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NAME : PROF.NARENDRA MALHOTRA
DESIGNATION: PROF. UNIVERSITY SARAJEVO SCHOOL OF SCIENCE & TECHNOLOGY (SSST)
V.P. WAPM(WORLD ASSOCIATION OF PRENATAL MEDICINE)
PRESIDENT ISPAT (2017-2019)
PAST PRESIDENT ISAR ( 2016 – 2017)
SEC GEN SAFOG (2015-2019)
PAST PRESIDENT FOGSI(2008)
MANAGING DIRECTOR GLOBAL RAINBOW HEALTH CARE, AGRA
DIRECTOR ART-RAINBOW –IVF (AGRA & DELHI)
CITY: AGRA, INDIA
AFFILIATIONS: M.D., F.I.C.O.G., F.I.C.M.C.H, F.R.C.O.G.,F.I.C.S., F.M.A.S., A.F.I.A.P.
AWARDS: BEST CITIZENS OF INDIA AWARD, MAN OF THE YEAR AWARD, CORION AWARD, AWARDED BEST PAPER AND BEST POSTER
AT FOGSI : 5 TIMES, ETHICON FELLOWSHIP, AOFOG YOUNG GYN. AWARD,
PUBLICATIONS: JEFFCOATE’S PRINCIPLES OF GYNECOLOGY, 9TH EDITION, OPERATIVE OBSTETRICS AND GYNECOLOGY 2ND EDITION, AN
INTRODUCTION TO GENETICS & FETAL MEDICINE, 2ND EDITION, FOGSI’S PRINCIPLES & PRACTICE OF OBSTETRICS &
GYNECOLOGY FOR PG’S 3RD EDITION, DONALD BOOK ON OBSTETRIC PRACTICAL PROBLEMS MANUAL 2ND EDITION,
ULTRASOUND IN OBSTETRICS & GYNAECOLOGY 5TH EDITION, EDITOR 25 BOOKS, MANY CHAPTERS, ON EDITORIAL
BOARD
OF MANY JOURNALS, EDITOR OF SERIES OF STEP BY STEP BOOKS.
OVER 35 PUBLISHED PAPERS, 165 CHAPTERS, 509 PRESENTATIONS, 28 ORATIONS.
SPECIAL INTRESTS: SPECIAL INTEREST IN HIGH RISK OBS., ULTRASOUND, ART & GENETICS.
TESTS FOR OVARIAN RESERVE
Narendra Malhotra
Neharika Malhotra
Jaideep Malhotra
Shally Gupta
ART-RAINBOW –IVF AGRA
GLOBAL RAINBOW HEALTH CARE
mnmhagra3@gmail.com
Natural Fertility
Reproductive aging
• Fertility reduced with increasing age of
women because of reduction in the number
of primordial follicles
• Biphasic decline: loss of follicles accelerates
around 37 years and precedes menopause by
10-12 years
Faddy & Gosden, 1995
• The chance of not conceiving a first child within
one year increases from under 5% in women in
their early 20s to approximately 30% or over in
the age group of 35 years and older
Abma et al., 1997.
• So, although the majority of women of older age
will obtain the desired pregnancy within a one-
year period, the chance of becoming subfertile
increases ∼6 fold in comparison with very young
women.
Concerns with Infertility treatment
(ART)
Poor ovarian responses
cycle cancellation
poor pregnancy rates
Excessive ovarian responses
risk of ovarian hyperstimulation syndrome
high E2 detrimental to the outcome (Ng et al.,
2000)
Physiology
• Follicular recruitment
• Selection of dominant follicle
• Follicular growth & Estradiol synthesis
• Endometrial proliferation
• Follicular rupture
• Luteal phase changes
Chronology of folliculogenesis in human
ovaries. Notice the time line at the periphery
Preantral period: It takes 300 days for a
recruited primordial to grow and develop to
the class 2/3 (0.4 mm) or cavitation (early
antrum) stage. Atresia can occur in preantral
class 1, 2, and 3 follicles.
Antral period: A class 4 (1 to 2 mm) follicle, if
selected, requires about 50 days to grow and
develop to the preovulatory stage. The
dominant follicle of the cycle appears to be
selected from a cohort of class five follicles,
and it requires about 20 days to develop to
the ovulatory stage. Atresia is common
during the antral period.
gc, number of granulosa cells; d, days
( Hum Reprod 1:81, 1986.)
Evolution of OR TESTS
• Over the past two decades, a number of
so-called ovarian tests have been studied for
their ability to predict outcome of IVF in terms
of oocyte yield and occurrence of pregnancy.
• Some of these tests have become part of the
routine diagnostic procedure for infertility
patients that will undergo assisted
reproductive techniques.
Ovarian Reserve
• OR can be considered normal in conditions
where stimulation with the use of exogenous
gonadotrophins will result in the development
of at least 8–10 follicles and the retrieval of a
corresponding number of healthy oocytes at
follicle puncture
Fasouliotis et al., 2000
Tests for ovarian reserve
Tests of ovarian reserve
Ultrasound markers
1. Ovarian volume
2. Antral follicle
count
3. Ovarian stromal
vascularity
Hormonal
markers
1. Anti-Mullerian
hormone
2. FSH
3. Inhibin
4. estradiol
SOME DYNAMIC TESTS :CC TEST,GAST etc ……….not used clinically
AGE
Age of the woman is a simple way of
obtaining information on the extent of
her OR, in terms of both quantity and
quality
Templeton et al., 1996.
However, in the view of the substantial
variation in the decline of reproductive
capacity with age
Te Velde and Pearson, 2002
.
