High P4 Research Articles

Progesterone alters the bovine uterine fluid lipidome during the period of elongation.

Successful bovine pregnancy establishment hinges on conceptus elongation, a key reproductive phenomenon coinciding with the period during which most pregnancies fail. Elongation is yet to be recapitulated in vitro, whereas in vivo it is directly driven by uterine secretions and indirectly influenced by prior circulating progesterone levels. To better understand the microenvironment evolved to facilitate this fundamental developmental event, uterine fluid was recovered on Days 12-14 of the oestrous cycle - the window of conceptus elongation initiation - from cycling heifers supplemented, or not, with progesterone. Subsequent lipidomic profiling of uterine luminal fluid by advanced high-throughput metabolomics revealed the consistent presence of 75 metabolites, of which 47% were intricately linked to membrane biogenesis, and with seven displaying a day by progesterone interaction (P ≤ 0.05). Four metabolic pathways were correspondingly enriched according to day and P4 - i.e. comprised metabolites whose concentrations differed between groups (normal vs high P4) at different times (Days 12 vs 13 vs 14). These were inositol, phospholipid, glycerolipid and primary bile acid metabolism. Moreover, P4 elevated total uterine luminal fluid lipid content on Day 14 (P < 0.0001) relative to all other comparisons. The data combined suggest that maternal lipid supply during the elongation-initiation window is primarily geared towards conceptus membrane biogenesis. In summary, progesterone supplementation alters the lipidomic profile of bovine uterine fluid during the period of conceptus elongation initiation.

Response to ovulation trigger is correlated to late follicular phase progesterone levels: A hypothesis explaining reduced reproductive outcomes caused by increased late follicular progesterone rise

Is there an association between progesterone (P4) levels on the day of hCG or GnRH trigger and on the day of oocyte retrieval in IVF/ICSI cycles? A significant positive correlation between P4 levels on the day of trigger and the day of oocyte retrieval is seen; HCG trigger induces a steeper P4 increase than GnRHa trigger. FSH induces LH receptor (LHR) expression on granulosa cells, and LHR produces progesterone when exposed to LH-like activity. FSH per se also to some extent induces P4 secretion. Late follicular phase progesterone rise has been associated with reduced reproductive outcomes. This study is based on data from a previously published RCT conducted from 2009 to 2011. A total of 384 participants were enrolled; 199 received 5000 IU hCG and 185 received buserelin 0.5 mg for triggering ovulation. P4 was measured on the day of ovulation induction and on the day of oocyte retrieval. FSH consumption and number of retrieved follicles were recorded. A significant linear relationship between P4 on the day of ovulation induction and oocyte retrieval was seen in the hCG trigger group (P < 0.00001) as well as in the GnRHa trigger group (P < 0.00001). The P4 ratio (the increase in P4 between ovulation induction and oocyte retrieval) was significantly higher in the group of patients with <5 follicles compared to those with 5-15 and >15 follicles (P < 0.0001). The FSH consumption per follicle was significantly higher in the group of patients with <5 follicles compared to those with 5-15 and >15 follicles (P < 0.0001). Although the study demonstrates a significant correlation between P4 levels before and after ovulation trigger, it does not demonstrate a causal relation to the number of LHRs present on granulosa cells. The findings of this study support the proposed hypothesis that follicles exposed to high levels of FSH during ovarian stimulation will respond with an inappropriately high LHR expression. This in turn causes a high P4 output in response to the trigger. This study further expands our understanding of the underlying mechanisms affecting reproductive outcomes in relation to ovarian stimulation. The authors received no specific funding for this work and disclose no conflicts of interest. N/A.

Ovarian hormones and antioxidant biomarkers in dromedary camels synchronized with new and re-used controlled intravaginal drug release (CIDR)/GPG (Ovsynch) program during breeding season.

This study aimed to investigate the effect of CIDR, re-used wCIDR, and Ovsynch protocols for the synchronization of follicular waves on ovarian hormones, oxidative stress, and antioxidant biomarkers during the breeding season. Dromedary camels (N = 18) were divided into three equal groups. The first group received CIDR. The second group received previously used wCIDR after thorough cleaning and disinfection. The third group was subjected to GPG protocol. Progesterone (P4), estradiol (E2) l, total antioxidant capacity (TAC), catalase (CAT), lipid peroxide product (MDA), nitric oxide (NO), and glutathione reduced (GSH) were measured. Days during CIDR affected P4 (P =0.0001), E2 (P =0.047), TAC (P =0.01), NO (P =0.028), and GSH (P =0.005). Days during re-used wCIDR effected P4, TAC, CAT, NO, GSH, and MDA (P ≤0.001). Days during GPG effected P4, E2, TAC, GSH (P =0.0001), MDA (P =0.036), and NO (P =0.02). CIDR-treated camels had high P4 (P =0.0001), E2 (P =0.0001), TAC (P =0.012), and NO (P =0.0001), with low GSH (P =0.001), and MDA (P =0.003). Exogenous progesterone improved ovarian hormones and the antioxidant capacity and minimized the oxidative stress than the GPG treatment and is recommended for future reproductive management of camels.

