Quantification of glucocorticoid and progestogen metabolites in bovine plasma, skimmed milk and saliva by UHPLC-HR-MS with polarity switching

Steroid metabolites are increasingly in focus when searching for novel biomarkers in physiological mechanisms and their disorders. While major steroids such as progesterone and cortisol are well-researched and routinely determined to assess the health, particularly the reproductive status of mammals, the function of potentially biologically active progestogen and glucocorticoid metabolites is widely unexplored. One of the main reasons for this is the lack of comprehensive, sensitive, and specific analytical methods. This is particularly the case when analyzing matrices like milk or saliva obtained by non-invasive sampling with steroid concentrations often below those present in plasma. Therefore, a new UHPLC-HR-MS method based on an Ultimate UHPLC system equipped with an Acquity HSS T3 reversed-phase column and a Q Exactive™ mass spectrometer was developed, enabling the simultaneous chromatographic separation, detection and quantification of eleven isobaric glucocorticoids (11-dehydrocorticosterone (A), corticosterone (B), cortisol (F), cortisone (E), the tetrahydrocortisols (THF): 3α,5α-THF, 3α,5β-THF, 3β,5α-THF, 3β,5β-THF, and the tetrahydrocortisones (THE): 3α,5α-THE, 3α,5β-THE, 3β,5α-THE) and twelve progestogens (progesterone (P4), pregnenolone (P5), the dihydroprogesterones (DHP): 20α-DHP, 20β-DHP, 3α-DHP, 3β-DHP, 5α-DHP, 5β-DHP, and the tetrahydroprogesterones (THP): 3α,5α-THP, 3α,5β-THP, 3β,5α-THP, 3β,5β-THP) in bovine plasma, skimmed milk, and saliva. A simple liquid-liquid extraction (LLE) with MTBE (methyl tert-butyl ether) was used for sample preparation of 500 μL plasma, skimmed milk, and saliva. Heated electrospray ionization (HESI) with polarity switching was applied to analyze steroids in high-resolution full scan mode (HR-FS). The method validation covered the investigation of sensitivity, selectivity, curve fitting, carry-over, accuracy, precision, recovery, matrix effects and applicability. A high sensitivity in the range of pg mL−1 was achieved for all steroids suitable for the analysis of authentic samples

Vascular Endothelial Growth Factor A and VEGFR-1 change during preimplantation in Heifers

Vascular endothelial growth factor A (VEGFA) plays a critical angiogenic role in the endometrium of placentalia during preimplantation. The role of VEGFA and its receptors is not fully characterised in bovine reproduction. We analysed the mRNA expression of VEGFA isoforms 121, 165 and 189, and VEGF receptors 1 and 2 in three experimental settings (A, B and C). We compared intercaruncular endometrium of cyclic to pregnant heifers at Days 12, 15 and 18 post insemination (Day 0), and between Day 15 and Day 18 conceptuses (A). We further compared caruncular versus intercaruncular endometrium at Day 15 (B), and endometrium of heifers carrying embryos originating from somatic cell nuclear transfer (SCNT) versus in vitro fertilisation (IVF) at Day 18 (C). Endometrial VEGFA protein was localised and quantified. Pregnant heifers displayed lower intercaruncular endometrial mRNA expression of VEGFA-121 (p = 0.045) and VEGFA-189 (p = 0.009) as well as lower VEGFA protein abundance (p < 0.001) at Day 15. The VEGFA protein was localised in intercaruncular luminal, glandular epithelium and in tunica muscularis of blood vessels. At Day 15, caruncular endometrium displayed higher VEGFA mRNA expression than intercaruncular endometrium (p < 0.05). Intercaruncular endometrial VEGFA protein at Day 18 was higher in abundance in SCNT than in IVF (p = 0.038). Therefore, during preimplantation in cattle, there may be a need for timely physiological reduction in intercaruncular endometrial VEGFA expression in favour of the caruncular area to facilitate a gradient towards the implantation sites. A higher expression of VEGFA in SCNT may predispose for later placentation abnormalities frequently observed following SCNT

