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In Dried Urine Spots by Liquid Chromatography-Mass Journal Pre-proof Direct Measurement of Pregnanediol 3-Glucuronide (PDG) In Dried Urine Spots by Liquid Chromatography-Mass Spectrometry to Detect Ovulation<!–<ForCover>Handelsman DJ, Nimmagadda R, Desai R, Handelsman TD, Whittle B, Skorupskaite K, Anderson RA, Direct Measurement of Pregnanediol 3-Glucuronide (PDG) In Dried Urine Spots by Liquid Chromatography-Mass Spectrometry to Detect Ovulation, Journal of Steroid Biochemistry and Molecular Biology, doi: 10.1016/j.jsbmb.2021.105900</ForCover>–> David J Handelsman, Rama Nimmagadda, Reena Desai, Timothy D Handelsman, Belinda Whittle, Karolina Skorupskaite, Richard A Anderson PII: S0960-0760(21)00093-5 DOI: https://doi.org/10.1016/j.jsbmb.2021.105900 Reference: SBMB 105900 To appear in: Journal of Steroid Biochemistry and Molecular Biology Received Date: 18 February 2021 Revised Date: 17 March 2021 Accepted Date: 7 April 2021 Please cite this article as: { doi: https://doi.org/ This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier. Direct Measurement of Pregnanediol 3-Glucuronide (PDG) In Dried Urine Spots by Liquid Chromatography-Mass Spectrometry to Detect Ovulation Short title: Urine PDG for Ovulation Detection 1David J Handelsman, 1Rama Nimmagadda, 1Reena Desai, 1Timothy D Handelsman 2Belinda Whittle, 3Karolina Skorupskaite, 3Richard A Anderson 1Andrology laboratory, ANZAC Research Institute, University of Sydney, Concord Hospital, NSW 2139 Australia 2MyHealthTest Pty Ltd, Canberra ACT, Australia 3 MRC Centre for Reproductive Health, Queens Medical Research Institute, University of Edinburgh, Scotland, UK *Correspondence: DJ Handelsman ANZAC Research Institute Hospital Road, Concord Hospital NSW 2139 E: djh @anzac.edu.au Words: 3830 Abstract: 247 words References: 49 Tables: 4 Figures: 3 + 1 supplementary figure Acknowledgements: This project received grant funding from the Australian Government (Entrepreneurs Programme - Innovation Connections: ICG000282 (proof of concept), Cooperative Research Centres Program: CRC-P57325) and the UK Medical Research Council (MR/N022556/1 to the MRC Centre for Reproductive Health) and the Wellcome Trust through Scottish Translational Medicine and Therapeutics Initiative 102419/Z/13/A. The authors would like to thank Agilent Technologies Australia for provision of an LC-MS instrument and technical support. Disclaimer: RAA has undertaken consultancy work for Roche Diagnostics, Merck, Ferring, NeRRe Therapeutics and Sojournix Inc. BW is an employee of MyHealthTest Pty Ltd. The other authors have nothing to disclose in relation to this work. Journal Pre-proof Abstract Page 1 of 17 Background: Non-invasive self-testing using an objective chemical method to detect ovulation is valuable for women planning conception, practising contraception or undergoing infertility investigations or treatment. Methods: Based on luteal phase secretion of progesterone (P4) and excretion of its major metabolite, pregnanediol glucuronide (PDG), we developed a novel direct liquid chromatography-mass spectrometry (LCMS) method to measure PDG and other steroid glucuronides in urine and in dried urine spots (DUS) without deconjugation or derivatization. Urine PDG by LCMS and immunoassay (P3G) and P4 by immunoassay with and without adjustment for creatinine were evaluated in daily first void urine samples from 10 women through a single menstrual cycle in which ovulation was confirmed by serial transvaginal ultrasound. Results: Urine PDG with and without creatinine adjustment was stable during the follicular phase with the expected striking rise in the luteal phase peaking at 5 days after ovulation. Using a single spot urine sample (100 µL) or a DUS (<20 µL urine) and an optimal threshold to distinguish pre- from post-ovulatory samples, in ROC analysis urine PDG adjusted for creatinine accurately identified ovulation in 92% of samples was comparable with P3G immunoassay and superior to urine P4 with or without adjustment for creatinine. Extending the analysis to two or three consecutive daily samples reduced the false negative rate from 8% to 2.6% for two and 1.9% for three urine samples. Conclusions: This method holds promise as a non-invasive self-test method for women to determine by an objective chemical method their ovulatory status. Keywords: ovulation, pregnanediol glucuronide, fertility, human, female, urine Introduction Human fertility requires release (ovulation) and fertilisation of an oocyte by sperm delivered through patent fallopian tube(s). These