There is a need to identify women of
relatively young age with clearly
diminished reserve, as well as women
around the mean age at which natural
fertility on average is lost (41 years) but
still with adequate OR.
In clinical terms
• we aim to identify women with a high risk of producing a
poor response to ovarian stimulation and/or a very low
probability of becoming pregnant through IVF,
• as well as those who still produce enough oocytes to have a
good chance of becoming pregnant even if female age is
advanced.
• If it appears possible to identify such categories of women,
• then management could be individualized, for instance by
stimulation dose or treatment scheme adjustments
(Tarlatzis et al., 2003),
• by counselling against initiation of IVF treatment or
pertinent refusal to accept initiation, or by indicating the
necessity of early initiation of treatment before reserve has
diminished too far.
OVARIAN RESERVE
• OR is currently defined as the number and quality
of the follicles left in the ovary at any given time.
An accurate measure of the quantitative OR
would involve the counting of all follicles present
in both ovaries, as is done in post-mortem studies
(Block, 1952).
• For obvious reasons, in OR testing, the true size
of the follicle pool has not been used as the
benchmark for evaluation
Lass et al., 1997a; Lambalk et al., 2004; Lass, 2004; Sharara and Scott,2004
Tests of ovarian reserve
• Ultrasound markers
1. Ovarian volume
2. Antral follicle count
3. Ovarian stromal
vascularity
• Hormonal markers
1. Anti-Mullerian
hormone
2. FSH
3. Inhibin
HOW TO SELECT POOR RESPONDERS
• D–3 FSH > 17 MIU/ML
• D—3 E2 <60pg/ml
• D—3 LH >3mIU/ML
• D—3 Inhibin low<45pg/ml
• Clom chal test FSH on D-10
• AMH and ANTIOV ANTIBODY
• USG Ov vol <3cms
• Antral follicle count and baseline color flows
Basal FSH
Systematic review
Tests for ovarian reserve
Tests for ovarian reserve
Tests for ovarian reserve
RELATION OF AMH WITH OTHER
BIOCHEMICAL PREDICTORS OF OR
Anti-Müllerian hormone
(TGF-β superfamily: Müllerian duct regression in male embryos)
Maximal expression occurs in
preantral and small antral follicles
Expression disappears in maturing
pre-ovulatory follicles (expression
restricted to GCs of the cumulus)
1. Laven et al. J Clin Endocrinol Metab 2004; 89: 318–323; 2. Weenen et al. Mol Hum Reprod 2004;10: 77–83;
3. Cook et al. Fertil Steril 2000; 73: 859–861; 4. La Marca et al. Hum Reprod 2004; 19: 2738–2741;
5. La Marca et al. Hum Reprod 2006; 21: 3103–3107
Tests for ovarian reserve
Possible actions of AMH
Tests for ovarian reserve
WHY AMH STANDS OUT ?
FSH, ESTRADIOL
&INHIBIN B are part
of feedback system→
serum levels are not
independent of each
other.
MOREOVER CHANGES OCCUR LATE
IN REPRODUCTIVE AGEING
PROCESS.
Mean serum AMH levels and the menstrual cycle
La Marca A et al. Hum. Reprod. Update 2010;16:113-130
AMH and the menstrual cycle:
re LH peak
AMH in ovarian reserve
• Serum AMH shows a progressive decrease with age and correlate
well with AFCs, age and FSH (de Vet et all 2002)
• AMH is the only marker of ovarian reserve showing a mean
longitudinal decline over time in younger women(<35yrs) and in
women over 40 years
(Van Rooij et al 2004)
• AMH reflects the continuous decline of the oocyte /Follicle pool
with age
AMH ASSAY
Until recently two different kits were in use.
• DSL assay
• Immunotech-Beckman assay
• Now, the combined single assay in vogue:
AMH GENII enzyme linked immunosorbent
assay kit by Beckman-Coulter, USA.
• 1ng/ml = 7.18 pmol/l in the previous kits.
Parameters of AMH Gen II kit
Kit
Methods
Cat. n.
Materials
Volume
of
sample
Sensitivity
Time of
incubation
Calibration range
AMH Gen II
ELISA
A79765
serum
plasma
20 µl 0,08 ng/ml
2x1h
+30+10min
0,16 – 22,5 ng/ml
Manufactured by: Beckman Coulter, Inc.
Specificity:
There are no cross-reaction with
Inhibin A, Activin A, FSH, LH
0.3
AMH
• Increasing age means a decreasing
AMH level.
• Lower AMH levels at any given time
irrespective of age predicts a poor
response to ART.
• High AMH levels – candidates prone
for OHSS.
Tests for ovarian reserve
AMH and PCOS
• AMH in small follicles is around 3 times
the large follicles (Fanchin HR 2005)
• AMH declines during follicular
maturation
• Peripheral AMH indicates that not only
are there more antral follicles but that
each follicle probably produces more
AMH
• AMH and PCOS :a mountain too high ?
Helen Mason RMA 2010
AMH pre and Post Endometrioma
surgery
• Ovarian endometriomas are commonly seen in women
with endometriosis.
• Laparoscopic surgery remains the treatment of choice,
however new evidence suggests that such surgery
negatively affects the ovarian reserve.
• Unfortunately there are serious flaws in surgical
technique which can bias the results of these studies.
We propose that using only experienced surgeons,
performing the appropriate surgical technique,
without using bipolar electrocoagulation, one can
preserve AMH levels in women undergoing
laparoscopic excision of ovarian endometriomas.
