of long-acting injectable progesterone (iP4) for estrus induction in ewes during non-breeding season. To evaluate the serum progesterone (P4) curve induced by the application of iP4 in sheep and the effect of its use prior to the cyclicity induction protocol, for Experiment 1, 39 ewes were allocated into three groups: no-treatment, control (n = 14); 37.5 mg of iP4 im (iP4 37.5, n = 13), and 75 mg of iP4 im (iP4 75, n = 12). P4 dosage in a subset of animals (5 per group) showed the highest P4 level at 48 h and reduction to basal levels 10 days after injection, when all females received an intravaginal device (IVD) containing 60 mg of medroxyprogesterone acetate (MPA) that remained for 11 days. At withdrawal the animals received 400 IU of eCG im, and rams remained for three days with ewes. On days 6 and 12 after IVD removal, blood samples were collected from a subset (n = 5 per group) of ewes that manifested estrous. iP4 75 group had lower luteal P4 concentration, and lower conception rate (35 days after mating) when compared to the other groups. To evaluate the possibility of replacing IVD with iP4, on experiment 2, 36 ewes were allocated into two treatments: control (n = 10), IVD for nine days and 300 IU of eCG at withdrawal; and iP4 75 (n = 26) 75 mg of iP4 im and, after 9 (n = 13) or 10 (n = 13) days, animals were injected with 300 IU of eCG. Estrus manifestation and conception rates were lower in groups treated with iP4. Eleven days after estrus, 93.3% of a subset of animals (5 from control and 10 from iP4 75) exhibited P4 concentrations above 1 ng/mL, indicating ovulations. In conclusion, the injection of 75 mg of long acting iP4 maintained levels above 1 ng/mL for at least 7 days, and negatively affected P4 luteal concentration and conception rate after a subsequent cyclicity induction protocol. Treatment with 75 mg of iP4 associated to eCG is suitable for inducing ovulations, although inhibits estrus manifestation.

P-298 The live birth rate of the endometriosis patient is significantly increased by high luteal phase serum progesterone in HRT-FET cycles - A cohort study

Abstract Study question What is the optimal serum progesterone (P4) cut-off level for endometriosis patients undergoing hormone replacement therapy frozen embryo transfer (HRT-FET) with intensive exogenous progesterone luteal phase support? Summary answer Endometriosis patients with luteal serum P4 levels≥118nmol/l had significantly higher live birth rates (LBR) compared to patients with lower P4 levels. What is known already A low luteal serum P4 level decreases the reproductive outcome of HRT-FET cycles in the “standard” patient, and an optimal cut-off level of 10ng/ml has been suggested. Interestingly, some studies reported a negative impact of too high serum P4 levels during HRT-FET. The pathology of endometriosis is dominated by an endometrial disruption of progesterone and oestrogen signalling pathways. This results in intra-endometrial oestrogen dominance, and leads to progesterone resistance which negatively impacts endometrial receptivity and function. Until now no study explored the optimal serum P4 level in the endometriosis patient undergoing HRT-FET. Study design, size, duration A cohort study including 262 HRT-FET cycles in 179 patients, undergoing transfer of warmed blastocysts, deriving from freeze all cycles from January 2016 to august 2019. Participants/materials, setting, methods Patients were diagnosed with endometriosis either by laparoscopy or by ultrasound in cases with visible endometriomas. Pre-treatment consisted of 42 days of oral contraceptive pill and 5 days' wash-out, followed by 6 mg oral oestrogen daily. Intensive exogenous progesterone supplementation, including vaginal progesterone gel 90mg/12h commenced when the endometrium was ≥7mm. From the 4th day of progesterone patients also received intramuscular progesterone 50mg. Blastocyst transfer was scheduled for the 6th day of progesterone. Main results and the role of chance The mean P4 level was 103.1 ±44.4nmol/l and the overall positive HCG, live birth (LBR) and total pregnancy loss rates (TPLR) were 60%, 39% and 34%, respectively. The optimal serum P4 cut-off level was 118nmol/l defined as the maximum of the Youden index. No significant differences were seen between patients above or below 118nmol/l as regards age, BMI, fertilisation method or blastocyst score. In a total of 33% of cycles (86/262) the P4 level was ≥118nmol/l, and the unadjusted LBR was significantly higher in the high P4 group 51% (44/86) versus 34% (59/176) (p = 0.006) in the low P4 group. Furthermore, a non-significant difference in TPLR was found: 41% vs 25 % in favour of high P4 (p = 0.066). A logistic regression analysis showed that patients with P4 levels ≥118nmol/l were more likely to achieve a live birth compared to patients with P4 levels &amp;lt;118nmol/l; (odds ratio 2.1 [95% confidence interval 1.2 - 3.7] after adjusting for age, BMI, blastocyst score, blastocyst age (day 5 or day 6) and number of blastocysts transferred. Limitations, reasons for caution Cohort study with data prospectively registered and retrospectively analyzed. The estimated serum cut-off for P4 of 118nmol/l is valid for vaginal progesterone gel 90mg/12h and intramuscular progesterone 50mg from the 4th progesterone day; whether the cut-off is applicable for other progesterone regimens needs to be explored. Wider implications of the findings The optimal luteal P4 level of the endometriosis patient undergoing HRT-FET is significantly higher than that estimated for the "standard" patient due to endometrial progesterone resistance. Luteal phase P4 monitoring is mandatory to obtain the highest LBR in the endometriosis patient undergoing HRT-FET. Trial registration number 1-10-72-4-17

