This document discusses immunological factors that can impact in vitro fertilization (IVF) success rates. It notes that maternal immune tolerance is required for pregnancy but may be insufficient in some cases, leading to issues like miscarriage. IVF is an alternative to bypass some fertility issues but has limitations. Immunological testing and immunotherapy during IVF, like treating certain antibodies, has increased IVF success rates to 40-50%, compared to typical averages. Precise microinjection techniques are important for IVF/ICSI procedures. Overall, considering immunological factors can improve IVF outcomes for patients with recurrent implantation failure or pregnancy loss.

1. IVF and Immunotherapy
Mohamed Labib salem, PhD
Prof. of Immunology
Faculty of Science, Tanta University
The Scientific Conference on Laboratory
Diagnosis and ICSI
‫والتشخيص‬ ‫المجهرى‬ ‫للحقن‬ ‫الدولى‬ ‫العلمى‬ ‫االمؤتمر‬
‫المعملى‬
15 March, 2013

3. Presentation
• Immunity and Reproductive Immunology
• Maternal tolerance to the fetus
• Breaking (insufficient) tolerance
• IVF as an alternative
• Limitation of IVF
• Immunological analyses for IVF
• Immunotherapy during IVF

5. Immunity: Active and Passive
Artificially acquired
Passive immunity
Active immunity
Naturally acquired Naturally acquired
Artificially acquired

6. IMMUNITY ?
ANERGY ?
TOLERANCE ?

9. INNATE AND ADAPTIVE IMMUNITY

10. WHY IMMUNE SYSTEM????
SELF FROM NON SELF
DANGER FROM NON DANGER
TOLERANCE VERSUS ACTIVATION

13. 1st signal
2nd signal
2nd signal
1st signal
Antigen-specific T Cell Responses

14. T cell Plasticity

21. Regulatory T Cells
Regulatory T
cells
Mature
dendritic
cell
Regulatory T
cell
Proliferation
T cells compete for
cytokine signals
T cells compete
for same antigen
Cytotoxic
T cell

22. Disorders of the Immune System:
Autoimmune Disease
Cytotoxic T cell
Pancreas
Beta cell

23. Disorders of the Immune System:
Immune Complex Disease
Glomerular basement
membrane of kidney
Large complex
Endothelial cell
Small complex

24. REPRODUCTIVE IMMUNOLOGY
Fertilization
(Migration versus Immigration)

30. Reproductive Immunology
• Refers to a field of medicine that studies interactions (or the
absence of them) between the immune system and
components related to the reproductive system, such as
maternal immune tolerance towards the fetus, or
immunological interactions across the blood-testis barrier.
• The concept has been used by fertility clinics to explain the
fertility problems, recurrent miscarriages and pregnancy
complications observed when this state of immunological
tolerance is not successfully achieved.
• Immunological therapy is the new up and coming method
for treating many cases of previously "unexplained
infertility" or recurrent miscarriage.
Pearson, H. Immunity’s Pregnant Pause. Nature Publishing Group 2002; 420: 265-266

31. A unique problem
• Pregnancy presents a complex immunological
problem for the mother.
• Foetus shares only 50% genetic relatedness
with the mother, the remainder being with
the father.
• Cells and molecules of the immune system
interact in such a way as to prevent the
rejection of the semiallogenic foetus and
support its growth and development.

32. A unique problem
In 1953 Sir Peter Medawar, a pioneer in the field of
transplantation biology, presented a lecture in
which he asked the following question:
“The immunological problem of pregnancy may be
formulated thus: how does the pregnant mother
contrive to nourish within itself, for many weeks or
months, a foetus that is an antigenically foreign
body?”
To this day, this question remains relevant and, as
yet, incompletely answered and is the foundation of
the field of reproductive immunology.

33. Immune cells at the maternal-foetal
interface
• A number of immune cells have been identified
within the maternal-foetal interface, including:
– uterine Natural Killer cells (70%),
– macrophages (20%),
– T cells (including CD4+, CD8+, gd T cells, regulatory T
cells) (10%),
– dendritic cells and B cells (few).
• The numbers of these cells and roles that they
play differs throughout the various stages of
pregnancy.