Marshal V et al Obs and Gyn Forum Feb 2014
Two types of phenotypes for POF
• 1. No remaining follicles
(genetic,chem,radiation,surgery etc)
• 2. Abundant follicles(autoimmune)
• Since the serum anti-müllerin hormone (AMH)
level is correlated with the number of
remaining primordial follicles some
researchers believe the above two phenotypes
can be distinguished by measuring serum
AMH levels.
Ultrasound parameters
1. Ovarian volume (Syrop et al., 1995; Lass et al.,
1997)
2. Antral follicle count (Tomas et al., 1997; Chang et
al., 1998a & 1998b; Ng et al., 2000; Fratarelli et
al., 2000; Hsieh et al., 2001; Nahum et al., 2001;
Kupesic and Kurjak, 2002; Popovic- Todorovic et
al., 2003 )
3. Ovarian stromal blood flow (Zaidi et al., 1996;
Engmann et al., 1999; Kupesic and Kurjak, 2002;
Kupesic et al., 2003; Popovic-Todorovic et al.,
2003, Ng et al., 2005 & 2006 )
Ovarian volume π/6 x length x height x width
Total ovarian volume and the volume of the
smallest ovary predictive of peak E2 levels,
no. of oocytes and cycle cancellation
(Syrop et al., 1995)
Mean ovarian volume prior to stimulation
predictive of poor ovarian response
(Lass et al., 1997)
Other tools for volume calculation .....
Antral Follicle count
• Ultrasonographic assessment of the total number of
antral follicles measuring 2–9 mm is a reliable
determinant of ovarian reserve
Jayaprakasan etal
2008.
• The pool of antral follicles comprises pre-antral and
early antral follicles (0.2–2.0 mm) that are largely
gonadotrophin-independent.
• Small antral follicles (1.0–6.0 mm) selectable due to
their responsiveness to gonadotrophins, and larger
antral follicles (>6.0 mm) that are gonadotrophin-
dependent
Gougeon
1989
• At any given timepoint during the menstrual cycle, the
ovaries contain follicles at different developmental
stages and antral follicle counts must be made during
the early follicular phase.
• The total antral follicle count (tAFC) is made by counting
the number of antral follicles measuring 2–9 mm in
both ovaries and can be estimated using two- or three-
dimensional ultrasound
Jayaprakasan et al 2008 .
Antral follicle count (AFC)
• Reflection of primordial follicle pool
• Counting in longitudinal plane and then in
transverse plane
AFC
• Various definitions of AFC
2–5 mm (Chang et al., 1998; Bansci
et al., 2002; Jarvela et al., 2003);
2–8 mm (Sharara and
McClammrock, 2000) and
 2–10 mm (Fratarelli et al., 2000;
Hsieh et al., 2001; Yong et al.,
2003).
• Prediction of the poor response based
on AFC is the same regardless of the
definition used for the size of antral
follicles. (Khairy et al., 2008)
AFC HOW ? AFC WHEN ?
Two-dimensional (2D) or 3-
dimensional (3D) USS
Counting by the 2D-equivalent,
3D multiplanar modes and
3D-rendered inversion mode
3D image displays and
rendering techniques do not
appear to offer any
advantage over a
conventional 2D display in
terms of AFC measurement
reliability. (Jayaprakasan et
al., 2006)
In the early follicular phase for those with
regular cycles or anytime for anovulatory
patients
Moderate interobserver and intercycle
variability of AFC (Hansen et al., 2003)
AFC tells WHAT ?
AFC significantly correlated with
age of women, serum FSH, total ovarian
volume, duration and dosage of
gonadotrophin, serum E2 on the day of hCG
and no. of oocytes
AFC achieved the best predictive value of the
number of oocytes obtained; followed by
basal FSH, body mass index and age of
women.
(Ng et al., HR, 2000)
Interobserver
variability of
AFC
Tend to be greater at
higher AF counts but the
difference is not
significant
Pooled SD (as an
estimate of average
interobserver variability)
was 1.6± 0.5 AFs.
(Hansen et al., 2003)
Pooled SD for
intercycle
variability, up to a
mean AF count of
15 was 3.0±0.3
AFs.
Intercycle variability of AFC
SonoAVC : antral follicle count
Sonography-based Automated Volume Count
Automatically calculates the
number and volume of hypoechoic
structures in a volume dataset.
Can significantly reduce time for
assessment and reporting.
From the calculated volume an average
diameter can be calculated.
It also lists the objects according to
their size
MOST COST EFFECTIVE SINGLE TEST
Advantages of sonoAVC
• Automated volume measurements are in very good
agreement with actual volumes of the assessed
structures or with other validated measurement
methods.
• The technique seems to provide reliable and highly
reproducible results under a variety of conditions.
• Automated measurements take less time than
manual measurements.
Baris Ata ;Togas Tulandi Fertility and Sterility
Volume 97 , Pages 2163-2170, June 2011
AFC Vs ovarian volume
Prediction of pregnancy
Prediction of poor response
• The predictive performance of ovarian
volume toward poor response is clearly
inferior compared with that of AFC.
• AFC may be considered the test of first
choice when estimating quantitative ovarian
reserve before IVF.
(Hendriks et al., 2007)
AFC Vs FSH
• The predictive performance of AFC toward
poor response is significantly better than that
of basal FSH.