120 Trilostane Effectively Reduces CL Progesterone Synthesis Prior to Acquisition of Luteolytic Sensitivity in Cyclic Gilts

Abstract Understanding the mechanisms of luteolytic sensitivity (LS) is critical for identifying novel methods of controlling the reproductive cycle of livestock species. Uniquely, porcine corpora lutea (CL) remain unresponsive to the luteolytic effects of prostaglandinF2a (PGF) until days 12-13 of the estrous cycle. Previous data from our laboratory has demonstrated that luteal macrophages (MACs) play an active role in this process. It was hypothesized that suppressing progesterone (P4) production prior to LS would activate MACs and initiate LS. The objective of this study was to determine the optimal dose of Trilostane, (TRIL) a commercially available 3β-hydroxysteroid dehydrogenase inhibitor, to reduce luteal and serum P4 by a minimum of 80% and induce LS. Gilts received Matrix® for 14 days and then checked twice daily for estrus. At estrus (day 0), gilts were randomly assigned to receive the TRIL treatment (0,3,5,7.5,or 10mg/kg, n=2 per treatment) orally, every 12h for 36h starting on day 7 of the estrous cycle. Blood samples were taken every 12 hours on days 6 through 9 post estrus to measure P4 following treatment. Gilts were euthanized on day 9 of the estrous cycle, and CL tissue was frozen for P4 analysis. Progesterone concentrations were determined via RIA. Data were analyzed using a MIXED procedure of SAS and statistical significance was determined at P &amp;lt; 0.05 and a tendency at 0.05&amp;lt; P &amp;lt; 0.10. Serum P4 was reduced by 50% by 12h post treatment in all TRIL groups compared to control (P &amp;lt; 0.05). However, only the 10mg/Kg dose effectively reduced P4 by 80% compared to control (P &amp;lt; 0.05) and suppressed P4 throughout the treatment period. Luteal P4 was reduced by 72.4,82.4,87.7, and 88.6% compared to controls (P &amp;lt; 0.05) in treated with 3,5,7.5 and 10 mg/kg of TRIL, respectively. Collectively, oral administration of TRIL effectively reduced both serum and luteal P4, and the optimal dose of 10mg/Kg TRIL will be utilized in future studies addressing progesterone’s regulation of luteal macrophage, and their role in controlling LS.

Direct effects of linoleic and linolenic acids on bovine uterine function using in vivo and in vitro studies.

The aim of the present study was to evaluate the effects of continuous administration of linoleic acid or linolenic acid into the intra-uterine horn, ipsilateral to the corpus luteum, on the duration of the estrous cycle and plasma progesterone (P4) concentration. The effects of linoleic and linolenic acids on bovine uterine and luteal functions were also studied using a tissue culture system. Intra-uterine administration of linoleic or linolenic acid (5 mg/10 ml of each per day) in cows, between days 12 and 21, resulted in a prolonged estrous cycle compared to the average duration of the last one to three estrous cycles before administration in each group (P < 0.05). Moreover, plasma P4 concentration in cows treated with linoleic or linolenic acid was high between days 19 and 21 (linoleic acid), or on day 20 (linolenic acid), compared to that of the control cows (saline administration; P < 0.05 or lower). Both linoleic (500 µg/ml) and linolenic (5 and 500 µg/ml) acids stimulated prostaglandin (PG) E2 but inhibited PGF2α production by cultured endometrial tissue (P < 0.01), while P4 production by cultured luteal tissue was not affected. These findings suggest that both linoleic and linolenic acids support luteal P4 production by regulating endometrial PG production and, subsequently, prolonging the duration of the estrous cycle in cows.