34. Sufficient Tolerance

35. Protection of the
conceptus (Fetus)
Low MHC on
trophoblast
Immuno-
suppressive
environment
Separate
Circulatory
system
HLA-G/E/C
on
tropgoblast
Suppressive
factors in
semen
Sensitization
by Semen
proteins
Th2
profile
1
2
3
4
5
6 7

36. Immune tolerance in pregnancy:
Placental mechanisms
The placenta functions as an
immunological barrier between the
mother and the fetus, creating an
immunologically privileged site

37. Figure 1.
The Placenta: The Maternal-Fetal Interface
Umbilical Cord
Decidua
Intervillous
Space
Spiral artery
Maternal
Tissue
Fetal
Tissue
Umbilical Cord
Decidua
Intervillous
Space
Spiral artery
Maternal
Tissue
Fetal
Tissue

38. • The placenta secretes Neurokinin B
containing phosphocholine
molecules.
• This is the same mechanism used by
parasitic nematodes to avoid
detection by the immune system of
their host.
Immune tolerance in pregnancy:
Placental mechanisms

39. • Presence of small suppressor T cells in
the fetus that inhibit maternal
cytotoxic T cells by inhibiting the
response to IL-2.
Immune tolerance in pregnancy:
Placental mechanisms

40. • Unlike most other cells in the body, the
placental trophoblast cells do not express
the classical MHC class I isotypes HLA-A
and HLA-B.
• This absence prevents destruction by
maternal cytotoxic T cells, which
otherwise would recognize the fetal HLA-A
and HLA-B molecules as foreign.
Immune tolerance in pregnancy:
Placental mechanisms

41. • The placental trophoblast cells do
express the atypical MHC class I isotypes
HLA-E and HLA-G, which is assumed to
prevent destruction by maternal NK cells,
which otherwise destruct cells that do
not express any MHC class I.
Immune tolerance in pregnancy:
Placental mechanisms

42. • The mother does produce Ab against foreign
ABO blood types, where the fetal blood cells
are possible targets.
• However, these preformed Ab are usually of the
IgM type, and therefore usually do not cross
the placenta.
• Rarely, ABO can give rise to IgG Ab that cross
the placenta, and are caused by sensitization of
mothers (usually of blood type 0) to antigens in
foods or bacteria
Immune tolerance in pregnancy:
Placental mechanisms

43. The Reasons for Miscarriages
Cause Percent
Infection 1%
Anatomy abnormal 5-10%
Progesterone level low 20%
Chromosome abnormal
Primary miscarrier (no live births) 7%
Secondary miscarrier (one or more live births) 50%
Immune mechanisms 50%
Unknown 15%
:

44. Insufficient Tolerance

45. Insufficient Tolerance:
Consequences
• Many cases of spontaneous abortion
may be described in the same way as:
– maternal transplant rejection
– infertility.
– Rh disease
– pre-eclampsia

46. Insufficient tolerance: Rh disease
• Rh disease is caused by the mother producing
Abs (including IgG Abs) against the Rhesus D
antigen on her baby's RBCs.
• It occurs if the mother is Rh negative and the
baby is Rh positive, and a small amount of Rh
positive blood from any previous pregnancy
has entered the mother's circulation to make
her produce IgG antibodies against the D
antigen (Anti-D).

47. Insufficient tolerance: Rh disease
• Maternal IgG is able to pass through the
placenta into the fetus and if the level of it is
sufficient, it will cause destruction of D
positive fetal red blood cells.
• This leads to development of Hemolytic
Disease of the Fetal-Newborn (HDFN), also
called Rh disease.
• Generally HDFN becomes worse with each
additional Rh incompatible pregnancy.