basic requirements (ovulation, sperm delivery, patent tube(s)) are routinely evaluated in couples undergoing fertility investigations. Evaluating ovulatory status is initially based on a history of regular menses potentially supplemented by characteristic mid-cycle vaginal mucus changes and biphasic basal body temperature monitoring, which are reliable methods to verify ovulation (1) but require reliable daily self-testing. However, biochemical confirmation is also sought, and is essential when menstrual cycles are irregular or when pharmacological ovulation induction is used. BiochemicalJournal confirmation of ovulation is based on midPre-proof-luteal phase serum progesterone (2) or urinary excretion of conjugated metabolites of ovarian steroids (pregnanediol 3-glucuronide (PDG) and/or estrone 3-glucuronide (EG)) measured by immunoassay (3-5). These urinary tests supersede traditional chemical methods pioneered in the 1950’s (6, 7), which were mainly deployed in natural fertility awareness methods of contraception (8). However, verifying ovulation has wider application for women Page 2 of 17 planning to become pregnant without needing formal medical assessment so that simpler, non-invasive tests suitable for home use are desirable. Progesterone (P4) is a universal steroidogenic precursor (9) produced in all steroidogenic tissues which release low but detectable levels into the circulation. It is secreted in large amounts by the corpus luteum following ovulation and from the placenta during pregnancy. In non-pregnant humans, elevated serum P4 concentrations are specific for the post-ovulatory luteal phase of the menstrual cycle leading to its wide use to confirm recent ovulation. However, this requires a venepuncture by a health care professional in the mid-luteal phase of the menstrual cycle so is not readily adaptable for self-testing at home. Alternative methods to measure the major urine metabolite of P4, pregnanediol (PD), have been developed based on measurement of PDG, the phase II (glucuronidated) excretory metabolite of PD in urine. The temporal profiles of serum P4 and urine PDG both peak at 7-8 days after ovulation so they are interchangeable biomarkers of ovulation(4). After the first chemical methods to measure urine PDG developed in the 1950’s (6), numerous immunoassay methods have been reported (10, 11). Originally these required cumbersome 24 hour urine collections, which was simplified by adapting to spot urine collections (12) including an adaptation to measurement from dried pH strips (13). Urine PDG immunoassays have the generic methodological limitations of direct (non-extraction) immunoassays, notably cross-reactivity from structurally related steroids and urine matrix interference (14). Gas chromatography-mass spectrometry methods to measure non-conjugated PD have been available since the 1960s (15, 16) and are used in anti-doping laboratories as an endogenous reference compound (17) but current methods require pre-assay enzymatic deconjugation, solvent extraction and derivatization. Similarly, an early liquid chromatography-mass spectrometry (LC-MS) method to measure urinary excretion of sex steroid metabolite conjugates (18) did not include PDG whereas the first mass spectrometry (MS) method to measure urine PDG used an older, obsolete hard ionization technique (19). More recent LC-MS methods are reported to measure PDG in methanol-extracted serum (20) and in unextracted urine, a matrix that impairs column performance and lifetime (21), but not in dried urine spots. We therefore developed a method to measure PDG by LC-MS in dried urine spots (DUS) without deconjugation or derivatization suitable for adaptation to a home self-test modality and avoids need for cold storage and transporting liquid urine samples. DUS sampling technology has been used in screening for drugs and intermediary metabolites (22-26), and most recently for unconjugated androgens (27) and glucocorticoids (28), but there are no reports of application to steroid glucuronides. As controls, we aimed to measure excretion of additional urinary steroid glucuronides, testosterone (T) 17-glucuronide (G), dihydrotestosterone (DHT) 17-G, 5α,3α-androstanediol (5α 3-G, androsterone (A) 3-G and etiocholanolone (Etio) 3-G to investigate their possible menstrual cycle variation. Journal Pre-proof Page 3 of 17 Materials and Methods Materials Steroid glucuronides (T 17-G (catalog # D507c), DHT 17-G (S006), 5α 3-G (S004), A 3-G (D572) and Etio 3- G (D607) and internal standards (IS) (d3-T-G
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