• AFC might be considered the test of first
choice in the assessment of ovarian reserve
prior to IVF. (Hendriks et al., 2005)
AFC Vs hormonal markers
AFC
• Result immediately
available
• Moderate interobserver
variability
• Moderate intercycle
variability
• Good correlation with
no. of oocyets obtained
in IVF
Hormonal markers (AMH)
• Results not immediately
available
• No interobserver
variability
• Less intercycle
variability
• Good correlation with
no. of oocytes obtained
in IVF
Ovarian stromal vascularity
• Adequate vascular supply to provide endocrine
and paracrine signals may play a key role in the
regulation of follicle growth
• Normal responders had higher peak systolic
velocity of ovarian stromal vessels than poor
responders (Zaidi et al., 1996; Engmann et al.,
1999)
• Women with RI >0.56 had longer stimulation
duration and lower number of oocytes. (Bassil et
al.,1997)
OVARIAN STROMAL BLOOD FLOW
&
ASSISTED REPRODUCTION
OVARIAN
STROMAL
PSV
GREAT DELIVERY OF
GONADOTROPINS TO
GRANULOSA-THECAL COMPLEX
PRODUCTION
OF GREATER
No OF
FOLLICLES
Ovarian stromal vascularity by
2D power Doppler
• Mean RI was positively correlated with AFC
(r=0.239; P=0.03)
• No difference in ovarian responses between
those with unilateral/bilateral absent ovarian
stromal flow and bilateral ovarian stromal flow
• Ovarian stromal vascularity indices by 2D power
Doppler had no predictive value for the ovarian
response.
(Ng et al., HR, 2005)
Stromal vascularity
• Even with same echogenecity, PCOS has more
stromal flow.
Ovarian stromal vascularity by
3D power Doppler
B (95% CI) Beta R² change P value
AFC 0.421
(0.204, 0.648)
0.329 0.170 <0.001
Age -0.516
(-0.809, -0.224)
-0.299 0.084 0.001
Body mass
index
-0.388
(-0.720, -0.057)
-0.189 0.036 0.022
Number of oocytes obtained
Basal FSH, mean ovarian volume, mean ovarian VI,
FI and VFI were excluded in the equation.
(Ng et al., RBMO, 2006)
Effect of age on ovarian stromal
vascularity in fertile women
< 20 yrs 21-30 yrs 31-40 yrs >40 yrs
Peak systolic
velocity
(cm/sec)
10.35
(8.40-14.00)
10.10
(7.05-15.70
12.10
(8.75-30.10)
11.45
(6.60-56.05)
(Ng et al., HR, 2003)
PCOS
• Ovarian function in
women with PCOS is
poorly understood
• Color Doppler evaluation
of the intraovarian vessels
can give us a fairly good
idea of resistance ovary or
an ovary likely to
hyperstimulate
Stromal flow in PCOS
• This observation may help to explain the excessive
response often seen during gonadotrophin
administration in women with PCOS. We believe
that a quantification study of the vascular flow,
including the VI, FI, and VFI of the entire ovarian
stroma using 3D power Doppler, is more accurate
than the previously reported quantification
analysis using 2D imaging, and may be a new
parameter to assist in the ultrasound diagnosis of
PCOS.
• Basal ovarian stromal blood flow does not differ
between women with PCOS and women with
normal ovulation, good ovarian reserve, and
infertility.
• Moreover, stromal flow has no predictive value, in
these patients, for clinical pregnancy achievement
in an IVF-ET setting
Younis JS et al Fertil steril 2011
low moderate impedance in
the stromal vessels,
a RI of 0.53 or less will signify
chances of OHSS with
gonadotrophin stimulation.
low impedance will deliver a
higher dose of injectable
gonadotrophins to the
granulosa theca complex
Risk factors:
• POLYCYSTIC OVARIAN DISEASE (PCO)
• “PCO” LIKE RESPONSE IN A PREVIOUS
TREATMENT CYCLE
• OHSS OR POSSIBLE SYMPTOMS IN A
PREVIOUS TREATMENT CYCLE
Predicting OHSS
• PCO like ovaries - >24 antral follicles in both ovaries
combined
• High number of medium/large follicles
• - ≥13 follicles ≥11 mm in diameter
• - >11 follicles ≥10 mm in diameter
Perifollicular
blood flows
• Good flows may be a predictor
of good oocyte
• These follicles with good flows
usually yield a competent
oocyte(fertilizable oocyte/M 2
oocyte)
• Currenty clinical utility of
detailed analysis of perifollicular
blood flow is limited
• 3 D blood flow patterns are also
now available and may be of
clinical significance in predicting
pregnancy outcome
PSV > 10 cm/s
COLLECTION RATE
EMBRYO DEVELOPMENT
IMPLANTATION RATES
•RI = 0.50-0.55 - hemodynamic
parameter of follicular growth
•RI = 0.42-0.48 - signifficant
reduction of vascular resistance
in the preovulatory phase
1.
2.
3.