48. Insufficient tolerance: Pre-eclampsia
• One cause of pre-eclampsia is an abnormal immune
response towards the placenta.
• Exposure to partner's semen can prevent pre-
eclampsia, largely due to the absorption of several
immune modulating factors present in seminal fluid.
• Pregnancies resulting from egg donation, where the
carrier is less genetically similar to the fetus than a
biological mother, are associated with a higher
incidence of pregnancy-induced hypertension and
placental pathology
• The local and systemic immunologic changes are also
more pronounced than in normal pregnancies.

49. Preeclampsia is associated with uterine NK cells of KIR
genotype AA and the presence of HLA-C2 on the surface of
extravillous trophoblast cells (EVT).
Parham P J Exp Med 2004;200:951-955

50. Antigenicity of the sperm
• The presence of anti-sperm antibodies in infertile men
was first reported in 1954 by Rumke and Wilson.
• The number of cases of sperm autoimmunity is higher
in the infertile population leading to the idea that
autoimmunity could be a cause of infertility.
• Anti sperm antigen has been described as three Ig (IgG,
IgA, IgM) each of which targets different part of the
spermatozoa.
• If more than 10% of the sperm are bound to anti-
sperm antibodies (ASA), then infertility is suspected.

51. Antigenicity of the sperm
• Infertility after anti-sperm antibody
binding can be caused by:
– autoagglutination,
– sperm cytotoxicity,
– blockage of sperm-ovum interaction,
– inadequate motility.

52. The Reproductive Autoimmune Failure
• The reproductive autoimmune failure
syndrome (RAFS) was first described in 1988.
• It is the association of pregnancy wastage,
infertility and endometriosis with circulating
autoantibodies.
• Patients with RAFS have polyclonal B-cell
activation; that is, their antibody producing
cells including those that manufacture
autoantibodies are very active.

53. When the immune system is the cause
of miscarriage, the chances of mother
having a successful pregnancy without
treatment is:
30% after 3 miscarriages,
25% after 4 miscarriages, and
5% after 5 miscarriages

54. MICROINJECTION
In Vitro Fertilization (IVF)

55. In vitro fertilization (IVF)
• IVF is a method of assisted reproduction that
involves combining an egg with sperm in a
laboratory dish.
• If the egg fertilizes and begins cell division, the
resulting embryo is transferred into the woman’s
uterus where it will hopefully implant in the
uterine lining and further develop.
• IVF bypasses the fallopian tubes and is usually the
treatment choice for women who have badly
damaged or absent tubes.

56. Microinjection
• It is a simple mechanical process in which a
needle (a glass micropipette) roughly 0.5 to 5
micrometers in diameter penetrates the cell
membrane and/or the nuclear envelope into a
single living cell.
• The desired contents are then injected into the
desired sub-cellular compartment and the
needle is removed.
• Microinjection is normally performed under a
specialized optical microscope setup called a
micromanipulator.

57. MICROINJECTION
IUSI ICSI IVF
Male failure Female failure

58. A specialized microinjection
workstation for microinjection.

60. A Workstation
for
microinjection

61. Microinjection
The goal is to have the blastocyst located
at the holding pipette with the inner cell
mass positioned at 180 degrees from the
cell injection needle.

62. All of these observations
provide evidence that:
• Immune problems can affect implantation.
• Immunotherapy can positively modulate this
problem, resulting in IVF success.

63. Cytokines in the Follicular Fluid and their
Influence on IVF-embryo transfer

64. Cytokines in the Follicular Fluid and their
Influence on IVF-ET
Current Women’s Health Reviews, 2005, 1, 49-60

65. Cytokines in the Follicular Fluid and their
Influence on IVF
Current Women’s Health Reviews, 2005, 1, 49-60

66. Recommendations for IVF candidates with a history
of recurrent pregnancy losses
PGS: Preimplantation genetic screening — also known as PGD.