Endocrine and local cellular signaling of hormones involved in the regulation of the transition from cyclicity to pregnancy in livestock

Early pregnancy loss is one of the biggest drawbacks in agriculture today and causes economic, ecologic and ethical burden on farms. In cattle, losses occur mainly during the preimplantation period and are hypothesized to be a result of an inadequate embryo maternal communication leading to failures in pregnancy recognition. Consequently, the regression of the corpus luteum (CL), which is the primary source for the pregnancy maintaining hormone progesterone (P4), is not inhibited. In cattle and swine, the regulation of the estrous cycle as the basis for reproduction and especially the initiation of luteolysis during the estrous cycle is not completely understood. In addition, the factors and mechanisms necessary for the inhibition of the latter during pregnancy as an essential part in the process of pregnancy recognition are not fully known to date. During the estrous cycle, estrogens trigger ovulation and P4 is produced by the CL during the subsequent luteal phase. If pregnancy does not establish, the luteal phase of the estrous cycle is terminated by luteolysis, which is triggered by pulses of prostaglandin F2α (PGF2α) resulting in the regression of the CL. Metabolites of the steroids P4 and estradiol-17β (E2) have for long been thought to serve only as excretory products. Nowadays, this assumption is outdated. It is hypothesized that steroid metabolites take part in regulating the estrous cycle and are potentially involved in the initiation of luteolysis. Therefore, we determined ten different P4 metabolites and estrogen conjugates during the estrous cycle of cattle and swine. We found that the profiles of P4 metabolites correlated well with P4 except for the period around luteolysis. During luteolysis, P4 levels dropped more rapidly leading to an increased P4 metabolite to P4 ratio thereafter. Estrogen conjugate levels in serum were found to be rather constant during the estrous cycle, whereas conjugated E2 levels in the endometrium were high at ovulation, decreased thereafter and began to transiently increase again beginning at day 12 post ovulation. The observed increase is probably due to an endometrial estradiol synthesis in swine and might be involved in the initiation of luteolysis. In case of pregnancy in cattle and swine, luteolysis must be inhibited to maintain P4 secretion by the CL. Since prostaglandin E2 (PGE2) is thought to play a luteoprotective role, it is suggested that a reorientation of PGF2α towards PGE2 production within the reproductive tract is a prerequisite for a successful establishment of pregnancy in ruminants and in swine. A previous study in cattle however revealed that intrauterine prostacyclin (PGI2) and PGF2α highly exceed intrauterine PGE2. Consequently, we hypothesized that in addition to PGE2, other prostaglandins might contribute to a successful establishment of pregnancy. Within the framework of this thesis, we aimed at determining the prostaglandin secretome of the bovine embryo during different stages of the preimplantation period from blastocyst hatching until embryo elongation. For this purpose, we developed a liquid chromatography–mass spectrometry (LC-MS) based approach for the quantification of 30 different prostaglandins and metabolites and measured prostaglandins in embryo culture supernatants. We found that bovine embryos predominantly secrete PGE2 and Δ12-PGD2 until day 12 post insemination. iv Thereafter, prostaglandins of the F-series (6keto-PGF1α, > PGF2α > PGE2) are the most abundant prostaglandins produced by the embryo. These findings suggest that the prostaglandin secretion of the bovine embryo changes dynamically with the advancing development. Thus, a complex mixture of prostaglandins influences endometrial secretion generating an optimal uterine environment for the developing embryo. The quantification of novel players during the estrous cycle and early pregnancy found the basis for a better understanding of the transition from cyclicity to pregnancy in livestock. Yet, their specific role in fine-tuning the endometrial function during the estrous cycle and the preimplantation period needs to be unraveled in follow-up studies

Reconsidering “low-dose”—Impacts of oral estrogen exposure during preimplantation embryo development