Mucosal Susceptibility to Human Immunodeficiency Virus Infection in the Proliferative and Secretory Phases of the Menstrual Cycle.

Factors underlying HIV acquisition in women remain incompletely understood. This study evaluated ex vivo mucosal HIV-1BaL infection (ectocervix, endocervix), T cell frequencies and phenotype (ectocervix, endocervix, peripheral blood), and HIV-1BaL-induced tissue immune responses (ectocervix) in the proliferative and secretory phases of the menstrual cycle using samples obtained from women undergoing hysterectomies. Tissue infectivity (number of productively infected explants) and infection level following 500 and/or fifty 50% tissue culture infectious dose (TCID50) HIV-1BaL challenge were similar in the proliferative and secretory phases. Although not associated with infection outcomes, higher frequencies of HIV target CD4+α4β7+ T cells, and stronger HIV-1BaL-induced proinflammatory responses were detected in ectocervix in the secretory versus proliferative phase. Independently of the cycle phase, serum E2 concentrations were inversely associated with ectocervical and endocervical tissue infection levels following high-dose 500 TCID50 HIV-1BaL challenge, with frequencies of CD4+α4β7+ T cells in endocervix, and with HIV-induced interleukin (IL)2R and IL4 in ectocervix. Although serum P4 concentrations and P4/E2 ratios were neither associated with tissue infection level nor infectivity, high P4 concentrations and/or P4/E2 ratios correlated with high frequencies of CD4+α4β7+ T cells in ectocervix, low frequencies of CD4+CD103+ blood T cells, low CD4+LFA-1+ T cells in endocervix, and high proinflammatory (IL1β, IL17, tumor necrosis factor α) ectocervical tissue responses to HIV-1BaL. The data suggest an inhibitory effect of E2 on mucosal HIV infection, provide insights into potential mechanisms of E2-mediated anti-HIV activity, and highlight P4-associated immune changes in the mucosa.

Long-term outcomes of freeze-all strategy: A retrospective analysis from a single ART center in Japan.

PurposeTo demonstrate the benefits of the freeze‐all strategy for in vitro fertilization treatment based on retrospective analyses.MethodsPost‐thaw embryo survival rates of slow‐frozen embryos in 294 cycles and vitrified embryos in 12 195 cycles were assessed. Progesterone (P4) and estradiol (E2) levels per mature oocyte by age category were assessed in 9081 cycles and pregnancy rates with fresh embryo transfer and frozen‐thawed embryo transfer by P4 level were assessed in 1535 cycles.ResultsThe survival rates of frozen‐thawed embryos were 92.5% with slow freezing and 99.1% with vitrification. P4 levels on the day of human chorionic gonadotropin (hCG) injection showed a trend toward an increase with age. The pregnancy rate per mature oocyte with fresh embryo transfer decreased dependently upon P4 level, while that with frozen‐thawed embryo transfer was not affected by P4 level. The pregnancy rates with frozen‐thawed embryo transfer were higher than those with fresh embryo transfer in patients aged 42 years or younger.ConclusionsThe freeze‐all strategy is a valuable treatment option which allows the separation of an embryo transfer cycle from an oocyte retrieval cycle, especially for patients with high P4 levels at oocyte retrieval and patients of advanced maternal age.

Impact of estradiol and progesterone levels during the late follicular stage on the outcome of GnRH antagonist protocols

The study aimed to assess the impacts and the targets of progesterone (P4) and estradiol (E2) levels on IVF outcomes in GnRH antagonist protocols. The study was retrospective and concerned patients for their first fresh embryo transfers, after stimulation by a recombinant FSH (rFSH)-GnRH antagonist protocol, between September 2012 and July 2017 in the Toulouse University Hospital. Multivariable analysis, taking into account female age and the ovarian stimulation index, showed that E2 levels had no impact on IVF outcomes, while high P4 levels (>1.10 ng/mL) were associated to low pregnancy rate. The P4 concentrations were significantly negatively correlated to the percentage of top embryos and to the implantation rate. Therefore, the deleterious effect of high levels P4 could to act mainly by impairing embryo quality, which questions the place of the freeze-all strategy in these cases.

Is embryogenesis and ART outcome different in polycystic ovary syndrome?