67. Immunological analyses for women with recurrent
pregnancy losses (RPL) or repeated implantation
failures (RIF)

68. Opinions of IVF physicians about the need for
immunologic analyses after failed IVF cycles

69. Some of the immune issues that are important
to the reproductive system
Antiphospholipid antibodies autoimmune
Antinuclear antibodies autoimmune
Natural killer cells autoimmune
Cytotoxic B-cells autoimmune
Blocking (protective) antibodies alloimmune

70. Miscarriages and Immunotherapy
Recommended medications for patients who have
experienced immune-mediated reproductive:
• Aspirin
• Heparin
• Prednisone
• IV mmunoglobulin G Infusion
• Enbrel (anti-TNF-alpha)
• Paternal Leukocyte Immunization
• Husbands Leukocyte Transfer (HLT)

71. Laboratory Testing
Blocking Antibody level
• Antiphospholipid Antibody Panel
• Antinuclear Antibody Panel
• Antihistone Antibody
• HLA Tissue Typing
• DQA1 DNA fingerprinting
• Immunophenotype
– Natural Killer Cell Activation
Assay
– Natural Killer Cell
Activation/IVIg Assay
– Intracellular Tumor Necrosis
Factor (TNF) Alpha Assay
– Quantitative Immunoglobulin
• Chromosome analysis
– Factor V Leiden Gene Mutation
– Factor II (Prothrombin) Gene
Mutation
– Methylene Tetrahydrofolate
Reductase (MTHFR) Gene
Mutation

72. Conclusion based on the use of
these immune markers
Immune testing is prudent, and when
recommendations are followed, the
combined IVF success rate is between 40%
and 50%, twice the recorded average!

73. Phospholipids
• Phospholipid molecules are normal components
of all cell membranes.
• At a very basic level, they help the fetus "stick" to
the uterus.
• Some phospholipid molecules have adhesion
properties i.e. glue like, and allow cells to fuse.
• The formation of the normal placenta involves
the fusion of small cells called cytotrophoblasts
into giant cells known as syncytiotrophoblasts.
• The syncytiotrophoblasts play a key role in the
regulation of nutrients going to the baby.

74. Anti-phospholipid Antibodies (APA)
• APA interfere with this process, so that
the transferred fetus has difficultly
implanting, i.e., attaching to the uterus.
• Furthermore, APA cause problems with
uterine and placental blood flow, making
the uterus unhealthy for successful
implantation.

76. Anti-phospholipid Antibodies
• Antibodies to phospholipid molecules
can damage the inside of the blood
vessel wall.
• This allows blood cells to stick to the
site of the injury and cause blood clots.

77. APA can cause blood vessels to constrict, causing decreased blood flow
throughout the circulatory system.
The combination of blood clots and constricted blood vessels may impair blood
supply to the fetus and placenta resulting in complete fetal demise or growth
retardation.
Anti-phospholipid Antibodies (APA)

78. Treatment of APA
• Antiphospholipid antibodies (APA) are treated
with low dose (baby) aspirin and a blood thinner
called Heparin.
• Heparin is a very large molecule and is unable to
cross the placenta.
• Aspirin is able to cross the placenta but the dose
used is so small that the fetus is unaffected.
• The effectiveness of treatment is much greater
when the medication, if indicated, is started prior
to conception and continued throughout the
pregnancy.

79. Antinuclear antibodies (ANA)
• Some people have antibodies to different nuclear
components, e.g. Systemic Lupus Erythematosus
(SLE).
• The miscarriage rate in SLE patients is much
higher than that of the general population.
• Although most women who suffer recurrent
miscarriages do not have clinical signs of SLE,
many exhibit autoimmune phenomena which is
similar to that seen in SLE patients.
• The placentas in these women are inflamed and
weakened.

80. Antinuclear antibodies (ANA)
• ANA cause inflammation in various tissues,
including the uterus.
• This inflammatory process prevents the uterus
from being able to host a proper implantation.
• CD56+CD16+ NK cells normally kill cancer cells
before they grow into large tumors.
• These NK cells may misinterpret the implanting
fetus as a cancer and kill it too.
• Women with ANA are treated with prednisone, a
corticosteroid, which suppresses the
inflammatory process and stabilizes the cell.

