Search

Immunoassays have been the preferred method for steroid hormone analysis for more than 50 years. Automated immunoassays (AIAs) offer high throughput, rapid data turnaround, and low cost for measuring steroid hormone concentrations. The application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for steroid quantification provides greater specificity and selectivity for individual steroids, the ability to simultaneously analyze multiple steroids, and high throughput and automation. We compared AIA and LC-MS/MS for analysis of 17beta-estradiol (E2) and progesterone (P4) over the course of several menstrual cycles in 12 rhesus macaques (Macaca mulatta). Serum samples were collected every 4 days across four menstrual cycles from each monkey. AIAs were performed on a Roche cobas e411 analyzer. LC-MS/MS analysis was performed on a Shimadzu-Nexera-LCMS-8060 instrument. Scatter plots with Passing-Bablok regression showed excellent agreement between AIA and LC-MS/MS for both E2 and P4. Bland-Altman plots revealed no bias for either method; however, AIA overestimated E2 at concentrations >140 pg/ml and underestimated P4 at concentrations >4 ng/ml compared to LC-MS/MS. A comparison of testosterone concentrations measured by AIA and LC-MS/MS in the same samples was also performed. In contrast to E2 and P4, AIA and LC-MS/MS yielded significantly different results for testosterone concentrations, with AIA consistently underestimating concentrations relative to those obtained by LC-MS/MS. Well-characterized automated immunoassays are an excellent tool for daily monitoring of monkey menstrual cycles or providing single data points requiring fast turnaround. In certain situations where AIAs may provide inaccurate estimations of E2 and P4 concentrations, LC-MS/MS assays are preferable., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: To develop a method to simultaneously quantify the synthetic contraceptive progestin segesterone acetate (Nestorone®, NES) and the endogenous steroid hormones estradiol (E2), progesterone (P4), and estrone (E1) in human serum samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS)., Study Design: We analyzed 615 serum samples collected from 67 reproductive-age women actively using a contraceptive vaginal ring (CVR) designed to release NES (200 mcg/d) and E2 (75-200 mcg/d). Samples were taken prior to and up to 30 days after CVR insertion and analyzed for concentrations of NES, E2, P4, and E1 in human serum using a Shimadzu Nexera-LCMS-8050 LC-MS/MS platform. Precision, accuracy, and sensitivity for all analytes were determined across multiple assays., Results: The assay ranges for NES, E2, P4, and E1 in this analytical method were 10 pg/mL to 10 ng/mL with a lower limit of quantification of 10 pg/mL for all targets. Assay precisions were less than or equal to 14.5% and accuracies ranged from 87.0% to 110.8%. When applied to the 615 clinical samples, 550 samples had quantifiable concentrations of NES (value range 0.014-1471 ng/mL). Similarly, 595 samples had quantifiable concentrations of E2 (0.010-0.312 ng/mL), 596 samples had quantifiable concentrations of P4 (0.010-5.791 ng/mL), and 609 samples had quantifiable concentrations of E1 (0.010-0.416 ng/mL)., Conclusions: The LC-MS/MS platform results in a robust, accurate, and sensitive method for the simultaneous quantification of NES and endogenous steroid hormones in human serum., Implications: The analytical method described allows for the simultaneous quantification of NES and endogenous steroids and can be used to monitor NES concentrations during clinical trials and subject adherence to treatment with NES., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: The objective was to develop a method to simultaneously quantify five commonly used hormonal contraceptives (HCs) and two endogenous sex steroids by liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS/MS) and apply this method to human serum samples., Study Design: We developed a method to simultaneously analyze ethinyl estradiol (EE2), etonogestrel (ENG), levonorgestrel (LNG), medroxyprogesterone acetate (MPA) and norethisterone (NET), along with estradiol (E2) and progesterone (P4), in human serum for a Shimadzu Nexera-LCMS-8050 LC-MS/MS platform. We analyzed serum collected from women self-reporting use of oral contraceptives, contraceptive implants or injectable contraceptives (n=14) and normally cycling women using no HC (n=15) as well as pooled samples from women administered various HCs (ENG, n=6; LNG, n=14; MPA, n=7; NET, n=5)., Results: Limits of quantitation were 0.010ng/mL for E2, EE2 and P4; 0.020ng/mL for ENG, LNG and MPA; and 0.040ng/mL for NET. Precisions for all assays, as indicated by coefficient of variation, were less than or equal to 12.1%. Accuracies for all assays were in the range of 95%-108%. Endogenous hormone values obtained from analysis of human serum samples are in agreement with levels previously reported in the literature for normally cycling women as well as for women taking the appropriate HC., Conclusions: We have developed a robust, accurate and sensitive method for simultaneously analyzing commonly used contraceptive steroids and endogenous sex steroids in human serum., Implications: This analytical method can be used for quantitating contraceptive steroid levels in women for monitoring systemic exposure to determine drug interactions, nonadherence, misreporting and proper dosing., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In pigs, endometrial functions are regulated primarily by progesterone and placental factors including estrogen. Progesterone levels are high throughout pregnancy to stimulate and maintain secretion of histotroph from uterine epithelia necessary for growth, implantation, placentation, and development of the conceptus (embryo and its extra-embryonic membranes). This study determined effects of long-term progesterone on development and histoarchitecture of endometrial luminal epithelium (LE), glandular epithelium (GE), and vasculature in pigs. Pigs were ovariectomized during diestrus (day 12), and then received daily injections of either corn oil or progesterone for 28 days. Prolonged progesterone treatment resulted in increased weight and length of the uterine horns, and thickness of the endometrium and myometrium. Hyperplasia and hypertrophy of GE were not evident, but LE cell height increased, suggesting elevated secretory activity. Although GE development was deficient, progesterone supported increased endometrial angiogenesis comparable to that of pregnancy. Progesterone also supported alterations to the apical and basolateral domains of LE and GE. Dolichos biflorus agglutinin lectin binding and α(v) integrin were downregulated at the apical surfaces of LE and GE. Claudin-4, α(2)β(1) integrin, and vimentin were increased at basolateral surfaces, whereas occludins-1 and -2, claudin-3, and E-cadherin were unaffected by progesterone treatment indicating structurally competent trans-epithelial adhesion and tight junctional complexes. Collectively, the results suggest that progesterone affects LE, GE, and vascular development and histoarchitecture, but in the absence of ovarian or placental factors, it does not support development of GE comparable to pregnancy. Furthermore, LE and vascular development are highly responsive to the effects of progesterone.