Introduction: As oogenesis is a continuum of months, oocyte quality will be dictated by events that occur during both the growth and maturation stages of development. Morphological variation of the oocyte and embryo result from intrinsic factors such as age and genetic defects or extrinsic factors such as stimulation protocol, culture condition, and nutrition. Controlled ovarian stimulation (COH) interferes with the balance of forces within the ovary and overrides their endogenous pattern of control to optimize normal selection process. Aim: 1. To compare the embryogenesis and outcome of assisted reproductive technology (ART) in polycystic ovary syndrome (PCOS) and non-PCOS women. 2. To determine whether embryo quality, implantation rate (IR) and clinical pregnancy rate (CPR) are related to estradiol (E2) or progesterone (P4) levels on the day of human chorionic gonadotropin (hCG). Materials and Methods: This was a retrospective case–control study of 425 women at a Tertiary Care Center from January 2017 to December 2018. Of these 183 were PCOS and 242 non-PCOS matched for body mass index and age. We compared the follicle stimulating hormone, antral follicle count (AFC), anti-Mullerian hormone, thyroid-stimulating hormone, total gonadotropin utilized, total days of stimulation, estradiol (E2), and progesterone (P4) on the day of hCG trigger between both groups. Before entering the in vitro fertilization program, patients were classified into PCOS and non-PCOS depending on the AFC at the baseline scan done on day 2/3 of the menstrual cycle. We calculated the fertilization, cleavage, blastocyst formation rate, and utilization rate of the embryos from the total oocytes retrieved and fertilized in four different groups classified according to the E2 levels on the day of the hCG (≤1000, 1001–2000, 2001–3000, >3000 pg/ml) in both PCOS and non-PCOS women. We also looked at the IR and CPR in the above four groups in fresh cycle and included the frozen embryo transfer cycles to calculate the utilization rate. Statistical comparison was done using the Mann–Whitney U test. Results: There was no statistically significant difference in the fertilization rate (P = 0.803), cleavage rate was higher in PCOS group (P = 0.001) and blastocyst formation rate was higher in non-PCOS group (P 3000 pg/ml. The IR was higher in the PCOS group as compared to non-PCOS in women with E2 between 2000 and 3000 (P = 0.020). No difference in the CPR across the E2 groups. The utilization rate of the embryos per egg retrieved and per 2PN was statistically higher in patients with an E2 Conclusion: As embryogenesis and endometrial receptivity can be affected by high E2 and P4 levels it is highly recommended to closely monitor COS cycles by measuring serum E2 and P4 levels on D2 and at the time of hCG trigger. The dose of gonadotropins dose should be chosen in such a way that the number of oocytes retrieved should not be more than 10 to 12. When E2 and P4 are elevated over a threshold value, one should cryopreserve the embryo and transfer them in the subsequent cycle to decrease the incidence of ovarian hyperstimulation syndrome and improve the IR.

108 Fecal metabolite monitoring as a tool to assess sexual maturation in polar bears

Polar bears (Ursus maritimus) in managed care generally are paired for breeding starting around the age of 5 or 6 years; however, DNA analyses of the wild population indicate that males as young as 2 or 3 may sire offspring and females as young as 4 can produce cubs. There are no reports describing longitudinal reproductive hormone parameters in juvenile polar bears. The objective of the current study was to determine if seasonal shifts in testosterone and progesterone (P4) metabolites are detectable in feces of polar bears 2-3 years old as part of a greater effort to characterise reproductive metabolites in a large cohort of juveniles throughout sexual maturation. Subjects were 2-year-old male (n=3) and female (n=3) polar bears residing at 5 zoological institutions in the USA. Individuals were monitored for 1 (1.1) or 2 (2.2) years. Fecal samples were collected noninvasively 3-4 times/week and hormone metabolites were extracted as previously described. Testosterone was evaluated as an indicator of gonadal activity in both sexes, whereas P4 was measured in samples collected from females only. Metabolites were quantified in duplicate using established enzyme immunoassay techniques. Student’s and paired t-tests were used to compare mean metabolite concentrations between seasons [breeding (BS; January-June) and nonbreeding (NBS; July-December)] by sex and within individual, respectively. All values are reported as mean concentration (ng metabolite/g dried feces±standard error of the means) and P-values less than 0.05 indicated statistical significance. Mean testosterone concentration of the 2-year-old males was 153.1±112.5. Overall, testosterone concentrations were higher in samples collected from 2-year-old males during BS versus NBS; however, when examined within individual, this held true for only 1 of 3 males. Mean testosterone of the 3-year-old male was 170.2±19.6 and values were significantly higher in BS (282.1±27.2) versus NBS (74.6±7.3). The overall mean testosterone and P4 concentrations of the 2-year-old females were 56.0±21.7 and 57.4±7.5, respectively. Two of the 3 females exhibited significantly higher testosterone concentrations during BS compared to NBS and all 3 exhibited higher P4 in the breeding versus the NBS. The 3-year-old female had significantly higher testosterone in BS (63.7±4.1) versus NBS (40.5±2.4) and showed evidence of regular ovarian cycles during BS. Despite no detectable differences in mean P4 between seasons (56.3±7.9 and 55.1±3.7), this female exhibited a distinct increase in P4 from October to November compared to the rest of NBS (85.7±8 v. 43.5±2.8), suggestive of pseudopregnancy. These results indicate that fecal reproductive hormone metabolite monitoring can be used to detect changes in metabolite excretion patterns associated with sexual maturation in polar bears and that males and females as young as 2 years old may exhibit seasonal variations in reproductive hormones. These data suggest bears in zoological institutions may be achieving sexual maturation earlier than believed previously and should be considered when managing bears in human care.