MULTIFOLLICULAR GROWTH
FOLLICULAR PROFILE
• difference between the sum of the points
obtained from each of the follicular rims
and the total number of the follicles
PREOVULATORY
BLOOD FLOW
OOCYTES OBTAINED
FROM HIGH
VASCULARIZED
FOLLICLES
• HIGHER QUALITY
OOCYTES
• HIGHER FERTILIZATION
& IMPLANTATION
RATES
Volume histogram
NORMAL OVARIAN FUNCTION IS CHARACTERIZED BY THE TYPICAL CYCLIC
BLOOD FLOW PATTERN
PERIOVULATORY
BLOOD FLOW
LUTEAL PHASE
REGRESSION
LUTEAL PHASE
MATURATION
ASSESSING OVARIAN RESERVE AND
RESPONSE
Deciding the stimulation protocol
Predictors of ovarian response are enumerated
as:
• Number of antral follicles
• Stromal flow: stromal FI
• Total ovarian stromal area
• Total ovarian volume
Kupesic S et al, Hum Reprod 2002; 17(4):950-55
• ACCURATE VOLUME MEASUREMENT
- FOLLICULAR VOLUME
- ENDOMETRIAL VOLUME
• DETECTION OF THE UTERINE
ABNORMALITIES
• EVALUATION OF THE FALLOPIAN TUBES
Ovarian reserve How to test
•AMH AND AFC
•OR ONLY AFC
thank you
WELCOME TO AGRA FOR THE INSUOG
CONGRESS 3-5 MAY
IF YOU WISH TO KNOW ALL ABOUT
ULTRASOUND FROM EXPERTS …U HAVE A
DATE WITH US

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Tests for ovarian reserve

  • 1. NAME : PROF.NARENDRA MALHOTRA DESIGNATION: PROF. UNIVERSITY SARAJEVO SCHOOL OF SCIENCE & TECHNOLOGY (SSST) V.P. WAPM(WORLD ASSOCIATION OF PRENATAL MEDICINE) PRESIDENT ISPAT (2017-2019) PAST PRESIDENT ISAR ( 2016 – 2017) SEC GEN SAFOG (2015-2019) PAST PRESIDENT FOGSI(2008) MANAGING DIRECTOR GLOBAL RAINBOW HEALTH CARE, AGRA DIRECTOR ART-RAINBOW –IVF (AGRA & DELHI) CITY: AGRA, INDIA AFFILIATIONS: M.D., F.I.C.O.G., F.I.C.M.C.H, F.R.C.O.G.,F.I.C.S., F.M.A.S., A.F.I.A.P. AWARDS: BEST CITIZENS OF INDIA AWARD, MAN OF THE YEAR AWARD, CORION AWARD, AWARDED BEST PAPER AND BEST POSTER AT FOGSI : 5 TIMES, ETHICON FELLOWSHIP, AOFOG YOUNG GYN. AWARD, PUBLICATIONS: JEFFCOATE’S PRINCIPLES OF GYNECOLOGY, 9TH EDITION, OPERATIVE OBSTETRICS AND GYNECOLOGY 2ND EDITION, AN INTRODUCTION TO GENETICS & FETAL MEDICINE, 2ND EDITION, FOGSI’S PRINCIPLES & PRACTICE OF OBSTETRICS & GYNECOLOGY FOR PG’S 3RD EDITION, DONALD BOOK ON OBSTETRIC PRACTICAL PROBLEMS MANUAL 2ND EDITION, ULTRASOUND IN OBSTETRICS & GYNAECOLOGY 5TH EDITION, EDITOR 25 BOOKS, MANY CHAPTERS, ON EDITORIAL BOARD OF MANY JOURNALS, EDITOR OF SERIES OF STEP BY STEP BOOKS. OVER 35 PUBLISHED PAPERS, 165 CHAPTERS, 509 PRESENTATIONS, 28 ORATIONS. SPECIAL INTRESTS: SPECIAL INTEREST IN HIGH RISK OBS., ULTRASOUND, ART & GENETICS.
  • 2. TESTS FOR OVARIAN RESERVE Narendra Malhotra Neharika Malhotra Jaideep Malhotra Shally Gupta ART-RAINBOW –IVF AGRA GLOBAL RAINBOW HEALTH CARE mnmhagra3@gmail.com
  • 4. Reproductive aging • Fertility reduced with increasing age of women because of reduction in the number of primordial follicles • Biphasic decline: loss of follicles accelerates around 37 years and precedes menopause by 10-12 years Faddy & Gosden, 1995
  • 5. • The chance of not conceiving a first child within one year increases from under 5% in women in their early 20s to approximately 30% or over in the age group of 35 years and older Abma et al., 1997. • So, although the majority of women of older age will obtain the desired pregnancy within a one- year period, the chance of becoming subfertile increases ∼6 fold in comparison with very young women.
  • 6. Concerns with Infertility treatment (ART) Poor ovarian responses cycle cancellation poor pregnancy rates Excessive ovarian responses risk of ovarian hyperstimulation syndrome high E2 detrimental to the outcome (Ng et al., 2000)
  • 7. Physiology • Follicular recruitment • Selection of dominant follicle • Follicular growth & Estradiol synthesis • Endometrial proliferation • Follicular rupture • Luteal phase changes
  • 8. Chronology of folliculogenesis in human ovaries. Notice the time line at the periphery Preantral period: It takes 300 days for a recruited primordial to grow and develop to the class 2/3 (0.4 mm) or cavitation (early antrum) stage. Atresia can occur in preantral class 1, 2, and 3 follicles. Antral period: A class 4 (1 to 2 mm) follicle, if selected, requires about 50 days to grow and develop to the preovulatory stage. The dominant follicle of the cycle appears to be selected from a cohort of class five follicles, and it requires about 20 days to develop to the ovulatory stage. Atresia is common during the antral period. gc, number of granulosa cells; d, days ( Hum Reprod 1:81, 1986.)
  • 9. Evolution of OR TESTS • Over the past two decades, a number of so-called ovarian tests have been studied for their ability to predict outcome of IVF in terms of oocyte yield and occurrence of pregnancy. • Some of these tests have become part of the routine diagnostic procedure for infertility patients that will undergo assisted reproductive techniques.