Perturbations of estrogen signaling during developmental stages of high plasticity may lead to adverse effects later in life. Endocrine-disrupting chemicals (EDC) are compounds that interfere with the endocrine system by particularly mimicking the action of endogenous estrogens as functional agonists or antagonists. EDCs compose synthetic and naturally occurring compounds discharged into the environment, which may be taken up via skin contact, inhalation, orally due to contaminated food or water, or via the placenta during in utero development. Although estrogens are efficiently metabolized by the liver, the role of circulating glucuro- and/or sulpho-conjugated estrogen metabolites in the body has not been fully addressed to date. Particularly, the role of intracellular cleavage to free functional estrogens could explain the hitherto unknown mode of action of adverse effects of EDC at very low concentrations currently considered safe. We summarize and discuss findings on estrogenic EDC with a focus on early embryonic development to highlight the need for reconsidering low dose effects of EDC.ISSN:1040-452XISSN:098-279

Conjugated estrogens in the endometrium during the estrous cycle in pigs

Estrogen metabolism results in the formation of inactive estrogen sulphates and glucuronides. Despite the lack of receptor binding, circulating conjugated estrogens might serve as a reservoir for the active form through the involvement of specific cleaving enzymes. In order to elucidate the potential role that estrogen conjugates play in the regulation of the estrous cycle, we determined the concentration of progesterone, estrogen and estrogen conjugates in serum and endometrial homogenates of cycling gilts. In addition, we determined the mRNA expression changes of enzymes (UDP glucuronosyltransferase (UGT), β-glucuronidase (GUSB), sulphotransferases (SULT) and steroid sulphatase (STS)) and transporters (multidrug resistance-associated protein (MRP), organic anion-transporting polypeptide (OATPs)) involved in the estrogen metabolism in the endometrium across the estrous cycle. GUSB displayed highest expression at estrous (day 0), decreasing expression during metestrus (day 3 and 6), minimal expression on day 10 and 12, and increasing expression towards proestrus (day 18), suggesting either a stimulation by estrogens or a negative impact of progesterone. The mRNA expression of the influx-transporter OATP1A2 significantly increased from day 0 to 6 and decreased again by day 10, while the efflux-transporters (MRP1, MRP2, and MDR1) displayed minimal expression at day 3 and 6. The mRNA expression of the UDP-glucuronsyltransferases followed a similar pattern, with minimal expression found at day 6. The analyses of the concentration of local and circulating steroid hormones points towards an interaction of the analyzed transporters and enzymes with steroid hormones, thereby possibly regulating the reservoir of active steroids contributing to the endometrial function

Quantification of glucocorticoid and progestogen metabolites in bovine plasma, skimmed milk and saliva by UHPLC-HR-MS with polarity switching

Steroid metabolites are increasingly in focus when searching for novel biomarkers in physiological mechanisms and their disorders. While major steroids such as progesterone and cortisol are well-researched and routinely determined to assess the health, particularly the reproductive status of mammals, the function of potentially biologically active progestogen and glucocorticoid metabolites is widely unexplored. One of the main reasons for this is the lack of comprehensive, sensitive, and specific analytical methods. This is particularly the case when analyzing matrices like milk or saliva obtained by non-invasive sampling with steroid concentrations often below those present in plasma. Therefore, a new UHPLC-HR-MS method based on an Ultimate UHPLC system equipped with an Acquity HSS T3 reversed-phase column and a Q Exactive™ mass spectrometer was developed, enabling the simultaneous chromatographic separation, detection and quantification of eleven isobaric glucocorticoids (11-dehydrocorticosterone (A), corticosterone (B), cortisol (F), cortisone (E), the tetrahydrocortisols (THF): 3α,5α-THF, 3α,5β-THF, 3β,5α-THF, 3β,5β-THF, and the tetrahydrocortisones (THE): 3α,5α-THE, 3α,5β-THE, 3β,5α-THE) and twelve progestogens (progesterone (P4), pregnenolone (P5), the dihydroprogesterones (DHP): 20α-DHP, 20β-DHP, 3α-DHP, 3β-DHP, 5α-DHP, 5β-DHP, and the tetrahydroprogesterones (THP): 3α,5α-THP, 3α,5β-THP, 3β,5α-THP, 3β,5β-THP) in bovine plasma, skimmed milk, and saliva. A simple liquid-liquid extraction (LLE) with MTBE (methyl tert-butyl ether) was used for sample preparation of 500 μL plasma, skimmed milk, and saliva. Heated electrospray ionization (HESI) with polarity switching was applied to analyze steroids in high-resolution full scan mode (HR-FS). The method validation covered the investigation of sensitivity, selectivity, curve fitting, carry-over, accuracy, precision, recovery, matrix effects and applicability. A high sensitivity in the range of pg mL⁻¹ was achieved for all steroids suitable for the analysis of authentic samples.ISSN:0003-2670ISSN:1873-432

