Measuring the Timing of Ovulation

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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 Universi
ty of Massachusetts at Boston
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This presentation is made possible by a grant
entitledShortcourses in Endocrinology at
Minority Undergraduate Institutionsfrom the
National Institute of General Medical Sciences
(NIGMS) to The Minority Affairs Committee of
the Endocrine Society
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SUMMER RESEARCH OPPORTUNITIES FOR MINORITY
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  research for 8 weeks in a cutting-edge endocrine
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  Endocrine Society with many benefits, e.g.,
  information about travel grants, scholarships,
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SUMMER RESEARCH OPPORTUNITIES FOR MINORITY
STUDENTS

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• You can apply if you are
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mer_research.cfm
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Classical markers especially in combinations are
useful delineators of the fertile period.
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• 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)

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• 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.

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• 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.

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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
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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/product
s/ prefs5.asp Calendars http//kidsdirect.net/BD/
tools/
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Zetek OvaCue Fertility Monitor (Salivary
Electrolytes)
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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.
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Current Biomarkers of Pregnancy Maintenance and Fetal Defects Current Biomarkers of Pregnancy Maintenance and Fetal Defects Current Biomarkers of Pregnancy Maintenance and Fetal Defects Current Biomarkers of Pregnancy Maintenance and Fetal Defects Current Biomarkers of Pregnancy Maintenance and Fetal Defects
Biomarker Biospecimen Biospecimen Biomarker for Reference
Alpha-Fetoprotein Alpha-Fetoprotein Serum Fetal Defects 57
hCG level hCG level Urine Pregnancy Maintenance 55
hCG glycoform ratio hCG glycoform ratio Urine Pregnancy Maintenance 56
Human placental lactogen Human placental lactogen Serum Placental Function 98
IGFBP- 4 protease IGFBP- 4 protease Serum Fetal Defects 60, 61, 62
Inhibin A Inhibin A Serum Fetal Defects Preeclampsia 59
Macrophage inhibitory cytokine -1 Macrophage inhibitory cytokine -1 Serum Predicting Miscarriage 63, 107
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Nobel Prize in Physiology or Medicine 1995
Edward Lewis, Christiane Nüsslein-Volhard Eric
Wieschaus work on genetic control of early
embryonic development
gt700 genes in the mouse associated with nidation
early embryogene-sis 895 genes expressed in
human embryonic stem carcinoma cells (Sperger
et al. 2003, PNAS 10013350).
Thomas W. Sadler 2004 Langman's Medical
Embryology, 9th Ed., Williams Wilkins
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Microarray analyses are already providing data.
Prenat Diagn 2003 23 410419. 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.
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Microarray analyses are already providing data.
At least 45 of these proteins are soluble,
extracellular, potential biomarkers.
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Some Biomarkers of Actual Potential Application in Assessing Endometrial Receptivity Some Biomarkers of Actual Potential Application in Assessing Endometrial Receptivity Some Biomarkers of Actual Potential Application in Assessing Endometrial Receptivity
Biomarker Biospecimen Reference
CA-125 Serum 70
Colony Stimulating Factor Serum 71
Cyclin E Endometrium 72
Endometrial bleeding-associated factor Endometrium, Serum 73
Glycodelin-A Endometrium, Serum 74, 75
Heparin binding-epidermal growth factor Endometrium 76
HoxA-10 and -11 Endometrium 77, 78
Insulin-like growth factor binding protein 1 Endometrium 79
Integrin alpha v beta 3 Endometrium 80
Integrin alpha 4 Endometrium 81
Integrin alpha 6 PBLs 82
Integrin beta 3 Endometrium 81
Interleukin 1 Uterine flushings 83
Leukaemia Inhibitory Factor Endometrium 84
Mucin-like glycoprotein Endometrium 85
Muc1 Endometrium 86
P27 Endometrium 72
Placenta protein 14 Serum 87
Targets used in current commercially available ER tests (service only) Targets used in current commercially available ER tests (service only) Targets used in current commercially available ER tests (service only)
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Biomarkers of Actual Potential Application in Assessing Fertilization or Implantation Biomarkers of Actual Potential Application in Assessing Fertilization or Implantation Biomarkers of Actual Potential Application in Assessing Fertilization or Implantation
Biomarker Biospecimen(s) Reference

Apolipoprotein D Serum 48
Dickkopf protein Endometrium 48
Early Pregnancy Factor Cervical mucus, Serum 43
Glycodelin A Serum 48
HCG Serum, Urine 55
Inhibin A level Inhibin A/B ratio Serum 59
IGF-binding serine protease 11 Endometrium 63
IGF-II Serum 88
IL-1ß Cervicovaginal secretions, Serum 89-93
IGFBP-1 Serum, Urine, Vaginal discharge 94, 95
MUC-1 Serum 96, 97
Osteopontin Endometrium 48
Pregnancy specific beta1 glycoprotein 1 Serum, Urine 63
Targets used currently in assessing fertilization/implantation Targets used currently in assessing fertilization/implantation Targets used currently in assessing fertilization/implantation
All listed biomarkers present in endometrium however, because of the invasiveness of obtaining endometrial samples, this biospecimen only listed in cases where there is no PubMed report of the biomarker being analyzed in more accessible biospecimens in the context of implantation/ pregnancy studies. All listed biomarkers present in endometrium however, because of the invasiveness of obtaining endometrial samples, this biospecimen only listed in cases where there is no PubMed report of the biomarker being analyzed in more accessible biospecimens in the context of implantation/ pregnancy studies. All listed biomarkers present in endometrium however, because of the invasiveness of obtaining endometrial samples, this biospecimen only listed in cases where there is no PubMed report of the biomarker being analyzed in more accessible biospecimens in the context of implantation/ pregnancy studies.
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New versions of old methods DNA methods applied
to urine sediments cornification is an apoptotic
process
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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?
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• 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 -- Proteinsgt2D gelsgtAbs
• Gestation or Gestation Loss Proteinsgt
  remove common proteins with Absgt2D gels of
  remaining unique proteinsgt unique Abs
• Microchips with Abs or Absgt evaluate sera or
  urines showing common or unique proteins
• HPLC or GC-MS/MS or MALDI-TOF for small molecule
  profiles in similar studies