Level of circulating concentrations of progesterone during ovulatory follicle development affects timing of pregnancy loss in lactating dairy cows

The objective of this experiment was to determine the effect of high versus low progesterone (P4) during the pre-dominance or dominance phase (or both) of ovulatory follicle development on follicular dynamics and fertility of lactating dairy cows. Progesterone (P4) was manipulated to reach high (H) or low (L) serum concentrations during the pre-dominance phase (d 0 to 4 of the wave) and dominance phase (d 5 to 7 of the wave) of a second follicular wave ovulatory follicle, creating 4 treatments: H/H, H/L, L/H, and L/L. Luteolysis was induced with PGF2α on d 7 of the wave and ovulation was induced with GnRH 56 h after PGF2α. Cows (n = 558) received artificial insemination (AI) 16 h following GnRH. Pregnancy was determined at 6 intervals during gestation and at calving to quantify pregnancy loss beginning at d 23 post-AI utilizing pregnancy-specific protein B (PSPB) in novel within-cow comparisons. Cows with single ovulations assigned to the L/L treatment had greater pre-ovulatory follicle diameter compared with cows assigned to the L/H or H/L treatments. Cows with single ovulations had greater pre-ovulatory follicle diameter compared with cows with double ovulations. Low P4 in H/L, L/H, and L/L increased double ovulation rate compared with H/H. Cows with double ovulations had greater pregnancies per AI (P/AI) on d 23 post-AI compared with cows with single ovulations but had greater losses if ovulations were unilateral. Cows with low P4 during the entire period of the ovulatory follicle development also had greater P/AI on d 23 post-AI compared with cows with high P4 during both phases. However, full-term P/AI was not different between treatments. This was a result of the greater incidence of pregnancy losses between d 35 and 56 of gestation for cows with unilateral double ovulations compared with bilateral double ovulations and single ovulatory cows. Cows with single ovulation and low circulating P4 during the dominance period of follicle development had increased pregnancy losses between d 35 and 56 of gestation compared with cows with single ovulations and high P4. The PSPB measurements on d 16 and 23 post-AI were highly accurate in the prediction of pregnancy at d 28. The PSPB differed on d 23 and 28 between cows that had versus cows that did not have pregnancy losses between d 28 and 35 of gestation. In summary, circulating concentrations of P4 during ovulatory follicle development affected numbers of follicles ovulated and timing of subsequent pregnancy losses.

Randomised controlled trial of the effect of concentration of progesterone before artificial insemination on fertility in ovulatory and anovulatory Bos indicus cattle.

To investigate the effect of concentration of progesterone (P4) before artificial insemination (AI) on fertility in ovulatory or anovulatory Bos indicus cattle. Randomised control study METHODS: The study included 162 heifers and 96 lactating cows. On days -10 to -12, animals were examined using transrectal ultrasound, administered PG and examined for a corpus luteum (CL). Those with a CL were allocated to Experiment 1. On day 0 they were administered an intravaginal progesterone-releasing device (IVD) containing progesterone (P4) (0.78 g), oestrodiol benzoate (ODB) and either saline or PG to induce high and low circulating P4 concentrations, respectively. Those without a CL were re-examined on day 0 and those without a CL at both examinations were allocated to Experiment 2. Cows and heifers were treated with an IVD containing P4 at 0.78 g or 1.56 g to induce low or high P4 concentrations, respectively. IVDs were removed on day 7 and PG and equine chorionic gonadotrophin (eCG) were administered. Females in oestrus on day 9 were inseminated; others were administered ODB and inseminated 22-26 h later. Greater concentrations of circulating P4 increased the odds of pregnancy to AI in anovulatory females (P = 0.008), but decreased the odds of pregnancy in one year but not another in ovulatory animals (P × year, P = 0.019). Manipulating P4 concentrations before AI has the potential to improve pregnancy outcomes to AI in B. indicus females, but treatment may need to vary between animals classified as anovulatory or ovulatory.

The impact of luteal serum progesterone levels on live birth rates-a prospective study of 602 IVF/ICSI cycles.

