1. Poor and hyper responders:
biomarkers management, strategies
C. Alviggi
University “Federico II”, Naples, Italy

2. Classical approach to OS is based on:
– Demographics and anthropometrics (Age, BMI, Race)
– Hormonal basal levels (FSH, LH, Estradiol)
– Health status
– Cause of Infertility
– Years on Infertility
– Previous IVF cycles
Last years: Individualized treatments
- reduce cancellations due to poor response
- reduce cancellation - hospitalization for OHSS
- reduce multiple pregnancies
- minimize stimulation burdens
- while increasing the chances of achieving pregnancy
-
Nineties: Main variables associated with OS

3. Maternal age remains the best predictor of outcome
of IVF

4. How can we use hormonal,
functional and genetic biomarkers?
AGE
MARKERS OF OVARIAN RESERVE
Hormonal Biomarkers:
FSH, Inhibin-B, AMH
Functional markers:
Antral Follicle Count (AFC)
Genetic Biomarkers:
Single Nucleotide Polymorphisms for FSH-
R/LH/LH-R/E2-R/AMH-R

5. La Marca et al., Hum Reprod 2009
AMH plays a key role in folliculogenesis
AMH levels are correlated with the number of
follicles at Gn independent stage AMH inhibits
• FSH mediated granulosa
proliferation
• Follicular growth
• Aromatase activity

6. Smeenk et al., Fertil Steril, 2007
AMH was found to be predictive of the number of oocytes and the
number of embryos, but not of embryo quality or the chance of
pregnancy
CAN AMH DIRECTLY PREDICT LIVE BIRTH RATE?
AMH for counselling purpose
CAN AMH DIRECTLY PREDICT ANEUPLOIDIES?
CAN AMH DIRECTLY PREDICT OHSS?

7. AMH models are not robust enough to counsel patients in
terms of chance of live birth
La Marca et al., Reprod Biomed Online 2011; Khader et al., Jornal of Ovarian Research, 2013
Area under ROC curve: 0.6639

8. Abnormal serum FSH and AMH correlated with rate of
embryonic aneuploidy
A Prospective cohort trial
372 IVF patients undergoing blastocyst biopsy and aneuploidy SCREENING
Group 1: normal ovarian reserve (n = 279)
Group 2: diminished ovarian reserve with day 2-3 FSH >10
IU/mL, AMH ≤1 ng/mL or both (n = 93)
RESULTS
Group 2 (diminished ovarian reserve) had a higher percentage of aneuploid
blastocysts (66% compared with 51.7%; P <0.05
The highest percentage of aneuploid blastocysts was in woman with
abnormal FSH and AMH (77.2%)
Katz et al., Obstet Gynecol., 2013

9. AMH is an accurate predictor of excessive response to
ovarian hyperstimulation
Broer et al., Hum Reprod Update, 2011

10. AMH for choosing protocol?
GnRH – Antagonist Protocol:
• Normal oocytes yield
• Very low excess response
• Low embryo cryopreservation
• High / maintained fresh CPR
GnRH Agonist Protocol:
• Very low cancellation of OPU &
OHSS
Antagonist Strategy
• Short stimulation
• Moderate cancellation
High
Responders
(150 IU)
Normal
Respondes
(150-225
IU)
Poor
Respondes
(300 IU)
S
A
F
E
&
E
F
F
E
C
T
I
V
E
I
N
D
I
V
I
D
U
A
L
I
Z
E
D
C
O
S
>15 pmol/L
> 2.1 ng/mL
5 – 15 pmol/L
0.7-2.1 ng/mL
<5 pmol/L
< 0.7 ng/mL
No benefit from higher FSH
dose
Reduced treatment
burdenAlviggi, Humaidan and Ezcurra, Reproductive Biology and Endocrinology, 2012, Adapted from Nelson, et al. Human Reproduction, 2009.

11. La Marca et al., BJOG, 2012
Age (years)
AMH (ng/ml)
Dosegram
FSH starting dose (IU/day)
FSH (IU/I)
A nomogram for the calculation of FSH starting dose based
of age, AMH and basal FSH

12. EFFECTIVE IN PREDICTING OVARIAN RESPONSE (POOR AND
HYPER)
AMH in the clinical IVF practice
NOT SUFFICIENT EVIDENCE FOR COUNSELLING ABOUT LIVE
BIRTH RATE, EMBRYONIC ANEUPLOIDIES
USEFUL IN CHOOSING PROTOCOLS AND REGIMENS; INCLUDING
USE OF GnRH-ANTAGONISTS AND r-hFSH DOSE (RCTs
REQUIRED)

13. Ultrasound determination of AFC is a useful technique for the
prediction of ovarian response. AFC did not predict the chance of
pregnancy, pregnancy loss, or live birth (retrospective analysis of
1049)
CAN AFC DIRECTLY PREDICT LIVE BIRTH RATE?
AFC for counselling purpose
Hsu et al., Fertil Steril, 2011
CAN AFC DIRECTLY PREDICT OHSS?