81. Most people have no ANA all the time (A,B).
Many women who miscarry have borderline (C,D,E) or abnormal levels
of ANA (F,G).
ANA levels

82. Anti-thyroid Antibodies (ATA)
• In 1990, Stagnaro-Green demonstrated in a
prospective analysis that thyroid antibodies were
markers for "at-risk" pregnancies.
• The two antibodies studied, anti-thyroid
peroxidase and anti-thyroglobulin antibodies, are
collectively referred to as anti-thyroid antibodies
(ATA).
• 67% of women with recurrent first trimester
losses had ATA, compared to 17% of controls with
no clinical manifestations of thyroid disease.

83. Anti-thyroid Antibodies (ATA)
• ATA are markers for abnormal T-lymphocyte
function and for polyclonal B-cell activation and
do not have a direct effect on implantation or the
fetus.
• Increases in the endometrial T-cell and IFNg have
been observed in infertile women with ATA.
• Infertile patients who demonstrate ATA can be
classified as having the reproductive autoimmune
failure syndrome (RAFS).
• In IVF patients, ATAs are treated with intravenous
immune globulin (IVIg) before the IVF transfer.

84. • Immunophenotype refers to the relative
amounts of T, B and NK cells in the
bloodstream.
• The immunophenotype assay involves labeling
a sample of blood with fluorescent dyes
directed to specific markers for each type of
lymphocyte: CD4 for T-helper cells, and CD56
for NK cells.
Immunophenotypes:
NK Cells and Cytotoxic B-Cells

85. Immunophenotypes:
NK Cells and Cytotoxic B-Cells
• The specimen is then placed into a cell flow
cytometer in which the cells pass in single file
across an argon laser that excites the dyes and
causes them to fluoresce.
• Intensity of the fluorescence is measured by
electronic tubes and digitized, allowing a
computer to calculate the relative percentages
of different lymphocyte subsets.

86. Flow Cytomter for
Immunophenotypes

87. Immunophenotypes:
NK Cells and Cytotoxic B-Cells
• There is a special class of NK cells (CD3-, CD16-,
CD56+) in the placenta that promotes cell
growth, secretes growth molecules for the
placenta and down regulates the mother's
immune response locally at the
maternal/placenta interface.
• Opposing is another group of NK cells (CD3-,
CD16+, CD56+), when activated by the cytokine
IL-2, are cytotoxic to placental trophoblast.
• The same cells secrete tumor necrosis factor
(TNF) which can destroy the placenta.

88. Immunophenotypes:
NK cells and Cytotoxic B-Cells
• Women with CD16+, CD56+ NK cells in excess of
20% are at risk for miscarriage despite optimal
immune treatment (paternal leukocyte
immunization, prednisone, aspirin and heparin).
• In a subset of women who have had multiple
failed IVFs, it is believed that TNF is secreted in
amounts that inhibit implantation and early
formation of the placenta resulting in an IVF cycle
which does not produce a clinical pregnancy.

89. Treatment of Immunophenotypes:
NK Cells and Cytotoxic B-Cells
• Women who have an elevation of NK cells are
candidates for immunologlobulin G infusion (IVIg).
• The dosage of IVIg is 400 mg/kg/day for three
consecutive days, monthly, until the NK cells become
normal or until the 28th week of pregnancy.
• In some studies, autoantibodies to phospholipid and
nuclear epitopes were demonstrably lower after IVIg.
• Some researchers have used Enbrel, a TNF alpha
inhibitor, instead of, or in addition to IVIg.

90. DQ Alpha Genotyping
• DQ Alpha genotyping refers to a specific kind of HLA (tissue)
typing done at the DNA level.
• The Class II HLA, found on the surface of white blood cells
(WBC), include HLA-DR and HLA-DQ.
• Each tissue type is made up of an "A" or alpha part and a "B"
or beta part.
• Mothers who are HLA-DQ alpha (DQA1) and/or DQ beta
(DQB1) compatible with their fetuses tend to have a high rate
of miscarriage before eight weeks of pregnancy.
• Mother's who are DQ alpha compatible with their fetuses can
develop an exacerbation of autoimmune processes, such as
rheumatoid arthritis and antiphospholipid antibody
syndrome, during the early portions of their pregnancy.