Is the chance of a live birth following IVF treatment and fresh embryo transfer affected by early and mid-luteal serum progesterone (P4) levels? Low as well as high serum P4 levels in the early and mid-luteal phase reduce the chance of a live birth following IVF treatment with fresh embryo transfer. Data from non-human studies and studies of frozen-thawed embryo transfer cycles indicate that low as well as high P4 levels during the mid-luteal phase decrease the chance of pregnancy. The altered P4 pattern may disrupt the endometrial maturation leading to asynchrony between embryonic development and endometrial receptivity, thereby, compromising implantation and early development of pregnancy. Prospective multicenter cohort study of 602 women undergoing IVF treatment. Patients were recruited from four Danish public Fertility Centers from May 2014 to June 2017. The study population was unselected, thus, representing a normal everyday patient cohort. Patients were treated in a long GnRH-agonist protocol or a GnRH-antagonist protocol and triggered for final oocyte maturation with either hCG or a GnRH-agonist. The same vaginal luteal support regimen was applied in all patients. Serum P4 levels from the early or mid-luteal phase were correlated to positive hCG and live birth rates (delivery > gestational week 20). Patients were divided into four P4 groups based on raw data of P4 serum levels and reproductive outcomes during early luteal phase (P4<60 nmol/l, P4 60-100 nmol/l, P4 101-400 nmol/l and P4>400 nmol/l) and during mid-luteal phase (P4<150 nmol/l, P4 150-250 nmol/l, P4 251-400 nmol/l and P4>400 nmol/l). The optimal chance of pregnancy was achieved with serum P4 levels of 60-100 nmol/l in the early luteal phase whereas the optimal P4 level during the mid-luteal phase was 150-250 nmol/l. Below, but most distinctly above these levels, the chance of pregnancy was consistently reduced. With an early luteal P4 level of 60-100 nmol/l, the chance of a positive hCG-test was 73%, 95% CI: [59, 84] following cleavage stage embryo transfer. In contrast, with P4 levels >400 nmol/l, the chance of a positive hCG-test was significantly reduced to 35%, 95% CI: [17, 57], thus, an absolute risk difference of -38%, P = 0.01. A similar negative association between early luteal P4 and live birth rate was found, although it did not reach statistical significance. During the mid-luteal phase, a P4 level of 150-250 nmol/l resulted in an optimal chance of live birth: 54%, 95% CI: [37, 70] compared to 38%, 95% CI: [20, 60] with a P4 level >400 nmol/l, thus, an absolute risk difference of -16%, P = 0.14. All estimates were adjusted for maternal age, maternal BMI, study site, final follicle count and late follicular P4 levels. This study is the first to explore the possible upper and lower thresholds for luteal P4 following IVF treatment and fresh embryo transfer, and the optimal P4 ranges found in this study should be corroborated in future clinical trials. Furthermore, the P4 thresholds in this study only apply to fresh IVF cycles, using vaginal luteal phase support, as the optimal P4 level in cycles using intramuscular P4 may be different. Future studies are necessary to explore whether additional exogenous luteal P4 supplementation in the low P4 group could increase the chance of a live birth following fresh embryo transfer, and whether patients with luteal P4 levels >400 nmol/l would benefit from segmentation followed by subsequent transfer in frozen/thawed cycles. NCT02129998 (Clinicaltrials.gov). L.H.T. received an unrestricted grant from Ferring Pharmaceuticals, Denmark, to support this study. P.H. received unrestricted research grants from MSD, Merck, Gedeon Richter and Ferring Pharmaceuticals outside of this work as well as honoraria for lectures from MSD, Merck and Gedeon Richter outside of this work. U.K. received honoraria for lectures from MSD and Ferring Pharmaceuticals outside of this work. C.A. received unrestricted research grants from MSD, IBSA, and Ferring Pharmaceuticals outside of this work as well as honoraria for lectures from MSD and IBSA. H.O.E. and B.B.P. received an unrestricted research grant from Gedeon Richter outside of this work. K.E., L.B., D.P. and B.H. have no conflict of interest. Furthermore, grants from 'The Health Research Fund of Central Denmark Region', 'The Research Foundation of the Hospital of Central Jutland', 'The Research Foundation of A.P. Møller', 'The Research Foundation of Aase & Ejnar Danielsen', 'The Research Foundation of Dagmar Marshall', 'The Research Foundation of Dir. Jacob Madsen & Hustru Olga Madsen', 'The Research Foundation of Fam. Hede Nielsen' and 'The Danish Medical Research Grant' supported conducting this study. The providers of funding were neither involved in the conduction of the study nor in the writing of the scientific report.

Role of progesterone concentrations during early follicular development in beef cattle: I. Characteristics of LH secretion and oocyte quality

Objective was to investigate the effect of different progesterone (P4) concentrations during early follicular development on luteinizing hormone (LH) secretion and oocyte characteristics in beef cows. Primiparous cows (n = 24) were estrous pre-synchronized and follicular ablation was performed (d 0) 6 days following the time of ovulation. At the time of follicular ablation, cows were assigned to either: 1) high P4 treatment - HiP4; a new CIDR was inserted on d 0 to supplement P4 from the existing corpus luteum [CL], or 2) low P4 treatment - LoP4; a previously-used CIDR and two doses of PGF 8 to 12 h apart were given on d 0. Concentrations of P4 were greater (P < 0.01) in the cows of the HiP4 than LoP4 group on d 1.5, 2.5, and 3.5. Peripheral concentrations of E2 were greater (P < 0.05) in the cows of the LoP4 than HiP4 group on d 2.5 and 3.5. Frequency of LH pulses was greater (P < 0.05) in the LoP4 than HiP4 group on d 2.5, but mean LH concentration and pulse amplitude did not differ between treatments. Number of follicles aspirated per cow, total oocytes recovered, recovery rate, percentage of oocytes graded 1 to 3, oocyte diameter, percentage BCB+ oocytes, and relative abundance of oocyte mRNA for FST did not differ (P > 0.10) between treatments. In conclusion, lower P4 concentrations during early follicular development resulted in increased LH pulse frequency and E2 concentrations, but did not affect characteristics of oocyte developmental competence.

