Measuring the Timing of Ovulation & Implantation: Old Ways & New

1. Medgar Evers College April 5, 2006 Measuring the Timing of Ovulation & Implantation: Old Ways & New Kenneth L. Campbell Professor of Biology; Associate Dean of Science & Mathematics University of Massachusetts at Boston

2. This presentation is made possible by a grant entitled“Shortcourses in Endocrinology at Minority Undergraduate Institutions”from the National Institute of General Medical Sciences (NIGMS) to The Minority Affairs Committee of the Endocrine Society

3. SUMMER RESEARCH OPPORTUNITIES FOR MINORITY STUDENTS • Are You Interested In: • Going away for the summer in 2007 to do research for 8 weeks in a cutting-edge endocrine lab in an area of your choice with a caring mentor? • Receiving a two-year free membership in The Endocrine Society with many benefits, e.g., information about travel grants, scholarships, online journals, etc.? http://www.endo-society.org/minorityactivities/summer_research.cfm

4. SUMMER RESEARCH OPPORTUNITIES FOR MINORITY STUDENTS • How do I qualify and What should I do? • You can apply if you are: • A full-time minority student beyond your sophomore year or if you are currently enrolled at a minority institution, and • A U.S. citizen or permanent resident. • For more details on eligibility and requirements, visit The Endocrine Society website at http://www.endo-society.org/minorityactivities/summer_research.cfm

5. Basic patterns of hormones & early embryonic stages There have been few markers for ovulation & the first 10 post-ovulatory days.

6. In hemochorial placentae trophoblastic antigens directly contact maternal blood; molecules <700D diffuse freely. Maternal Blood Syncytiotrophoblast cells Fetal Mesenchyme Cytotrophoblast cells Fetal Endothelium Fetal Blood Chorionic villus

7. Plasma Inhibin Urinary forms not yet found. europe.obgyn.net/nederland/mp/overgang/images/overgang14x.gif

8. Monitoring Ovarian Function and Predicting Ovulation Family Health International, Research Triangle Park, NC 12 - 15 Nov. 1984, K. Campbell & W. Collins, Co - Chairs Predictors of ovulation & the fertile period have not changed much in 20 years. LH, estradiol, progesterone, & steroid effects remain key indices. Peak Campbell, KL (1985) Monitoring ovarian function and predicting ovulation: summary of a meeting, Research Frontiers in Fertility Regulation 3:1-16.

9. Classical markers especially in combinations are useful delineators of the fertile period.

10. Cervical mucus & basal body temperature, well-tested biomarkers, are mainstays of the Billings method of fertility monitoring. Progesterone dominant Estradiol dominant Fertility Disorders and the Billings Ovulation Method P. Vigil, Faculty of Biological Sciences Pontifical Catholic University of Chile reproduced at http://www.woomb.org/omrrca/vigil/ fertilityDisorders.htm

11. Advances with Potential Impact on Biomarker Use Since 1984 • Human & animal genome projects • DNA & protein databases & computerized search techniques • Animal cloning & associated techniques • Molecular monitoring of development • DNA biotechnology; PCR, microarray • Mass spectrometry: MALDI-TOF, MS/MS; coupling to HPLC • High throughput immunoassay, fluorescence & luminescence detection; sensitivity at or below attomolar levels (down to single molecules)

12. Biomarker Progress Since 1984 • Wilcox, Weinberg, Baird, Dunson, et al. refined algorithms for using E1G/PdG ratios as indices for ovulation, the fertile period, establishment of pregnancy, & continuation of pregnancy. • Early pregnancy factor (EPF, heat shock protein 10, HSPE1) cloned& characterized: = extracellular chaperonin 10, a 3.1 kb gene at locus 2q33.1, encodes a 10.8 kD, protein of 101 amino acids with no signal sequence; involved in mitochondrial protein folding; produced by the ovary & platelets within 24 h of fertilization. • Portable miniaturized ultrasound units, e.g., Renco Pregtone II, are now available.

13. Biomarker Progress Since 1984 (cont.) • Commercial kits to evaluate serum, urinary, & salivary hormones & sperm numbers • Software to track calendars, cervical mucus, BBT, & urinary or salivary hormones • Devices to monitor direct & indirect hormonal effects on cervical mucus, salivary ferning, & salivary, vaginal, & cervical electrolytes • hCG field test improvements Holman et al. (1998) A commercial pregnancy test modified for field studies of fetal loss, Clinica Chimica Acta 271(1):25-44. • Molecular screens now identifying genes specifically expressed during placentation & early embryogenesis.