Galectin-1 induces gene and protein expression related to maternal-conceptus immune tolerance in bovine endometrium†.

Conceptus secretory factors include galectins, a family of carbohydrate binding proteins that elicit cell adhesion and immune suppression by interacting with intracellular and extracellular glycans. In rodents, galectin-1 (LGALS1) promotes maternal-fetal immune tolerance in the decidua through expansion of tolerogenic cluster of differentiation 11c (CD11c) positive dendritic cells, increased anti-inflammatory interleukin (IL)-10, and activation of forkhead box P3 (FOXP3) positive regulatory T cells (Treg). This study characterized galectin expression in early ruminant conceptuses and endometrium. We also tested the effect of recombinant bovine LGALS1 (rbLGALS1) and progesterone (P4) on endometrial expression of genes and protein related to maternal-conceptus immune tolerance in cattle. Elongating bovine and ovine conceptuses expressed several galectins, particularly, LGALS1, LGALS3, and LGALS8. Within bovine endometrium, expression of LGALS3, LGALS7, and LGALS9 was greater on Day 16 of pregnancy compared to the estrous cycle. Within ovine endometrium, LGALS7 was greater during pregnancy compared to the estrous cycle and endometrium of pregnant sheep tended to have greater LGALS9 and LGALS15. Expression of endometrial LGALS4 was less during pregnancy in sheep. Treating bovine endometrium with rbLGALS1 increased endometrial expression of CD11c, IL-10, and FOXP3, within 24h. Specifically, within caruncular endometrium, both rbLGALS1 and P4 increased FOXP3, suggesting that both ligands may promote Treg expansion. Using IHC, FOXP3+ cells with a leukocyte phenotype were localized to the bovine uterine stratum compactum near the uterine surface and increased in response to rbLGALS1. We hypothesize that galectins have important functions during establishment of pregnancy in ruminants and bovine conceptus LGALS1 and luteal P4 confer mechanisms of maternal-conceptus immune tolerance in cattle.

Changes in Porcine Corpus Luteum Proteome Associated with Development, Maintenance, Regression, and Rescue during Estrous Cycle and Early Pregnancy.

Corpus luteum (CL), a transitory gland, undergoes rapid growth in a limited time to produce progesterone (P4) followed by its regression. A complex molecular signaling is involved in controlling luteal P4 production. In the present study, 2D gel electrophoresis-based proteomics and in silico functional analysis were used to identify changes in key proteins and pathways in CL along the different stages of the estrous cycle as its development progresses from early (Day 3) to mid-luteal phase (Day 9), effective functioning (Day 12) followed by regression (Day 15) or, in the case of pregnancy, rescue of function (Day 15). A total of 273 proteins were identified by MALDI-MS/MS analysis that showed significant changes in abundances at different stages of CL development or regression and rescue. Functional annotation of differentially abundant proteins suggested enrichment of several important pathways and functions during CL development and function maintenance including cell survival, endocytosis, oxidative stress response, estradiol metabolism, and angiogenesis. On the other hand, differentially abundant proteins during CL regression were associated with decreased steroid synthesis and metabolism and increased apoptosis, necrosis, and infiltration of immune cells. Establishment of pregnancy rescues CL from regression by maintaining the expression of proteins that support steroidogenesis as pathways such as the super-pathway of cholesterol biosynthesis, RhoA signaling, and functions such as fatty acid metabolism and sterol transport were enriched in CL of pregnancy. In this study, some novel proteins were identified along CL development that advances our understanding of CL survival and steroidogenesis.

Impact of manganese amino acid complex on tissue-specific trace mineral distribution and corpus luteum function in gilts.