  • 10. Ovarian Reserve • OR can be considered normal in conditions where stimulation with the use of exogenous gonadotrophins will result in the development of at least 8–10 follicles and the retrieval of a corresponding number of healthy oocytes at follicle puncture Fasouliotis et al., 2000
  • 12. Tests of ovarian reserve Ultrasound markers 1. Ovarian volume 2. Antral follicle count 3. Ovarian stromal vascularity Hormonal markers 1. Anti-Mullerian hormone 2. FSH 3. Inhibin 4. estradiol SOME DYNAMIC TESTS :CC TEST,GAST etc ……….not used clinically
  • 13. AGE Age of the woman is a simple way of obtaining information on the extent of her OR, in terms of both quantity and quality Templeton et al., 1996. However, in the view of the substantial variation in the decline of reproductive capacity with age Te Velde and Pearson, 2002 . There is a need to identify women of relatively young age with clearly diminished reserve, as well as women around the mean age at which natural fertility on average is lost (41 years) but still with adequate OR.
  • 14. In clinical terms • we aim to identify women with a high risk of producing a poor response to ovarian stimulation and/or a very low probability of becoming pregnant through IVF, • as well as those who still produce enough oocytes to have a good chance of becoming pregnant even if female age is advanced. • If it appears possible to identify such categories of women, • then management could be individualized, for instance by stimulation dose or treatment scheme adjustments (Tarlatzis et al., 2003), • by counselling against initiation of IVF treatment or pertinent refusal to accept initiation, or by indicating the necessity of early initiation of treatment before reserve has diminished too far.
  • 15. OVARIAN RESERVE • OR is currently defined as the number and quality of the follicles left in the ovary at any given time. An accurate measure of the quantitative OR would involve the counting of all follicles present in both ovaries, as is done in post-mortem studies (Block, 1952). • For obvious reasons, in OR testing, the true size of the follicle pool has not been used as the benchmark for evaluation Lass et al., 1997a; Lambalk et al., 2004; Lass, 2004; Sharara and Scott,2004
  • 16. Tests of ovarian reserve • Ultrasound markers 1. Ovarian volume 2. Antral follicle count 3. Ovarian stromal vascularity • Hormonal markers 1. Anti-Mullerian hormone 2. FSH 3. Inhibin
  • 17. HOW TO SELECT POOR RESPONDERS • D–3 FSH > 17 MIU/ML • D—3 E2 <60pg/ml • D—3 LH >3mIU/ML • D—3 Inhibin low<45pg/ml • Clom chal test FSH on D-10 • AMH and ANTIOV ANTIBODY • USG Ov vol <3cms • Antral follicle count and baseline color flows
  • 22. RELATION OF AMH WITH OTHER BIOCHEMICAL PREDICTORS OF OR
  • 23. Anti-Müllerian hormone (TGF-β superfamily: Müllerian duct regression in male embryos) Maximal expression occurs in preantral and small antral follicles Expression disappears in maturing pre-ovulatory follicles (expression restricted to GCs of the cumulus) 1. Laven et al. J Clin Endocrinol Metab 2004; 89: 318–323; 2. Weenen et al. Mol Hum Reprod 2004;10: 77–83; 3. Cook et al. Fertil Steril 2000; 73: 859–861; 4. La Marca et al. Hum Reprod 2004; 19: 2738–2741; 5. La Marca et al. Hum Reprod 2006; 21: 3103–3107
  • 27. WHY AMH STANDS OUT ? FSH, ESTRADIOL &INHIBIN B are part of feedback system→ serum levels are not independent of each other. MOREOVER CHANGES OCCUR LATE IN REPRODUCTIVE AGEING PROCESS.
  • 28. Mean serum AMH levels and the menstrual cycle La Marca A et al. Hum. Reprod. Update 2010;16:113-130 AMH and the menstrual cycle: re LH peak
  • 29. AMH in ovarian reserve • Serum AMH shows a progressive decrease with age and correlate well with AFCs, age and FSH (de Vet et all 2002) • AMH is the only marker of ovarian reserve showing a mean longitudinal decline over time in younger women(<35yrs) and in women over 40 years (Van Rooij et al 2004) • AMH reflects the continuous decline of the oocyte /Follicle pool with age
  • 30. AMH ASSAY Until recently two different kits were in use. • DSL assay • Immunotech-Beckman assay • Now, the combined single assay in vogue: AMH GENII enzyme linked immunosorbent assay kit by Beckman-Coulter, USA. • 1ng/ml = 7.18 pmol/l in the previous kits.
  • 31. Parameters of AMH Gen II kit Kit Methods Cat. n. Materials Volume of sample Sensitivity Time of incubation Calibration range AMH Gen II ELISA A79765 serum plasma 20 µl 0,08 ng/ml 2x1h +30+10min 0,16 – 22,5 ng/ml Manufactured by: Beckman Coulter, Inc. Specificity: There are no cross-reaction with Inhibin A, Activin A, FSH, LH
  • 32. 0.3
  • 33. AMH • Increasing age means a decreasing AMH level. • Lower AMH levels at any given time irrespective of age predicts a poor response to ART. • High AMH levels – candidates prone for OHSS.
  • 35. AMH and PCOS • AMH in small follicles is around 3 times the large follicles (Fanchin HR 2005) • AMH declines during follicular maturation • Peripheral AMH indicates that not only are there more antral follicles but that each follicle probably produces more AMH • AMH and PCOS :a mountain too high ? Helen Mason RMA 2010
  • 36. AMH pre and Post Endometrioma surgery • Ovarian endometriomas are commonly seen in women with endometriosis. • Laparoscopic surgery remains the treatment of choice, however new evidence suggests that such surgery negatively affects the ovarian reserve. • Unfortunately there are serious flaws in surgical technique which can bias the results of these studies. We propose that using only experienced surgeons, performing the appropriate surgical technique, without using bipolar electrocoagulation, one can preserve AMH levels in women undergoing laparoscopic excision of ovarian endometriomas. Marshal V et al Obs and Gyn Forum Feb 2014
  • 37. Two types of phenotypes for POF • 1. No remaining follicles (genetic,chem,radiation,surgery etc) • 2. Abundant follicles(autoimmune) • Since the serum anti-müllerin hormone (AMH) level is correlated with the number of remaining primordial follicles some researchers believe the above two phenotypes can be distinguished by measuring serum AMH levels.