14. Clearblue (ClearPlan) Easy Fertility Monitor, Unipath Limited The product comprises a number of test sticks and a hand held Fertility Monitor, and monitors the levels of luteinising hormone (LH) and the estrogen metabolite, estrone-3 glucuronide (E3G), in urine. From the start of the menstrual cycle, the user performs a daily test stick reading over 10 or 20 consecutive days, according to the length of the cycle and the timing of the LH surge. From these readings, the monitor will display the fertility status over the course of the cycle – low, high or peak – signalling when successful conception is most likely to occur. The Clearblue Easy Fertility Monitor is proven to be 99% accurate in detecting LH surges in laboratory tests. Logitudinal sampling & integration of LH & urinary estrogen signals

15. Male fertility tests

16. Cycle tracking software & online calculators Are women willing to share existing information with this project? You provide Babycomp with the information it requires by means of simple input and regular BBT measurements which are highly accurate to within 1/100th of a degree. By analyzing this information Babycomp is able to detect ovulation, and calculate in advance when your best opportunity for conceiving is. The Fertility Monitor is a series of interrelated computer programs which assist you in using the Sympto-Thermal Method while Ovusoft Fertility Software is an software package to do the same. BBT & Cervical Mucus: http://www.tcoyf.com/products/ prefs5.asp Calendars: http://kidsdirect.net/BD/tools/

17. Salivary ferning: indirect estrogen biomarker Fertility Tracker® Saliva Fertility Monitor Fertile-Focus Ovuscope The salt or electrolyte crystals that are present in dried female saliva rise significantly prior to or during a normal ovulation period to form miniature patterns which resemble fern leaves. During your fertile period, a sample of your saliva, when placed in … [a] viewing scope, will present this unique pattern.

18. Zetek OvaCue® Fertility Monitor (Salivary Electrolytes) At the same time [as estradiol is rising], other hormones are changing the amounts of ... (electrolytes) that are kept or discarded by the body. This is what produces the Cue Peak, a high point in the salivary readings of electrolytes. Why salivary electrolyte readings are better than urine LH A few days after the Cue Peak, the luteinizing hormone (LH) in the blood increases and decreases very sharply over a period of 24 hours (the LH peak). LH is the hormone that triggers the release of the ovum from the ovary. The egg is released within 24 hours after the LH is at its highest in the blood. Some time (as much as 12 hours) after the peak of LH in the blood, LH is also present in the urine. It is this increasing concentration of urine LH that can be detected by the different brands of urine-based ovulation predictors. Prediction using this method depends on the exact relationships between the time of the highest blood LH, the time the urine stick begins to see the LH, and the time of the measurement. At best, the time between the urine signal and actual ovulation cannot be more than 24 hours in advance. At worst, it is seen only after ovulation has already occurred, offering no chance of conception for the current cycle.

20. hCG field test improvements: increasing sample volumes & reaction times & use of a reflectance reader greatly improved assay sensitivity Holman et al. (1998) A commercial pregnancy test modified for field studies of fetal loss, Clinica Chimica Acta 271(1):25-44.

22. HoxD4 Nobel Prize in Physiology or Medicine 1995: Edward Lewis, Christiane Nüsslein-Volhard & Eric Wieschaus; work on “genetic control of early embryonic development” HoxD2 >700 genes in the mouse associated with nidation & early embryogene-sis; 895 genes expressed in human embryonic stem & carcinoma cells (Sperger et al. 2003, PNAS 100:13350). Thomas W. Sadler 2004 Langman's Medical Embryology, 9th Ed., Williams & Wilkins

23. Microarray analyses are already providing data. Prenat Diagn 2003; 23: 410–419. Published online in Wiley InterScience (www.interscience.wiley.com). Identification of expressed sequence tags preferentially expressed in human placentas by in silico subtraction David Miner and Aleksandar Rajkovic* Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas, USA Conclusion In silico subtraction identified 44 previously studied genes involved in placental physiology as well as 63 EST clusters preferentially expressed in placental tissue, which may serve as targets for future studies seeking novel markers for prenatal diagnosis or to better understand placental genetics. Should be extended to all early embryonic tissues.

24. Microarray analysis of trophoblast differentiation: gene expression reprogram-ming in key gene function categories. Bruce J. Aronow, Brian D. Richardson, & Stuart Handwerger. Departments of Endocrinology and Molecular and Developmental Biology, Children’s Hospital Research Foundation and Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-2029. Physiol Genomics 6: 105–116, 2001. … DNA microarray analysis to characterize the process by which human cytotrophoblast cells differentiate into syncytiotrophoblast cells in a purified cell culture system. Of 6,918 genes analyzed, 141 genes were induced and 256 were downregulated by more than 2-fold. Dynamically regulated genes were divided by the K-means algorithm into 9 kinetic pattern groups, then by biologic classification into 6 overall functional categories: cell and tissue structural dynamics, cell cycle and apoptosis, intercellular communication, metabolism, regulation of gene expression, and expressed sequence tag (EST) and function unknown. Gene expression changes within key functional categories were tightly coupled to morphological changes. In several key gene function categories, such as cell and tissue structure, many gene members of the category were strongly activated while others were strongly repressed. These findings suggest that differentiation is augmented by “categorical reprogramming” in which the function of induced genes is enhanced by preventing the further synthesis of categorically related gene products. Microarray analyses are already providing data. At least 45 of these proteins are soluble, extracellular, & potential biomarkers.