Pregnancy outcomes are not improved by administering gonadotropin-releasing hormone at initiation of a 5-day CIDR-Cosynch resynchronization protocol for lactating dairy cows

Using a 5-d controlled internal drug-release (CIDR)-Cosynch resynchronization protocol, the objective of this study was to determine the effect of the initial GnRH injection on pregnancy per artificial insemination (P/AI) to the second artificial insemination in lactating Holstein dairy cows. On 37 ± 3 d (mean ± standard deviation) after the first artificial insemination, and upon nonpregnancy diagnosis (d 0 of the experiment), lactating cows eligible for a second artificial insemination (n = 429) were enrolled in a 5-d CIDR-Cosynch protocol. On d 0, all cows received a CIDR insert and were assigned randomly to receive the initial GnRH injection (GnRH; n = 226) of the protocol or no-GnRH (n = 203). Blood samples were collected from a sub-group of cows (n = 184) on d 0 and analyzed for progesterone (P4) concentration. On d 5, CIDR inserts were removed, and all cows received 1 injection of PGF2α. On d 6 and 7, cows were observed once daily by employees for tail-chalk removal, and cows detected in estrus on d 6 or 7 received artificial insemination that day (EDAI), and did not receive the final GnRH injection. The remaining cows not detected in estrus by d 8 received GnRH and timed artificial insemination (TAI). Pregnancy status was confirmed by transrectal palpation of uterine contents at 37 ± 3 d (mean ± standard deviation) after the second artificial insemination. Eliminating the initial GnRH injection had no effect on P/AI compared with cows receiving GnRH (27 vs. 21%), respectively. Similarly, method of insemination (EDAI vs. TAI) and its interaction with treatment had no effect on P/AI. Primiparous cows had greater P/AI than multiparous cows (31 vs. 21%). Mean P4 concentrations (n = 184) at the initiation of the protocol did not differ between treatments (4.51 ± 0.35 ng/mL no-GnRH vs. 3.96 ± 0.34 ng/mL of GnRH). When P4 concentrations were categorized as high (≥1 ng/mL) or low (<1 ng/mL), P/AI tended to be greater for high P4 concentrations (n = 136) compared with low (n = 48) P4 concentrations (26 vs. 16%, respectively). No differences were observed in the proportion of cows with high or low P4 between treatments. Collectively, these results provide evidence that eliminating the initial GnRH in a 5-d CIDR-Cosynch resynchronization protocol for lactating dairy cows did not reduce P/AI in this study.

Update on Multiple Ovulations in Dairy Cattle

Simple SummaryMultiple ovulations (MOV) in cattle can lead to twin pregnancies, which negatively affects the health, production, and reproduction of cows. Despite many studies, the causal mechanisms behind MOV are still not well understood. There is a general agreement that MOV are more likely during periods of low progesterone (P4), which may increase the luteinizing hormone (LH) release at the time of selection, resulting in more than one follicle becoming dominant. The MOV rate also increases in older cows and when the selection of a dominant follicle occurs concurrently with a high milk yield. Additional risk factors for MOV are ovarian cysts, diet, season, and genetics. A better understanding of the mechanisms underlying MOV may help to mitigate twinning, perhaps through the appropriate reproductive management protocols or genetic selection.This review updates the causal mechanisms and risk factors for multiple ovulations (MOV) in cattle. Clearly, MOV can lead to twin pregnancies, which negatively affects the health, production, and reproduction of cows. Therefore, a better understanding of the factors causing MOV may help to reduce twinning. Multiple ovulations occur after two or more follicles deviate and achieve codominance. The MOV rate is influenced by a complex network of hormones. For example, MOV is more common during periods of low progesterone (P4), that is, in anovulatory cattle or when luteolysis coincides with the selection of the future ovulatory follicle. There is also strong evidence for the luteinizing hormone (LH) being the primary factor leading to codominance, as high P4 concentrations suppress the transient LH surges and can reduce the ovulation rate in cattle or even inhibit deviation. Rates of MOV are increased in older and higher-producing dairy cows. Increased milk production and dry matter intake (DMI) increases hormone clearance, including P4; however, the association between milk yield and MOV has not been consistent. Additional risk factors for MOV include ovarian cysts, diet, season, and genetics.