Functional corpora lutea (CL) are required for pregnancy establishment and gestational maintenance in swine, and CL function is susceptible to environmental influences. Manganese (Mn) could be critical in regulating CL function since it is a component of the antioxidant enzyme Mn superoxide dismutase (MnSOD) as well as enzymes involved in cholesterol and steroid hormone synthesis. We hypothesized that a more bioavailable dietary Mn source would increase Mn content in the CL thereby influencing luteal function during the mid-luteal phase of the estrous cycle. Postpubertal gilts (n = 32) were assigned to one of four gestation diets. The control diet (CON) met or exceeded National Research Council (2012) requirements and was formulated to contain 20 parts per million (ppm) of added Mn in the form of Mn sulfate. Three additional diets included 20 (treatment [TRT]1), 40 (TRT2), or 60 (TRT3) ppm of added Mn from a Mn-amino acid complex (Availa-Mn; Zinpro Corporation) instead of Mn sulfate. Dietary treatment began at estrus synchronization onset and continued through 12 days post estrus (dpe) of the ensuing estrous cycle. Blood samples were collected at estrus onset, which was assigned as 0 dpe, as well as 4, 8, and 12 dpe. Gilts were euthanized and tissues were collected at 12 dpe. Serum progesterone (P4) increased (P < 0.01) from 0 to 12 dpe but was unaffected by dietary treatment (P = 0.15) and there was no effect of the interaction between day and treatment (P = 0.85). Luteal Mn content increased (P ≤ 0.05) by 19%, 21%, and 24% in gilts fed TRT1, TRT2, and TRT3, respectively, compared to CON. Luteal P4 concentrations decreased (P = 0.03) 25%, 26%, and 32% in gilts fed TRT1, TRT2, and TRT3, respectively, compared to CON. Relative to CON gilts, CL calcium content decreased (P = 0.02) by 36%, 24%, and 34% for TRT1, TRT2, and TRT3 gilts, respectively. Collectively, these data support the hypothesis that feeding a more bioavailable Mn source increases Mn accumulation in CL tissue. If and how this influences CL function may be related to altered luteal P4 concentrations.

29 Influence of dietary manganese source on the corpus luteum proteome in pigs

Abstract Functional corpora lutea (CL) are required for pregnancy establishment and gestational maintenance in swine. Manganese (Mn) could be critical in regulating CL function since it is a cofactor for the enzyme Mn superoxide dismutase (Mn-SOD) as well as enzymes involved in progesterone (P4) synthesis. We hypothesized a more bioavailable dietary Mn source would increase CL Mn content thereby influencing luteal function during the mid-luteal phase of the estrous cycle. Post-pubertal gilts (n = 32) were assigned to one of four gestation diets. The control diet (CON) met or exceeded NRC requirements and was formulated to contain 20 ppm added Mn in the form of Mn sulfate. Three additional diets included 20 (TRT1), 40 (TRT2) or 60 (TRT3) ppm Mn from a Mn-amino acid complex (Availa-Mn; Zinpro Corporation) in place of Mn sulfate. Dietary treatment began at estrus synchronization onset and continued through D12 of the ensuing estrous cycle when gilts were euthanized. Mn content increased (P ≤ 0.06) 19, 21 and 24% in CLs of gilts fed TRT1, TRT2, and TRT3, respectively, and luteal P4 concentration decreased (P ≤ 0.03) 25, 26, and 32% in gilts fed TRT1, TRT2, and TRT3, respectively, compared to CON. Total CL protein was extracted and liquid chromatography with tandem mass spectrometry (LC-MS/MS) was performed to assess global protein abundance. Compared to CON, 29, 105, and 118 proteins were differentially abundant (P &amp;lt; 0.01) in TRT1, TRT2, and TRT3, respectively. KEGG pathway analysis revealed proteins involved in P4 signaling (membrane-associated P4 receptor component 2) and cholesterol synthesis and transport (mevalonate kinase, diphosphomevalonate decarboxylase, low density lipoprotein receptor) were downregulated in response to Availa-Mn. Collectively, these data support the posit that dietary Mn source affects Mn accumulation and P4 concentration in CL tissue and influences protein abundance which may affect CL function.