Pigs experience significant conceptus loss near mid-gestation, correlating with increasing glandular epithelial (GE) development and secretory activity. Secreted phosphoprotein 1 (SPP1, osteopontin) increases in GE between days 30 and 40 of pregnancy and is expressed in the GE of day 90 pseudopregnant pigs, suggesting that progesterone (P(4)) from corpora lutea is responsible for induction of SPP1 in GE. In this study, pigs were ovariectomized and treated daily with P(4) to assess effects of 40 days of P(4) exposure on SPP1, P(4) receptor (PGR), uteroferrin (ACP5), and fibroblast growth factor 7 (FGF7) expression in porcine endometria. PGR mRNA decreased in pigs injected with P(4) compared with pigs injected with corn oil (CO), and PGRs were downregulated in the luminal epithelium (LE) and GE. ACP5 mRNA increased in pigs injected with P(4) compared with pigs injected with CO, and ACP5 was induced in the GE of P(4)-treated pigs. FGF7 mRNA increased in pigs injected with P(4) compared with pigs injected with CO, and FGF7 was induced in the LE and GE of P(4)-treated pigs. SPP1 mRNA was not different between pigs injected with P(4) compared with pigs injected with CO, and SPP1 was not present in the GE of P(4)-treated pigs. Therefore, long-term P(4), in the absence of ovarian and/or conceptus factors, does not induce SPP1 expression in GE. We hypothesize that a servomechanism involving sequential effects of multiple hormones and cytokines, similar to those for sheep and humans, is required for GE differentiation and function, including the synthesis and secretion of SPP1.

Secreted phosphoprotein one (SPP1, osteopontin) may regulate conceptus implantation and placentation. We investigated effects of progesterone (P(4)) and the conceptus on expression and localization of SPP1 in the ovine uterus. Steady-state levels of SPP1 mRNA in the endometrium of unilaterally pregnant ewes did not differ significantly between nongravid and gravid horns within their respective days of pregnancy; however, levels did increase as pregnancy progressed. SPP1 mRNA was detectable in the glandular epithelium (GE) of both nongravid and gravid horns via in situ hybridization. SPP1 protein was localized to the apical surface of the luminal epithelium of both nongravid and gravid uterine horns. Gravid horns exhibited extensive stromal SPP1 on Days 40 through 120, whereas SPP1 was markedly lower in the stroma of nongravid uterine horns through Day 80 of pregnancy. By Day 120, stromal expression of SPP1 between nongravid and gravid horns was similar. Long-term P(4) treatment of ovariectomized ewes induced SPP1 in the uterine stroma and GE. A bioactive 45-kDa SPP1 fragment was purified from uterine secretions and promoted ovine trophectoderm cell attachment in vitro. Interestingly, increased stromal cell expression of SPP1 was positively associated with vascularization as assessed by von Willebrand factor staining. Finally, ovine uterine artery endothelial cells produced SPP1 during outgrowth into three-dimensional collagen matrices in an in vitro model system that recapitulates angiogenesis. Collectively, P(4) induces and the conceptus further stimulates SPP1 in uterine GE and stroma, where SPP1 likely influences histotrophic and hematotrophic support of conceptus development.

Fibroblast growth factor 7 (FGF7) stimulates cell proliferation, differentiation, migration and angiogenesis. The consensus is that FGF7, expressed by mesenchymal cells, binds FGF receptor 2IIIb (FGFR2) on epithelia, thereby mediating epithelial-mesenchymal interactions. The pig uterus is unique in that FGF7 is expressed by the luminal epithelium (LE) and FGFR2 is expressed by the LE, glandular epithelium (GE), and trophectoderm to effect proliferation and differentiated cell functions during conceptus development and implantation. FGF7 expression by the uterine LE of pigs increases between Days 9 and 12 of the estrus cycle and pregnancy, as circulating concentrations of progesterone increase, progesterone receptors (PGR) in the uterine epithelia decrease, and the conceptuses secrete estradiol-17beta (E(2)), for pregnancy recognition. Furthermore, E(2) increases the expression of FGF7 in pig uterine explants. The present study investigates the relationships between progesterone, E(2), and their receptors and the expression of FGF7 in the pig uterus in vivo. Pigs were ovariectomized on Day 4 of the estrus cycle and injected i.m. daily from Day 4 to Day 12 with either corn oil (CO), progesterone (P4), P4 and ZK317,316 (PZK), E(2), P4 and E(2) (PE), or P4 and ZK and E(2) (PZKE). All gilts (n = 5/treatment) were hysterectomized on Day 12. The results suggest that: 1) P4 is permissive to FGF7 expression by down-regulating PGR in LE; 2) P4 stimulates PGR-positive uterine stromal cells to release an unidentified progestamedin that induces FGF7 expression by LE; 3) E(2) and P4 can induce FGF7 when PGR are rendered nonfunctional by ZK; and 4) E(2) from conceptuses interacts via estrogen receptor alpha, but not estrogen receptor beta in LE to induce maximal expression of FGF7 in LE on Day 12 of pregnancy in pigs.