Fertilization and
Unexplained Failure
Catherine Combelles, Ph.D.
Faculty, Biology Department
Middlebury College
Middlebury, Vermont U.S.A
Disclosure: I have no commercial relationships, nor am I engaged in other activities
that might be perceived as a potential conflict of interest
Learning Objectives
 To highlight the need to focus increased attention on
cases of fertilization and unexplained failures
 To define the types of failures and present potential
etiologies (oocyte- or sperm-borne defects)
 To discuss a few select informative case reports
 To propose recommendations for early interventions
and management strategies
The Types of Failures
= unavailability of embryos from failures prior to and
during fertilization
~ Despite unremarkable cycles and ‘normal’ oocytes/sperm
~ Partly-explained or not yet explained
 How defined?
 ~20-30% failure
routinely
 Elevated
 100% (total fertilization failure, TFF)
‘rarely’
The Incidence of TFFs
Cycle
Type
Incidence
Study Design
Study
IVF
5% (n=206)
RCT, >20% past fert.,
non-male factor
Bhattacharya et al., 2001
IVF
11% (n=70)
(n 70)
Unexplained infertility,
mild endo.
Ruiz et al., 1997
IVF
16% (n=320)
Normal sperm
Barlow et al., 1990
ICSI
2% (n=209)
RCT, >20% past fert.,
non-male factor
Bhattacharya et al., 2001
ICSI
1.3% (n=1779)
Various diagnoses
Esfandiari et al., 2005
ICSI
3% (n=2732)
Various diagnoses
Liu et al., 1995
Various diagnoses
Flaherty et al., 1998
ICSI
3% (n=1343)
Recurring Cases of TFFs
 29% likelihood of recurrence with IVF (Barlow et al., 1990)
 3/33 couples with recurrence with ICSI (Flaherty et al., 1998)
 Case reports/series, e.g.:
 IVF+sperm donor: 3 patients (Ezra et al., 1992)
 ICSI: 1 patient (Rosenbusch and Schneider, 1999)
 IVF+ICSI: 2 patients (Combelles et al., 2010; 2011)
The Poignancy of Failure Cases
Often unpredictable
Canceled cycles
Loss of time and resources with perhaps
unnecessarily repeated attempts
Distress from all parties involved
Identify specific defects
Understand underlying causes
Implement prognosis tools
Devise treatment strategies
Counsel/manage patients
How May Cases Present Themselves?
Light Microscopy at Fertilization Check
1 PN
0 PN
≥ 3PN
1 or 2 PB
1 or 2 PB
Other
1 or 2 PB
2 PN, 2 PB
No further progression
What Further Defect(s) May Be Identified?
Fluorescence Microscopy of Failed Fertilized Oocytes
pb
pb
 Technical considerations
 Diagnostic and clinical utilities
pb
 Other types of analyses
pb
Machtinger et al.
others
Potential EtiologiesDuring Fertilization
6-11% (IVF/ICSI)
20-23% (IVF/ICSI)
PN
formation,
dynamics
6% (IVF)
sperm aster
formation
57% (IVF)
sperm
penetration
activation
sperm
decondensation
8% (IVF/ICSI)
40% (ICSI)
Asch et al., 1995
Rawe et al., 2000
Potential EtiologiesOocyte Determinants
Cellular and molecular elements necessary for
full fertilization competence
Dominant
follicle
Ovulation
Oocyte DevelopmentA Long Journey to Fertilizability
Developmental Transitions During Oocyte Development
Potential EtiologiesSperm Determinants
acrosome
nucleus
centriole
tail
other factors (protamine, mRNA’s….)
oocyte-activating factor
Different clinical relevancies with IVF vs. ICSI
Post-ovulatory aging
Window of maximal
fertilizability
Maturation
Growth
Growing
antral
follicles
Prematuration
Early
atretic
follicles
Post-maturation
Fertilization Potentiall
in oviduct or laboratory
Intrinsic Failure Cases Identified To Date
Oocyte Maturation Blocks
Spermiogenesis
and/or Centrosome
Defect
+
GV
M-I
M-II
Spontaneous Activation
X
Oocyte Activation Failure
(sperm or oocyte defect)
2 PN, 2 PB
Combelles, 2012
Case Series #1: Maturation Arrest Blocks
Types and Incidence
GV
M-I
M-II
Oocyte Maturation
Type of Arrest
Number
of
Patients
Number of
Reports
Diagnostic Evaluations
Beyond Light
Microscopy
GV
4
3
0/3
M-I
17
10
7/10
Mixed (GV+M-I)
9
4
1/4
Maturation Arrest Blocks
Diagnostic Efforts
• M-I arrest cases with
differing spindle &
chromosome abnormalities
• Need for timely evaluation
Combelles et al., 2003; Heindryckx et al., 2011
Maturation Arrest Blocks
Treatment Attempt
Heindryckx et al., 2011
Pilot Study
Patient Case 1
Donated M-I
Patient Case 2
2 Attempts
M-I arrested
(n=25)
30h IVM
Iono.