27. Other Prospects? New Possibilities? New versions of old methods: DNA methods applied to urine sediments; cornification is an apoptotic process

28. Hormones, cornification/apoptosis, Y-Chromosomal DNA, microbial DNA PCR & Real Time PCR Adequate insemination? What do sperm washout profiles look like? What types of sperm are lost or washed out first? Is quantitative probing of peri-ovular, peri-implantation microbial environment possible?

29. Ways to identify new biomarkers • MALDI-TOF or HPLC-MS/MS examination of urines or sera from women with & without known gestation or gestational loss (improved with prior 2-D gel electrophoresis) • Differential microchip screening of proteins from urines or sera from women with known gestation or gestational loss. • Gestation -- : Proteins>2D gels>Abs • Gestation + or Gestation Loss +: Proteins> remove common proteins with Abs>2D gels of remaining unique proteins> unique Abs • Microchips with Abs or Abs> evaluate sera or urines showing common or unique proteins • HPLC or GC-MS/MS or MALDI-TOF for small molecule profiles in similar studies

30. Conclusions The processes of interest are occult. We have reasonably good, well- tested methods to monitor many key events & some new devices to do so. New approaches to old methods & use of new genomic data indicate more markers do exist. Development & use of such markers would open important additional windows on the events central to predicting & understanding ovulation, fertilization, implantation, & failure of implantation.

33. Direct Biomarkers of Ovulation, Fertilization, Implantation, Early Embrogenesis & Pregnancy Loss • E2, P, LH, FSH, PdG/E1G Ratios • hCG patterns, IGFBP-I • Classical neural defect markers: PPAP-A, α-FP, urinary E3 • Markers identified in vitro or via genetic experiments & protein expression microarray studies in the 1st week of gestation: WNT genes, LIF, interferons, ...

34. Molecular Markers • Endometrium: Indian Hedgehog & its signalling path, protease inhibitors, matrix proteins, cytokines, growth factors, IL-1 • Trophoblast/synctiotrophoblast: hCG, inhibin A, IGF, VEGF, PDGF, bFGF, TGF, EGF, leptin, mucins, integrins, trophonin (specific cell membrane adhesion protein), matrix metalloproteases, IGFBP-1, hPL, vasopressinase, glycosylated free  subunit, ET-1, TNF-, angiotensin II, LIF,…

35. N. Takamoto et al. (2002) Identification of Indian Hedgehog as a Progesterone-Responsive Gene in the Murine Uterus, Molecular Endocrinology 16(10):2338–2348.

36. BioSense Vaginal electrical properties The electrolyte shifts have also been exploited in the cervical/vaginal environment.

37. Wednesday, September 25, 2002 FDA Approves Pheromone Sciences' PSC Fertility Monitor The PSC Fertility Monitor is a watch like fertility cycle monitoring device for the consumer and professional medical market. The PSC Fertility Monitor looks to set a new benchmark in providing a reliable "predictive" approach to natural, cycle-based family planning through the measurement of perspiration ion changes on the surface of the skin. These changes in female perspiration allow for the prediction of ovulation up to 4 days prior to the day of ovulation without the need for inconvenient urine or blood testing. [A] trial, … of 105 subjects, reported results showing ... 73% of the ovulatory women had their ovulation date correctly predicted to ... +/-2 days of the ovulation date determined by blood serum levels. [The] results are consistent with those … reported (76%) [in] a pilot study ... at Toronto General Hospital ... In the Toronto trial, the average deviation reported for the PSC Fertility Monitor was +/- 1.38 days between the actual date of ovulation and the predicted ovulation date. In the USA trial, the average deviation reported for the PSC Fertility Monitor was +/- 1.48 days between the actual date of ovulation and the predicted ovulation date. Fertilité-OV

38. Suggestions for this study • Begin by sampling for existing markers: LH, FSH, E2, P, E1G, PdG, hCG, IGFBP-1, α-FP, PPAP-A, uE3, inhibin A, inhibin B • Keep a reserve of samples for retrospective evaluation of urine sediment DNA & not - yet - identified soluble markers in urine or serum • Begin a significant program of research to uncover new biomarkers using updated methods and database sources