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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.
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Chronology of Endpoints Predictive of Limits of Fertile Period Timing of Ovulation in Days Relative to Ovulation ( Serum LH Peak 0.7 Days)a Chronology of Endpoints Predictive of Limits of Fertile Period Timing of Ovulation in Days Relative to Ovulation ( Serum LH Peak 0.7 Days)a Chronology of Endpoints Predictive of Limits of Fertile Period Timing of Ovulation in Days Relative to Ovulation ( Serum LH Peak 0.7 Days)a Chronology of Endpoints Predictive of Limits of Fertile Period Timing of Ovulation in Days Relative to Ovulation ( Serum LH Peak 0.7 Days)a Chronology of Endpoints Predictive of Limits of Fertile Period Timing of Ovulation in Days Relative to Ovulation ( Serum LH Peak 0.7 Days)a Chronology of Endpoints Predictive of Limits of Fertile Period Timing of Ovulation in Days Relative to Ovulation ( Serum LH Peak 0.7 Days)a
Endpoint Meanb SD Range Range Reference
  (days)   Min Max  
First day of mucus -7.3 2.5 -13 -3 24
Rise of E1G/PdG -7.2 2.8 -15 -2 24
Rise of urinary E1G -5.6 1.9 -11 -2 24
First day of fertile mucus -5.1 2.6 -12 -1 24
Rise of salivary E2 -5 --- --- --- 99
Rise of serum E2 -3.4c 0.9d -7 -2 36
Follicle size 15 2 mm -2.7 0.5 --- --- 100
Peak of E1G/PdG -2.5 2.3 -10 0 24
Peak volume of mucus -2 1 -4.5 -1.5 101
Peak of salivary E2 -1.5 --- --- --- 99
Peak of urinary E1G -1.3 1.9 -9 4 24
Rise of serum LH -1.3c 0.3d -2.3 -1 29
Peak of serum E2 -1.0 c 0.3d -2 0 29
Peak of serum LH -0.7 c 0.2d -1.9 -0.3 29
Peak of fertile mucus -0.4 2.2 -10 5 24
Rise of serum P -0.3c 0.3d -1.3 0 29
Rise of urinary PdG 0.2 3 --- --- 26
Rise of BBT 1.1 2 -2 7 26
Rise of salivary P 1.5 --- --- --- 99
a Modified from Table 1 of reference 7. Abbreviations E1G estrone-3-glucuronide PdG pregnanediol-3a-glucuronide E2 estradiol LH luteinizing hormone P progesterone BBT basal body temperature. a Modified from Table 1 of reference 7. Abbreviations E1G estrone-3-glucuronide PdG pregnanediol-3a-glucuronide E2 estradiol LH luteinizing hormone P progesterone BBT basal body temperature. a Modified from Table 1 of reference 7. Abbreviations E1G estrone-3-glucuronide PdG pregnanediol-3a-glucuronide E2 estradiol LH luteinizing hormone P progesterone BBT basal body temperature. a Modified from Table 1 of reference 7. Abbreviations E1G estrone-3-glucuronide PdG pregnanediol-3a-glucuronide E2 estradiol LH luteinizing hormone P progesterone BBT basal body temperature. a Modified from Table 1 of reference 7. Abbreviations E1G estrone-3-glucuronide PdG pregnanediol-3a-glucuronide E2 estradiol LH luteinizing hormone P progesterone BBT basal body temperature. a Modified from Table 1 of reference 7. Abbreviations E1G estrone-3-glucuronide PdG pregnanediol-3a-glucuronide E2 estradiol LH luteinizing hormone P progesterone BBT basal body temperature.
b Recomputed from listed sources by adjusting for relative average time of ovulation after the serum LH peak and by subtraction of any constants added in the original sources. b Recomputed from listed sources by adjusting for relative average time of ovulation after the serum LH peak and by subtraction of any constants added in the original sources. b Recomputed from listed sources by adjusting for relative average time of ovulation after the serum LH peak and by subtraction of any constants added in the original sources. b Recomputed from listed sources by adjusting for relative average time of ovulation after the serum LH peak and by subtraction of any constants added in the original sources. b Recomputed from listed sources by adjusting for relative average time of ovulation after the serum LH peak and by subtraction of any constants added in the original sources. b Recomputed from listed sources by adjusting for relative average time of ovulation after the serum LH peak and by subtraction of any constants added in the original sources.
c median c median c median c median c median c median
d 95 confidence intervals of the estimated median d 95 confidence intervals of the estimated median d 95 confidence intervals of the estimated median d 95 confidence intervals of the estimated median d 95 confidence intervals of the estimated median d 95 confidence intervals of the estimated median
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• E2, P, LH, FSH, PdG/E1G Ratios

• hCG patterns, IGFBP-I

• Classical neural defect markers PPAP-A, a-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, ...

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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,

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N. Takamoto et al. (2002) Identification of
Indian Hedgehog as a Progesterone-Responsive Gene
in the Murine Uterus, Molecular Endocrinology
16(10)23382348.
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Vaginal electrical properties
The electrolyte shifts have also been exploited
in the cervical/vaginal environment.
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Fertilité-OV
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• Begin by sampling for existing markers LH, FSH,
  E2, P, E1G, PdG, hCG, IGFBP-1, a-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