(n=15)
M-I
Ca2+ ionophore
+ 16h IVM
(n=13)
M-II
(n=3)
+PB (n=4)
Abn. (n=9)
AOA
+ ICSI
M-I arrested
(n=13 and 11)
Culture
(n=10)
Normal spindle
by PolScope
M-I
(n=12)
Iono.
Culture
Staining
M-I
No fertilization
Case Series #2: Oocyte Activation Failures
Testing of Parental Origin
Combelles et al., 2010
Rybouchkin et al., 1996
Mouse Oocyte Activation Test
(MOAT)
Split Fertilization
Sibling oocytes
Mouse
Patient
Partner’s
sperm
w/ control injections
Donor
sperm
No Fert.
Sperm
defect
Fert.
No Fert.
Oocyte
defect
No Fert
OR
0%
~% to controls
Sperm defect
Oocyte defect
Oocyte Activation Failures
Potential Gamete Defects
Phospholipase C-PLC-
Ca2+
oscillations
Ca2+ response machinery
Activation
Ca2+ ionophore, other
artificial activation methods
Oocyte Activation Failures
Assisted Oocyte Activation (AOA) Treatment
MOAT Results
0-20%
20-85%
Fert. 6 (0-18)
No AOA
Preg.
0
Preg.
14 (0-50)
0
Fert. 75 (50-92)
AOA
85-100%
22 (8-42)
0
73 (67-100)
34
75 (67-100)
43
17
 Not a universally applicable therapy
 Safety to be further established
Heindryckx et al., 2008
Oocyte Activation Failures
Other Diagnostic and Treatment Strategies
PLC-
Ca2+
Ca2+
oscillations
PLC-
Rogers et al., 2004
Ca2+ ionophore
response machinery
Activation
Human sperm with
mutant forms of PLC-
Other molecular
abnormalities?
Activation
deficiencies
Gasca et al., 2008
Yoon et al., 2008; Heytens et al.,
2009; Kashir et al., 2012
Case Report #3: Case-Specific Failure
History: 3 miscarriages; IVF Cycle 1 with abn. PN #, premature cleavages
ICSI/PGD Cycle 2:
Combelles et al., 2011
Case Report #3: Spontaneous Oocyte Activation
Light Microscopy Evaluation  Abn. cleavage patterns
SNP Microrray and PCR  Maternal origin
FISH  Aneuploidy
Cytoskeletal and Chromatin Analysis  Spontaneous activation
Chromatin
Microfilaments
Microtubules
Combelles et al., 2011
Case Report #4: Unexplained Recurrent TFF
History and Cycle Management
1. IVF Cycle: 0/16 fertilized
1. ICSI Cycle: 0/17 fertilized
1. Structural and functional analyses:
- Electron microscopy- no anomalies
- MOAT- normal activation potential
2. Split ICSI:
- ± ionophore
- ± donor sperm
- Cellular analysis
Combelles et al., 2010
Case Report #4: Unexplained Recurrent TFF
Oocyte-Specific Defect
23 +PB oocytes
ICSI
Donor
sperm
n=8
Partner s
Partner’s
sperm
with AOA
n=15
pb
0 PN
Chromatin &
cytoskeletal
assessment
pb
Combelles et al., 2010
Failures in Post-Activation Events
Sperm Structural Defects
 Centrosome dysfunction
 Failed syngamy and cleavage
Diagnostic:
- electron microscopy
- heterologous ICSI in bovine oocytes
Sperm selection
Sperm centrosome
Sperm proteasome
Sperm aster formation
Syngamy
Simerly et al., 1995; Van Blerkom, 1996, Nakamura et al., 2002; Rawe et al., 2002; 2008
Early Interventions and Management
Cycle 1
TFF from IVF
Cycle 2
Cycle 2
Repeat IVF
Split IVF/ICSI or ICSI
FF
Abnormalities at
retrieval
No FF but no
pregnancy
p
g
y
Assess all FF oocytes
Activation issues?
•Rescue IVM
•Analyze some oocytes
Yes
No
MOAT
Centrosome test
Explore gamete defects
Diagnose
gamete
defects
Cycle 3
Oocyte
defect
Sperm
defect
Split diagnostic/
treatment based on results
Cycle 3
Cycle 3
ICSI + AOA
Split ICSI + AOA/diagnostic
Evidence  Recommendations
Complex etiology
Augment basic understanding of
fertilization and gametogenesis
Uncertain etiology
Implement diagnostic strategies
Diagnosis and
prognosis
Counsel patients
Limited
treatment options
Increase research
Test novel therapies
Few cases
Increase reporting
Adopt a collaborative approach
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