14. AFC show significant association with live
birth rate
AFC (n)
A retrospective study
2495 cycles of ICSI
Lukaszuk et al., European Journal of Obstetric and Gynecology and
Reproductive Biology, 2013

15. Broer et al., Hum Reprod Update, 2011
AFC is an accurate predictor of excessive response to
ovarian hyperstimulation

16. AFC for choosing protocols?
Nomogram for the calculation of FSH starting dose based of age, AFC and bFSH
Age (years)
AFC (n)
Dosegram
FSH starting dose (IU/day)
FSH (IU/I)
La Marca et al., Journal of Ovarian Research, 2013

17. EFFECTIVE IN PREDICTING OVARIAN RESPONSE (POOR – HYPER)
AFC in the clinical IVF practice
NOT SUFFICIENT EVIDENCE FOR COUNSELLING ABOUT LIVE BIRTH
RATE
POTENTIALLY USEFUL IN CHOOSING PROTOCOLS?

18. At least two of the following three features must be present:
• Advanced maternal age (≥40 years) or any other risk factor for
POR (Turner syndrome, X-fragile mutations, hystory of chemotherapy etc.)
• A previous poor ovarian response (POR) (≤3 oocytes with a conventional
stimulation protocol)
• An abnormal ovarian reserve test (i.e., AFC 5–7 follicles or
AMH 0.5–1.1 ng/ml)
o Two episodes of POR after maximal stimulation are sufficient to define a patient as a
poor responder
o Patients over 40 years age with an abnormal ovarian reserve test should be more
properly defined as expected poor PORs patient
ESHRE consensus on the definition of “poor response” to ovarian stimulation for
in vitro fertilization: the Bologna criteria
A.P. Ferraretti, A. La Marca, B.C.J.M. Fauser, B. Tarlatzis, G. Nargunds and L. Gianaroli on behalf
of the ESHRE working group on Poor Ovarian Response Definition

19. Hypo-response to r-hFSH
 Young, normogonadotrophic women: normal ovarian
reserve and apparently normal response at OS (5 or
more oocytes retrieved – estradiol levels >1000 pg/mL)
BUT…
Increase in the cumulative FSH dose (i.e.
>2500-3000 IU) and in the stimulation length
(hypo-sensitivity to FSH)
De Placido, et al. Hum Reprod 2001; Clin Endocrinol 2004; Hum Reprod 2005; Drugs 2008.
Ferraretti, et al. Fertil Steril 2004. Kailasam, et al. Hum Reprod 2004.
Alviggi, et al. RBMOnline 2006; RBMOnline 2009; Reprod Biol Endocrinol 2009; 2011.
Devroey, et al. Hum Reprod Update 2009 (EVAR) Workshop Group 2008.

20. The cumulative FSH consumption
is higher in carriers of v-betaLH
v-betaLH homozygosis (n = 3); v-betaLH
heterozygosis (n = 21); LH wild type (n = 196). r-hFSH: recombinant human follicle-stimulating hormone; LH
luteinizing hormone; v-betaLH: variant
beta subunit luteinizing hormone.
Alviggi et al. Reproductive Biology and Endocrinology, 2013

21. Greb, et al. JCEM, 2005; Gromoll & Simoni TEM 2005.
Asn/Asn Asn/Ser Ser/Ser
0
10
20
30
40
50
n=46 n=72 n=43
FSHampoules(n)
*
*
p < 0.05
*
Asn/Asn Asn/Ser Ser/Ser
0.0
2.5
5.0
7.5
10.0
n=46 n=72 n=43
*
*
p < 0.05
FSH(IU/l)
FSH-R: Ser680 genotype and sensitivity to FSH
Physiological menstrual cycle:
higher serum FSH is in Ser680 carriers
-25 -20 -15 -10 -5 0
0
5
10
15
Asn/Asn
Ser/Ser
menstruation
day relative to midcycle LH peak
FSH(IU/L)
FSH receptor genotype and
ovarian response to FSH
Women with Ser/Ser have normal or increased AFC
But…
During COS they show hypo-sensitivity to FSH and
require higher FSH doses!
Perez Mayorga, et al. 2000; Sudo, et al. 2002;
Choi, et al. 2004; Falconer, et al. 2005.

22. Conclusions 1
Lessons from last decades
 Same age, BMI and hormonal profile: same protocols
 Same age, BMI and hormonal profile: different ovarian reserve
(different strategies, analogues, FSH starting dose)
 Same ovarian reserve: different sensitivity to FSH and LH
(different FSH starting dose and eventual LH supplementation)
Nineties
Nowadays

23. Patients are counseled appropriately, have realistic expectations of the outcome
of their ovarian stimulation and clinicians can choose the optimal stimulation
strategy even in that very first treatment cycle
That AFC and AMH can predict ovarian response accurately enables clinicians and
thereby patients to be informed about all these critical steps
AMH and AFC: the two
faces of the moon
Nelson
Fertil Steril, 2013
Conclusions 2
Markers of ovarian reserve are crucial…

24. combination between hormonal, functional and genetic
biomarkers to secure the right treatment for the right patient
– Normal genetic profile for FSH-R LH/LH-R with very low AMH/AFC, no
dose will compensate
– Bad genetic profile for FSH-R with normal AMH/AFC, increase the dose
of FSH
– Bad genetic profile for LH/LH-R with normal AMH/AFC, increase dose
of FSH and add LH
Conclusions 3
… But should be integrated with genetics