Transcript Biomarkers of Ovulation, Fertilization, & Pregnancy

Measuring the Timing of Ovulation
& Implantation: Old Ways & New
Kenneth L. Campbell
Professor of Biology
University of Massachusetts at Boston
Basic patterns of
hormones & early
embryonic stages
There have been
few markers for
ovulation & the
first 10 postovulatory days.
In hemochorial placentae trophoblastic antigens directly
contact maternal blood; molecules <700D diffuse freely.
Maternal
Blood
Syncytiotrophoblast
cells
Cytotrophoblast
cells
Fetal
Mesenchyme
Fetal
Endothelium
Fetal
Blood
Chorionic villus
Plasma Inhibin
Urinary
forms not
yet found.
europe.obgyn.net/nederland/mp/ov
ergang/images/overgang14x.gif
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.
Peak
LH, estradiol,
progesterone, &
steroid effects remain
key indices.
Campbell, KL (1985) Monitoring
ovarian function and predicting
ovulation: summary of a
meeting, Research Frontiers in
Fertility Regulation 3:1-16.
Classical markers especially in combinations are
useful delineators of the fertile period.
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
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)
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.
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.
Clearblue (ClearPlan) Easy Fertility Monitor,
Unipath Limited The product comprises a number of test
Logitudinal
sampling &
integration of LH
& urinary
estrogen signals
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.
Male fertility tests
Cycle tracking software & online calculators
Are women willing to share existing
information with this project?
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.
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.
BBT & Cervical Mucus: http://www.tcoyf.com/products/ prefs5.asp
Calendars: http://kidsdirect.net/BD/tools/
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.
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.
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.
Current Biomarkers of Pregnancy Maintenance and Fetal Defects
Biomarker
Biospecimen
Biomarker for:
Reference
Alpha-Fetoprotein
Serum
Fetal Defects
57
hCG level
Urine
Pregnancy Maintenance
55
hCG glycoform ratio
Urine
Pregnancy Maintenance
56
Human placental lactogen
Serum
Placental Function
98
IGFBP- 4 protease
Serum
Fetal Defects
60, 61, 62
Inhibin A
Serum
Fetal Defects; Preeclampsia
59
Macrophage inhibitory cytokine -1
Serum
Predicting Miscarriage
63, 107
Nobel Prize in Physiology or
Medicine 1995: Edward Lewis,
Christiane Nüsslein-Volhard &
Eric Wieschaus; work on
“genetic control of early
embryonic development”
HoxD2
Thomas W. Sadler 2004 Langman's Medical
Embryology, 9th Ed., Williams & Wilkins
HoxD4
>700 genes in the
mouse associated
with nidation &
early embryogenesis; 895 genes
expressed in
human embryonic
stem & carcinoma
cells (Sperger et
al. 2003, PNAS
100:13350).
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.
Microarray analyses are already providing data.
Microarray analysis of trophoblast differentiation: gene expression reprogramming 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 2fold. 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.
At least 45 of these proteins are soluble,
extracellular, & potential biomarkers.
Some Biomarkers of Actual & Potential Application in Assessing Endometrial Receptivity
Biomarker
CA-125
Colony Stimulating Factor
Cyclin E
Endometrial bleeding-associated factor
Glycodelin-A
Heparin binding-epidermal growth factor
HoxA-10 and -11
Insulin-like growth factor binding protein 1
Integrin alpha v beta 3
Integrin alpha 4
Integrin alpha 6
Integrin beta 3 *
Interleukin 1
Biospecimen
Serum
Serum
Endometrium
Endometrium, Serum
Endometrium, Serum
Endometrium
Endometrium
Endometrium
Endometrium
Endometrium
PBLs
Endometrium
Uterine flushings
Leukaemia Inhibitory Factor
Endometrium
Mucin-like glycoprotein *
Endometrium
Muc1
Endometrium
P27
Endometrium
Placenta protein 14
Serum
* Targets used in current commercially available ER tests (service only)
Reference
70
71
72
73
74, 75
76
77, 78
79
80
81
82
81
83
84
85
86
72
87
Biomarkers of Actual & Potential Application in Assessing Fertilization or Implantation
Biomarker
Apolipoprotein D
Dickkopf protein
Early Pregnancy Factor*
Glycodelin A
HCG*
Inhibin A level; Inhibin A/B ratio*
IGF-binding serine protease 11
IGF-II
IL-1β
IGFBP-1
MUC-1
Osteopontin
Pregnancy specific beta1 glycoprotein 1
Biospecimen(s)†
Reference
Serum
Endometrium
Cervical mucus, Serum
Serum
Serum, Urine
Serum
Endometrium
Serum
Cervicovaginal secretions, Serum
Serum, Urine, Vaginal discharge
Serum
Endometrium
Serum, Urine
48
48
43
48
55
59
63
88
89-93
94, 95
96, 97
48
63
* 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.
Other Prospects?
New Possibilities?
New versions of old methods:
DNA methods applied to urine
sediments; cornification is an
apoptotic process
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, periimplantation microbial
environment possible?
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
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.
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
First day of mucus
Rise of E1G/PdG
Rise of urinary E1G
First day of fertile mucus
Rise of salivary E2
Rise of serum E2
Follicle size 15 ± 2 mm
Peak of E1G/PdG
Peak volume of mucus
Peak of salivary E2
Peak of urinary E1G
Rise of serum LH
Peak of serum E2
Peak of serum LH
Peak of fertile mucus
Rise of serum P
Rise of urinary PdG
Rise of BBT
Rise of salivary P
b
Mean
(days)
-7.3
-7.2
-5.6
-5.1
-5
c
-3.4
-2.7
-2.5
-2
-1.5
-1.3
c
-1.3
c
-1.0
c
-0.7
-0.4
c
-0.3
0.2
1.1
1.5
SD
2.5
2.8
1.9
2.6
--d
0.9
0.5
2.3
1
--1.9
d
0.3
d
0.3
d
0.2
2.2
d
0.3
3
2
---
Range
Min
-13
-15
-11
-12
---7
---10
-4.5
---9
-2.3
-2
-1.9
-10
-1.3
---2
---
Reference
Max
-3
-2
-2
-1
---2
--0
-1.5
--4
-1
0
-0.3
5
0
--7
---
24
24
24
24
99
36
100
24
101
99
24
29
29
29
24
29
26
26
99
a
Modified from Table 1 of reference 7. Abbreviations: E1G = estrone-3-glucuronide; PdG = pregnanediol-3αglucuronide; 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.
c median
d
95% confidence intervals of the estimated median
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, ...
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,…
N. Takamoto et al. (2002) Identification of Indian Hedgehog as a
Progesterone-Responsive Gene in the Murine Uterus, Molecular
Endocrinology 16(10):2338–2348.
Vaginal
electrical
properties
The electrolyte
shifts have also
been exploited in
the cervical/vaginal
environment.
BioSense
Fertilité-OV
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.
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