Luteotropic effects of human chorionic gonadotropin administered 7.5 days after synchronous estrous induction in Morada Nova ewes

This study was conducted in ewes to assess effects of human chorionic gonadotropin (hCG) administration after imposing an estrous induction treatment regimen. Ewes (n = 115) were treated with a 60 mg medroxyprogesterone-intravaginal-sponge for 6 d plus 200 IU of equine chorionic gonadotropin (eCG) im and 37.5 μg d-cloprostenol im 36 h before sponge removal (Day 0). After natural mating, ewes having at least one corpus luteum (CL; n = 108) were administered either 1 mL of saline (G-Control; n = 53) or 300 IU of hCG (G-hCG; n = 55) on Day 7.5 after sponge removal (Day 0). Ovarian ultrasonography and blood collection were performed on Days 7.5, 13.5, 17.5, 21.5, and 30.5. Accessory CL (aCL) were observed in 81.5 % (G-hCG) and 0.0 % (G-Control) of ewes (P = 0.0001). Diameter, area, and volume of luteal tissue were greater (P < 0.05) in G-hCG from Day 13.5 to 30.5. Progesterone (P4) concentrations were greater (P < 0.05) on Days 13.5, 17.5, 21.5 and 30.5 for ewes of the G-hCG group. Pregnancy percentage was similar (P = 0.25) between groups [47.1 % (G-control) compared with 60.0 % (G-hCG)], although total number of lambs produced by estrous synchronized ewes was greater (P = 0.005) in ewes of the G-hCG group (90.9 % compared with 66.0 %). In conclusion, hCG administration 7.5 days after sponge removal from Morada Nova ewes during the non-breeding season is an effective treatment to induce aCL formation, improve luteal tissue biometry and P4 concentrations, and to enhance the total number of lambs born.

In vivo response of the corpus luteum to progesterone treatment of gilts during early gestation

Supplementation of progesterone (P4) in pregnant gilts increases concentrations of circulating P4 and stimulates the secretory activity of the endometrium. In this study, there was examination of the consequences of exogenous P4 administration on luteal P4 content and the expression of genes related to the corpus luteum (CL) function. Gilts with gonadotropin-induced estrus were administered daily injections of corn oil (n = 8) or P4 (n = 8) on days 3 through 10 after insemination. The animals were slaughtered on day 12 of pregnancy to obtain corpora lutea for real-time polymerase chain reaction analyses of selected genes and for enzyme immunoassay of P4. Injections with P4 had no effect on the concentration of P4 and the relative abundance of mRNA transcripts of cholesterol transport-related proteins, steroidogenic enzymes, and receptors for luteotropic factors in the luteal tissue. The abundance of prostaglandin (PG) endoperoxide synthase 2, PGI2 synthase, PGI2 receptor, fibroblast growth factor 2, peroxisome proliferator-activated receptor γ, and tumor necrosis factor α receptor type I transcripts increased after P4 treatment. In contrast, the relative abundance of angiopoietin 2 mRNA decreased in response to P4 administration. In summary, P4 supplementation in pregnant gilts does not affect luteal steroidogenesis but modulates the abundance of factors related to vascular function. Given that the endometrium is the main target tissue for P4, an indirect uterine-mediated effect of exogenous P4 on CL function is likely.

Canine conceptus-maternal communication during maintenance and termination of pregnancy, including the role of species-specific decidualization

Among domestic animal species, the reproductive biology of the dog belongs to the most peculiar. This includes the conceptus-maternal communication and endocrine mechanisms involved in maintenance of pregnancy. Dogs fully depend on luteal progesterone (P4) throughout pregnancy, with similar steroid secretion patterns in pregnant and non-pregnant bitches until prepartum luteolysis. Thus, dogs lack the classical recognition of pregnancy. The luteal P4 is the most important hormone regulating the onset and maintenance of pregnancy in previously estrogenized bitches. Although the canine uterus is exposed to high P4 levels, decidualization is not spontaneous but induced by the presence of embryos. Following implantation, decidualization continues, associated with development of the invasive endotheliochorial placenta, leading to establishment of maternal decidual cells expressing the nuclear P4 receptor (PGR). Consequently, although not producing steroids, the canine placenta remains highly sensitive to circulating ovarian steroids. The placental conceptus-maternal communication is responsible for the maintenance of pregnancy, with functional withdrawal of PGR evoking a luteolytic cascade with prepartum PGF2α release. The fetal trophoblast is the major source of prepartum placental prostaglandins. This conceptus-maternal communication is unique to the dog and has clinical implications. Due to luteal steroids, there is no prepartum estradiol increase. Elevated cortisol levels are observed irregularly. This emphasizes the unique character of canine reproductive physiology and the challenges in transferring translational research to the dog. Further research is needed for better understanding of canine reproduction and improving clinical protocols, including the latest results obtained from applying modern laboratory technologies such as the transcriptomic approach.