Relationship between Progesterone and Oestradiol-17beta Concentrations and Embryonic Loss in Frisian Cows under Egyptian Condition

The objective of this study was to determine the association between circulating (Progesterone, oestradiol-17β and P4/E2 ratio) concentrations and embryonic loss in Friesian cows under Egyptian condition. On day 25 by both progesterone (P4) level and sonography, 65.71% (69/105) cows were continued their gestation normal and 17.14% (18/105) cows not conceived. While, 17.14% (18/105) of inseminated cows showed high P4 level firstly and decline before day 25 suspecting early pregnancy loss, EPL. Also, 5 cows (7.25%) of the pregnant cows lost their feti from day 25 to day 45 (late pregnancy loss, LPL) were detected. Progesterone concentrations was noticed that increased P4 level starting from day 4 to day 7after AI in cows diagnosed pregnant (P

Nitric oxide activation by progesterone suppresses ATP-induced ciliary activity in oviductal ciliated cells.

Ciliary beat frequency (CBF) regulates the oviductal transport of oocytes and embryos, which are important components of the reproductive process. Local release of ATP transiently increases CBF by increasing [Ca2+]i. Ovarian hormones also regulate ciliary activity and oviductal transport. Progesterone (P4) induces nitric oxide (NO) production and high P4 concentrations induce ciliary dysfunction. However, the mechanism by which P4 affects CBF has not been elucidated. To evaluate the role of P4 in NO production and its effect on ATP-induced increases in CBF, we measured CBF, NO concentrations and [Ca2+]i in cultures of oviductal ciliated cells treated with P4 or NO signalling-related molecules. ATP induced a [Ca2+]i peak, followed by an increase in NO concentrations that were temporally correlated with the decreased phase of the transiently increased CBF. Furthermore, P4 increased the expression of nitric oxide synthases (iNOS and nNOS) and reduced the ATP-induced increase in CBF via a mechanism that involves the NO signalling pathway. These results have improved our knowledge about intracellular messengers controlling CBF and showed that NO attenuates oviduct cell functions. Furthermore, we showed that P4 regulates neurotransmitter (ATP) actions on CBF via the NO pathway, which could explain pathologies where oviductal transport is altered and fertility decreased.

αvβ3 Integrin and fibronectin expressions and their relation to estrogen and progesterone during placentation in swine

Mammalian pregnancy requires specific interactions between the conceptus and its mother that involve the endocrine system and adhesion molecules. The relation between adhesion molecules and their ligands at the fetal–maternal interface is crucial for developing a successful implantation. Progesterone (P4) and estrogen (E2) secreted by the porcine conceptus are required for the relation to be established. We investigated the expression of αvβ3 integrin and its ligand, fibronectin (FN), at the placental interface, and E2 and P4 concentrations in both serum and maternal and fetal placental extracts during placentation in swine. Placental and serum samples of crossbred sows at 17, 30, 60, 70, and 114 days gestation and no pregnant uteri were used. The presence of αvβ3 and FN were determined by immunohistochemistry, and E2 and P4 by chemiluminescence in homogenates of nonpregnant uterus (HoU), swine maternal placenta (HoPM), swine fetal placenta (HoPF) and serum. The expression of αvβ3 and FN increased at the interface at 17, 30 and 60 days gestation. Immunostaining decreased by 70 days. Serum E2 levels peaked at 17 days, then decreased, then increased again near term. The highest concentration of P4 occurred in HoPF at 70 days gestation, then decreased coincident with a decline in integrin and FN expression at the placental interface. High P4 levels during swine gestation may regulate the expression of αvβ3 integrin and FN at the placental interface for up to 70 days gestation. Other adhesion molecules and their ligands likely maintain the fetal–placental interface after 70 days.

EFFECT OF SELENIUM YEAST AND/OR VITAMIN E SUPPLEMENTED RATIONS ON SOME PHYSIOLOGICAL RESPONSES OF POST-LAMBING OSSIMI EWES UNDER TWO DIFFERENT HOUSING SYSTEMS