  • 38. Ultrasound parameters 1. Ovarian volume (Syrop et al., 1995; Lass et al., 1997) 2. Antral follicle count (Tomas et al., 1997; Chang et al., 1998a & 1998b; Ng et al., 2000; Fratarelli et al., 2000; Hsieh et al., 2001; Nahum et al., 2001; Kupesic and Kurjak, 2002; Popovic- Todorovic et al., 2003 ) 3. Ovarian stromal blood flow (Zaidi et al., 1996; Engmann et al., 1999; Kupesic and Kurjak, 2002; Kupesic et al., 2003; Popovic-Todorovic et al., 2003, Ng et al., 2005 & 2006 )
  • 39. Ovarian volume π/6 x length x height x width Total ovarian volume and the volume of the smallest ovary predictive of peak E2 levels, no. of oocytes and cycle cancellation (Syrop et al., 1995) Mean ovarian volume prior to stimulation predictive of poor ovarian response (Lass et al., 1997)
  • 40. Other tools for volume calculation .....
  • 41. Antral Follicle count • Ultrasonographic assessment of the total number of antral follicles measuring 2–9 mm is a reliable determinant of ovarian reserve Jayaprakasan etal 2008. • The pool of antral follicles comprises pre-antral and early antral follicles (0.2–2.0 mm) that are largely gonadotrophin-independent. • Small antral follicles (1.0–6.0 mm) selectable due to their responsiveness to gonadotrophins, and larger antral follicles (>6.0 mm) that are gonadotrophin- dependent Gougeon 1989 • At any given timepoint during the menstrual cycle, the ovaries contain follicles at different developmental stages and antral follicle counts must be made during the early follicular phase. • The total antral follicle count (tAFC) is made by counting the number of antral follicles measuring 2–9 mm in both ovaries and can be estimated using two- or three- dimensional ultrasound Jayaprakasan et al 2008 .
  • 42. Antral follicle count (AFC) • Reflection of primordial follicle pool • Counting in longitudinal plane and then in transverse plane
  • 43. AFC • Various definitions of AFC 2–5 mm (Chang et al., 1998; Bansci et al., 2002; Jarvela et al., 2003); 2–8 mm (Sharara and McClammrock, 2000) and  2–10 mm (Fratarelli et al., 2000; Hsieh et al., 2001; Yong et al., 2003). • Prediction of the poor response based on AFC is the same regardless of the definition used for the size of antral follicles. (Khairy et al., 2008)
  • 44. AFC HOW ? AFC WHEN ? Two-dimensional (2D) or 3- dimensional (3D) USS Counting by the 2D-equivalent, 3D multiplanar modes and 3D-rendered inversion mode 3D image displays and rendering techniques do not appear to offer any advantage over a conventional 2D display in terms of AFC measurement reliability. (Jayaprakasan et al., 2006) In the early follicular phase for those with regular cycles or anytime for anovulatory patients Moderate interobserver and intercycle variability of AFC (Hansen et al., 2003)
  • 45. AFC tells WHAT ? AFC significantly correlated with age of women, serum FSH, total ovarian volume, duration and dosage of gonadotrophin, serum E2 on the day of hCG and no. of oocytes AFC achieved the best predictive value of the number of oocytes obtained; followed by basal FSH, body mass index and age of women. (Ng et al., HR, 2000)
  • 46. Interobserver variability of AFC Tend to be greater at higher AF counts but the difference is not significant Pooled SD (as an estimate of average interobserver variability) was 1.6± 0.5 AFs. (Hansen et al., 2003) Pooled SD for intercycle variability, up to a mean AF count of 15 was 3.0±0.3 AFs. Intercycle variability of AFC
  • 47. SonoAVC : antral follicle count Sonography-based Automated Volume Count Automatically calculates the number and volume of hypoechoic structures in a volume dataset. Can significantly reduce time for assessment and reporting. From the calculated volume an average diameter can be calculated. It also lists the objects according to their size MOST COST EFFECTIVE SINGLE TEST
  • 48. Advantages of sonoAVC • Automated volume measurements are in very good agreement with actual volumes of the assessed structures or with other validated measurement methods. • The technique seems to provide reliable and highly reproducible results under a variety of conditions. • Automated measurements take less time than manual measurements. Baris Ata ;Togas Tulandi Fertility and Sterility Volume 97 , Pages 2163-2170, June 2011
  • 49. AFC Vs ovarian volume Prediction of pregnancy Prediction of poor response • The predictive performance of ovarian volume toward poor response is clearly inferior compared with that of AFC. • AFC may be considered the test of first choice when estimating quantitative ovarian reserve before IVF. (Hendriks et al., 2007)
  • 50. AFC Vs FSH • The predictive performance of AFC toward poor response is significantly better than that of basal FSH. • AFC might be considered the test of first choice in the assessment of ovarian reserve prior to IVF. (Hendriks et al., 2005)
  • 51. AFC Vs hormonal markers AFC • Result immediately available • Moderate interobserver variability • Moderate intercycle variability • Good correlation with no. of oocyets obtained in IVF Hormonal markers (AMH) • Results not immediately available • No interobserver variability • Less intercycle variability • Good correlation with no. of oocytes obtained in IVF
  • 52. Ovarian stromal vascularity • Adequate vascular supply to provide endocrine and paracrine signals may play a key role in the regulation of follicle growth • Normal responders had higher peak systolic velocity of ovarian stromal vessels than poor responders (Zaidi et al., 1996; Engmann et al., 1999) • Women with RI >0.56 had longer stimulation duration and lower number of oocytes. (Bassil et al.,1997)
  • 53. OVARIAN STROMAL BLOOD FLOW & ASSISTED REPRODUCTION OVARIAN STROMAL PSV GREAT DELIVERY OF GONADOTROPINS TO GRANULOSA-THECAL COMPLEX PRODUCTION OF GREATER No OF FOLLICLES
  • 54. Ovarian stromal vascularity by 2D power Doppler • Mean RI was positively correlated with AFC (r=0.239; P=0.03) • No difference in ovarian responses between those with unilateral/bilateral absent ovarian stromal flow and bilateral ovarian stromal flow • Ovarian stromal vascularity indices by 2D power Doppler had no predictive value for the ovarian response. (Ng et al., HR, 2005)
  • 55. Stromal vascularity • Even with same echogenecity, PCOS has more stromal flow.