Simultaneous quantification of progestogens in plasma and serum by UHPLC-HRMS employing multiplexed targeted single ion monitoring

Progesterone is the predominant gestagen in most mammals studied so far. It plays a substantial role in the regulation of the female reproductive cycle and in providing support for pregnancy maintenance. Despite its known functions, gaps in knowledge are present regarding its reduced metabolites that potentially exert biological activity. Therefore, a new UHPLC-HRMS method based on a Q Exactive™ mass spectrometer was developed to detect and quantify simultaneously progesterone, its hormone precursor pregnenolone and 10 reduced progestogens (20α-DHP, 20β-DHP, 3α,5α-THP, 3α,5β-THP, 3β,5α-THP, 3β,5β-THP, 3α-DHP, 3β-DHP, 5α-DHP and 5β-DHP) in plasma and serum samples. Purification was achieved by an optimized solid phase extraction (SPE) and the analysis was conducted in positive electrospray ionization (ESI) mode with the application of multiplexed selected ion monitoring (msx-t-SIM). The method validation included the study of sensitivity, selectivity, curve fitting, carry-over, accuracy, precision, recovery and matrix effects. Despite the poor ionization properties of underivatized steroids, a high sensitivity in the range of pg/mL was achieved.ISSN:0039-9140ISSN:1873-357

Progestogen Profiling Over the Course of Diapause and Resumption of Embryo Development in the European Roe Deer

Progesterone (P4) plays a pivotal role in maintenance of pregnancy in many mammalian species. Species-specific P4 metabolites have been shown to function as primary acting progestogen and the receptor binding capacity varies between species. The European roe deer (Capreolus capreolus) displays a 4-5 month period of embryonic diapause, which decouples fertilization from implantation. The majority of roe deer have two corpora lutea that secrete P4. No changes in P4 concentrations have been observed during pre-implantation embryo development. As 5α-DHP is known to play a major role during pregnancy in elephants and horses, we hypothesized that 5α-DHP functions as additional progestogen facilitating embryo reactivation. The profile of 11 progestogens was quantified in roe deer plasma over the course of diapause and resumption of embryo development including P4, 3α- and 3β-DHP, 20α- and 20β-DHP, 5α- and 5β-DHP, 3α,5α- and 3α,5β-THP, as well as 3β,5α- and 3β,5β-THP. While P4 was most abundant during diapause and resumption of development, 20α-DHP was the most abundant P4 metabolite. This is different than in pregnant elephants, where 5α-DHP was most abundant, and the luteal phase in cattle, where 3α,5α-THP was most abundant. With the exception of a weak correlation of 3β,5α-THP, none of the progestogens significantly correlated with embryonic development in the roe deer. Thus, plasma 5α-DHP does not seem to play a role in embryo reactivation. We propose that progestogens might contribute to priming the endometrium for supporting embryo development and preparation for implantation