This study was carried out to investigate the effect selenium yeast and/or vitamin E supplemented rations under two different housing types on reproductive performance of post-lambing Ossimi ewes. Along thirty days pre-lambing, eighty pregnant Ossimi ewes averaged 40.38±0.93 and 39.49±1.12kg live body weight were housed under two different housing systems, the first (semi open with concrete floor and roofed with west-east direction, 1st H) and the second (fully shaded roofed with single asbestos sheets and was topped with rice straw bales and natural earthen ground with north-south direction, 2nd H) housing type, respectively. Ewes were divided into two equal groups (40 ewes each) according to age, body weight and parity. Each group was settled in one of the housing type and randomly divided into four subgroups (ten ewes per each); group A was kept as a control and was fed the basal diet consist of roughage (rice straw) and concentrate mixture, group B selenium yeast (SY) was fed the basal diet supplemented with 0.3 mg of SY per kg of diet, group C vitamin E (VE) received the same basal diet supplemented with 40 mg VE per kg of diet and group D fed the basal diet supplemented with both SY and VE. Results reported that semi open type house (1stH) had significantly higher (P<0.01) Air temperature (AT) at morning and afternoon than that in fully shaded type house (2nd H). Daily average AT was lowest value (24.82 °C) in 2nd H than 1stH (30.90 °C). Relative humidity (RH) was significantly higher at morning than afternoon in two housing types. THI values ranged from 61.20 to 64.17and 87.10 to 91.34 in the morning and afternoon, respectively in the two hosing systems. However, the THI average, at morning and the afternoon values in 2nd house was lower (P< 0.05) THI than that of semi open type (1stH). The average values of thermo-regulatory parameters of ewes post-lambing in the morning and afternoon as affected by SE and VE supplemented rations under two different housing systems presented were significantly decreased in SY+VE group followed by SY and VE groups compared to control group in the morning and afternoon in two different housing types. Present data showed significantly (P<0.01) higher in most of hematological parameters (Hgb, RBCs, HCT, PLT, WBCs and differential leucocyte count) under the conditions of subsistence in the 2nd H compared with 1st H. antioxidant enzymes levels showed higher values in SY+VE group compared with the level observed another groups specially control group. Treated ewes by both SY and VE reached to first estrus post-lambing at earlier time and recorded heavier body weight and shorter estrus duration than the other ewes in first and second house. There was significant (P<0.01) increased in mean serum P4 concentration in all treated groups compared with control group at all time under two hosing systems, indicating the presence of functional corpus luteum in some of the animals. Also, that administration of SY plus VE gave significantly (P<0.01) higher P4 and E2 concentrations at day 8, 12, 16, 20 and 24 post-lambing. It could be concluded that SY and/or VE supplemented rations can improve physiological responses post-lambing of ewes under different housing systems.

Mucoadhesive tablets for the vaginal delivery of progesterone: in vitro evaluation and pharmacokinetics/pharmacodynamics in female rabbits

Objective: To develop mucoadhesive tablets for the vaginal delivery of progesterone (P4) to overcome its low oral bioavailability resulting from drug hydrophobicity and extensive hepatic metabolism.Methods: The tablets were prepared using mixtures of P4/Pluronic® F-127 solid dispersion and different mucoadhesive polymers. The tablets physical properties, swelling index, mucoadhesion and drug release kinetics were evaluated. P4 pharmacokinetic and pharmacodynamic properties were evaluated in female rabbits and compared with vaginal micronized P4 tablets and intramuscular (IM) P4 injection, respectively.Results: The tablets had satisfactory physical properties and their swelling, in vitro mucoadhesion force and ex vivo mucoadhesion time were dependent on tablet composition. Highest swelling index and mucoadhesion time were detected for tablets containing 20% chitosan-10% alginate mixture. Most tablets exhibited burst release (∼25%) during the first 2 h but sustained the drug release for ∼48 h. In vivo study showed that chitosan-alginate mucoadhesive tablets had ∼2-fold higher P4 mean residence time (MRT) in the blood and 5-fold higher bioavailability compared with oral P4. Further, same tablets showed 2-fold higher myometrium thickness in rabbit uterus compared with IM P4 injection.Conclusion: These results confirm the potential of these mucoadhesive vaginal tablets to enhance P4 efficacy and avoid the side effects associated with IM injection.

Co-culture of JEG-3, BeWo and syncBeWo cell lines with adrenal H295R cell line: an alternative model for examining endocrine and metabolic properties of the fetoplacental unit

Monocultures of different placental cells are used for many physiological and toxicological studies; however, they are not a true reflection of the interaction between placenta and fetus. To develop the most appropriate model to study endocrine and metabolic properties of fetoplacental unit we used three co-culture models of placental cells nonfusogenic JEG-3, unsyncytialised BeWo (BeWo) and syncytialised BeWo (syncBeWo) cultured with adrenal (H295R) cells. As an end point of endocrine properties we investigated steroids receptors expression and steroid secretion, while as metabolic properties AhR, CYP1A1and COMT expression. Progesterone (P4), estradiol (E2) and human chorionic gonadotropin (hCG) secretion (ELISA) and 3βHSD, CYP19, estrogen (ERα/β), progesterone (PR) and aryl hydrocarbon (AhR) receptors, CYP1A1 and COMT protein expression (Western blot) were evaluated. Comparing three co-culture models we observed: (1) there were no differences between JEG-3 and BeWo in the PR expression, however it was higher in BeWo compared to syncBeWo; (2) there were no differences in ERα protein expression in all models, while profile of ERβ expression was the highest in syncBeWo; (3) high P4 secretion in JEG-3 and BeWo while low in syncBeWo; (4) high E2 levels in JEG-3 and syncBeWo, while low E2 secretion in BeWo; (5) the highest hCG secretion in the JEG-3 and syncBeWo than in BeWo (6) the highest AhR, CYP1A1 and COMT expression in syncBeWo. Based on the results showing higher hCG secretion in the JEG-3 than in BeWo, representing villous and extravillous phenotype we suggest that JEG-3 model could be used to study fetoplacental steroidogenesis at the 1st, while BeWo model at the 3rd. Results showing comparable profiles of AhR, CYP1A1 and COMT expression in JEG-3 and BeWo models and the significantly higher expression in synBeWo points to synBeWo as a good model for study the metabolic properties.