91. Blocking Antibodies
• Early in pregnancy, the mother's immune system
receives signals from the tiny fetus.
• Many of the signals are hormonal, but others come
directly from genetic messages that the father has
contributed.
• Some of the messages involve the tissue type, also
known as the human leukocyte antigens (HLA) and the
white blood cell (leukocyte) type.
• HLA are expressed on white blood cells.
• They are unique to each individual and allow the body
to identify anything foreign to it such as infections,
cancers, transplanted organs and fetuses.

92. Blocking Antibodies
• One half of the fetus's HLA type is contributed by
mother and the other half by father.
• When a woman becomes pregnant, her body's
immune system usually recognizes the father's HLA as
different from her own, and the white blood cells in
her uterus produce protective, blocking antibodies.
• These antibodies coat the baby's cells and protect the
fetus from mother's killer cells.
• If father's HLA is too similar to mother's, her cells may
not recognize differences that are vital to the
production of blocking antibodies.

93. Blocking Antibodies

94. Group
Blocking Ab Present
Prior to Pregnancy
Delivered
women with no miscarriages YES (82 /100) 100/100 (100%)
Women with Miscarriages (RSA) NO (6 /175) 0 /175 (0%)
Comparison of Blocking Ab level in women
with vs. women without repeated failure

95. Treatment for Blocking Antibodies:
Paternal white cell immunizations
• Immunizing the mother with concentrates of the father's
white blood cells amplifies the HLA signal.
• Approximately 50% of patients show increase in the
blocking antibody level after 2 treatments.
• The other 50% require additional white cell
immunizations.
• To determine if additional preparations will be required,
the blocking antibody level should be measured 3 to 4
weeks after the second and all subsequent immunizations.
• When blocking levels are elevated, prior to conception,
the rate of successful pregnancy is nearly 80%.

96. Treatment for Blocking Antibodies: IVIg
• IVIg is an alternative to white cell preparations.
• The doses vary between 10 and 60 grams per month
or with lower doses of IVIg (10 grams) for patients
who have only the blocking antibody problem.
• The main differences between white cell transfusion
and IVIg are that IVIg has rapid onset of action and is
more versatile.
• However, IVIg is more expensive and provides
temporary blocking.
• In multiple studies, the success rate is the same
provided that the blocking antibody levels are
adequate.

97. Husbands Leukocyte Transfer (HLT)
• HLT is based on separating lymphocytes from
about 20ml of husbands blood and then inject
in to the lymphatics at least 5 times.
• This treatment can give a very good results in
terms of live births in recurrent IVF failures
and repeated abortions.

98. Success rate with varying treatment modalities in
women with history of RSA
Group
Received
PLI
APA & / or
ANA
Meds
Given *
Delivered
PT. NO. (%)
I NO NO NO 22%
II YES NO NO 82%
III YES YES NO 19%
IV YES YES YES (early) 81%
V YES YES YES (late) 46%
Medication for APAs and ANAs include low dose aspirin, heparin and
prednisone when indicated.
ANA - Antinuclear antibodies
APA - Antiphospholipid antibodies
PLI - Paternal leukocyte immunization

99. Why Reproductive Immunotherapy
• Patients that suffer from failed IVF, 11/12 studies have
revealed an increased prevalence of APA.
• Three controlled studies, two of which were randomized,
have demonstrated a significant increase in pregnancy
outcome when intravenous immune globulin (IVIg) was
administered to patients with a previous history of multiple
failed IVFs.
• A recent randomized prospective study concluded that up
to 54% of patients with failed IVFs had successful
pregnancies when given prednisone and aspirin prior to the
IVF if they have antinuclear antibodies.
• Women who have received immunotherapy prior to IVF
were twice as likely to have multiple births.