  • 56. Ovarian stromal vascularity by 3D power Doppler B (95% CI) Beta R² change P value AFC 0.421 (0.204, 0.648) 0.329 0.170 <0.001 Age -0.516 (-0.809, -0.224) -0.299 0.084 0.001 Body mass index -0.388 (-0.720, -0.057) -0.189 0.036 0.022 Number of oocytes obtained Basal FSH, mean ovarian volume, mean ovarian VI, FI and VFI were excluded in the equation. (Ng et al., RBMO, 2006)
  • 57. Effect of age on ovarian stromal vascularity in fertile women < 20 yrs 21-30 yrs 31-40 yrs >40 yrs Peak systolic velocity (cm/sec) 10.35 (8.40-14.00) 10.10 (7.05-15.70 12.10 (8.75-30.10) 11.45 (6.60-56.05) (Ng et al., HR, 2003)
  • 58. PCOS • Ovarian function in women with PCOS is poorly understood • Color Doppler evaluation of the intraovarian vessels can give us a fairly good idea of resistance ovary or an ovary likely to hyperstimulate
  • 59. Stromal flow in PCOS • This observation may help to explain the excessive response often seen during gonadotrophin administration in women with PCOS. We believe that a quantification study of the vascular flow, including the VI, FI, and VFI of the entire ovarian stroma using 3D power Doppler, is more accurate than the previously reported quantification analysis using 2D imaging, and may be a new parameter to assist in the ultrasound diagnosis of PCOS. • Basal ovarian stromal blood flow does not differ between women with PCOS and women with normal ovulation, good ovarian reserve, and infertility. • Moreover, stromal flow has no predictive value, in these patients, for clinical pregnancy achievement in an IVF-ET setting Younis JS et al Fertil steril 2011 low moderate impedance in the stromal vessels, a RI of 0.53 or less will signify chances of OHSS with gonadotrophin stimulation. low impedance will deliver a higher dose of injectable gonadotrophins to the granulosa theca complex
  • 60. Risk factors: • POLYCYSTIC OVARIAN DISEASE (PCO) • “PCO” LIKE RESPONSE IN A PREVIOUS TREATMENT CYCLE • OHSS OR POSSIBLE SYMPTOMS IN A PREVIOUS TREATMENT CYCLE
  • 61. Predicting OHSS • PCO like ovaries - >24 antral follicles in both ovaries combined • High number of medium/large follicles • - ≥13 follicles ≥11 mm in diameter • - >11 follicles ≥10 mm in diameter
  • 62. Perifollicular blood flows • Good flows may be a predictor of good oocyte • These follicles with good flows usually yield a competent oocyte(fertilizable oocyte/M 2 oocyte) • Currenty clinical utility of detailed analysis of perifollicular blood flow is limited • 3 D blood flow patterns are also now available and may be of clinical significance in predicting pregnancy outcome
  • 63. PSV > 10 cm/s COLLECTION RATE EMBRYO DEVELOPMENT IMPLANTATION RATES
  • 64. •RI = 0.50-0.55 - hemodynamic parameter of follicular growth •RI = 0.42-0.48 - signifficant reduction of vascular resistance in the preovulatory phase
  • 66. MULTIFOLLICULAR GROWTH FOLLICULAR PROFILE • difference between the sum of the points obtained from each of the follicular rims and the total number of the follicles
  • 68. OOCYTES OBTAINED FROM HIGH VASCULARIZED FOLLICLES • HIGHER QUALITY OOCYTES • HIGHER FERTILIZATION & IMPLANTATION RATES
  • 70. NORMAL OVARIAN FUNCTION IS CHARACTERIZED BY THE TYPICAL CYCLIC BLOOD FLOW PATTERN PERIOVULATORY BLOOD FLOW LUTEAL PHASE REGRESSION LUTEAL PHASE MATURATION
  • 71. ASSESSING OVARIAN RESERVE AND RESPONSE Deciding the stimulation protocol
  • 72. Predictors of ovarian response are enumerated as: • Number of antral follicles • Stromal flow: stromal FI • Total ovarian stromal area • Total ovarian volume Kupesic S et al, Hum Reprod 2002; 17(4):950-55
  • 73. • ACCURATE VOLUME MEASUREMENT - FOLLICULAR VOLUME - ENDOMETRIAL VOLUME • DETECTION OF THE UTERINE ABNORMALITIES • EVALUATION OF THE FALLOPIAN TUBES
  • 74. Ovarian reserve How to test •AMH AND AFC •OR ONLY AFC
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