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IVF and the ‘added extras’

FNA conference, Christchurch,New Zealand, 2017

Assoc Prof M. Louise Hull

Declarations

• Founding member, FertilitySA IVF Unit, Adelaide

• Board member, Genea Oxford Fertility, Christchurch, NZ

• Medical Advisory Board - Vifor Pharma

• Medical Advisory Board –Endometriosis Australia

• Travel awards - Merck Serono, MSD, Ferring, Origio

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When IVF isn’t working

Couples seek IVF units and fertility specialists whooffer additional treatments to enhance their

chance of pregnancy

Adoption Cycle

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The difference between ‘add ons’ andtreatments of the future?

Science

TechnologyMedicine

‘future treatments will have a proven benefit’

Does it have abiological basis

Does it address theclinical problem?

Does it benefitpatients?

‘Added extras’ often address the ‘too hard’ basket

• Low ovarian reserve

– Growth Hormone

– DHEAS

– testosterone

• Improving the quality of eggs

– Melatonin

– CoQ10

– L-arginine

• Enhancing implantation

– Glucocorticoids

– IVIG

– Intralipids

We can’t make new eggs

We can’t fix damaged oocytes

We can’t alter abnormal embryos

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Number ofeggs(Log scale)

Age related decline in egg number

Age from conception to menopause

Can Adjuvants improve egg numbers and quality?

estrogenandrogens

Follicle Stimulating HormoneLuteinizing Hormone

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estrogenandrogens

Follicle Simulating Hormone

Growth HormoneDHEAS

Luteinizing Hormone

Testosterone

Nagels HE, Rishworth JR, Siristatidis CS, Kroon B. Androgens (dehydroepiandrosterone ortestosterone) for women undergoing assisted reproduction. Cochrane Database of SystematicReviews 2015, Issue 11. Art. No.: CD009749.

Pre-treatment with DHEAmoderate quality evidenceAssociated with higher rates of live birth or ongoing pregnancyBetween 15% and 26% chance of live birth compared to 12%

Removal of high risk of bias studiesThe effect size was reduced and didn’t reach significance

Cochrane Review DHEA

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Nagels HE, Rishworth JR, Siristatidis CS, Kroon B. Androgens (dehydroepiandrosterone ortestosterone) for women undergoing assisted reproduction. Cochrane Database ofSystematic Reviews 2015, Issue 11. Art. No.: CD009749.

Pre-treatment with testosteroneAssociated with higher live birth ratesBetween 10% and 32%chance of livebirth compared to 8%

Removal of high risk of bias studiesThe remaining study showed no evidence of a difference

Cochrane Review Testosterone

E.M. Kolibianakis et al. Hum. Reprod. Update 2009;15:613-622

Live birth rate after growth hormonesupplementation

Small studies with low numbers of birth events$200 a day

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The Light study – multicentered, doubleblind placebo randomised controlled trial

Rob NormanRoger HartLuk RombautsLouise HullPeter IllingworthRichard HenshawMark BowmanBill LedgerHoward SmithJohn YovichRob MacLachlanLyndon HaleMary Birdsall

Aim and endpoints

Assessment of recombinant Growth hormone as an IVF adjunctin the unexpected poor responder IVF patient

primaryendpoint

secondaryendpoints

• livebirth

• Clinical pregnancy• Number of oocytes• FSH stimulation• Safety profile

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Inclusion criteria

Age < 40

BMI < 32

FSH < 15 IU

Cycle 25 – 35 days

Ovaries two

Uterus normal

Pap smear normal

BGL (fasting) normal

Poor responder on one or more IVF cycles

< 5 oocytes stimulation > 250 IU FSH

Poor responder on one or more IVF cycles

< 5 oocytes stimulation > 250 IU FSH

Ejaculatory sperm (may be frozen)Ejaculatory sperm (may be frozen)

Exclusion criteria

Clinically significant systemic disease

Radiotherapy / chemotherapy

Current /history of malignant disease

Pituitary / hypothalamic tumours

Current ovarian cyst > 3cm

Chronic infectious disease

PCOS (Rotterdam criteria)

PV bleeding unknown aetiology

PGD this cycle

Smoker in last 30 days

PGD this cycle

Smoker in last 30 days

Steroid use this cycleDHEAprednisolone

Steroid use this cycleDHEAprednisolone

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Study design

Day 2/3

•Start r-FSH•Start study drug

Day 5/6

• Start GnRH antagonist

Day 8/9/10

• Monitoring cycle

ET /Luteal support

• Clinic standard

Day OPU

• IVF/ICSI

Schedule OPU

• Cease study drug

100%

88.5

74.0

19.1

13.0

N = 131

HGH Placebo

65 65

Flow of study

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Primary endpoint

HGH(n=65)

Placebo(n=65)

SignificantDifference

Egg recovery per started cycle 62 (95%) 51(78%) No

Embryo transfer per started cycle 53 (82%) 42 (65%) No

Early pregnancy loss n = 8 4 4 No

Livebirth (deliveries per started cycle) n=17 9 (14%) 7 (11%) No

Livebirth (deliveries per ET) n = 17 9* (17%) 7** (17%) No

FET births n = 3 2 1

• * 3 sets of twins ie 11 babies delivered• ** includes 1 spontaneous pregnancy

Major adverse events

Event GrowthHormone

Placebo Outcome

Large infantPatent ductus arteriosisGenetic consult ? Syndrome (FET cycle)

Ongoing review

Trisomy 21 Termination

Tongue tie (FET cycle) Surgical release

Systolic heart murmur Spontaneousresolution

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Summary

• These results show neither efficacy nor a lack ofefficacy

• Limitations of this study– Enrolment not reached– Availability of study drug off protocol limited

recruitment

• Safety of new interventions paramount

What should be our current clinical practice?

• Understand the literature and explain it to patients• Be aware of the biases in some studies• Uncertain of benefits – use in low ovarian reserve

In my practice• Generally don’t use GH (expense, injectable, side effects)• If low ovarian reserve consider testosterone (gel) over DHEA

as not compounded

In Europe• Current trial of testosterone for 3 months prior to IVF

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Oxidative stress and egg quality

Broi et al 201458% of oocytes had meiotic abnormalities when matured in endometriosis PF21% when cultured in normal PF (P<0.01)

Endometriotic Peritoneal FluidControl Peritoneal Fluid

Borges et al 2015

Patient Number

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Cum

ulus

cel

l RED

OX

(mea

n flu

ores

cent

pix

els)

0

20

40

60

80

100

LOW

INTERMEDIATE

HIGH

Oxidative stress in granulosa cells1. Increased levels of antioxidant enzymesin peritoneal fluid (PF):-glutathione peroxidase (Szczepanska et al. (2003)

-superoxide dismutase (Szczepanska et al. (2003)-lipid peroxidase (Liu et al 2001)-catalase (Ota et al 2002)-xanthine oxidase (Ota et al 2002)

2. lower total antioxidant potential in PF(Szczepanska et al., 2003)

Dianne Feil 2010 –PhD thesis

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Antioxidants to improve egg quality

• Melatonin• N-acetyl-cysteine• L-arginine• carnitine• selenium• vitamin B complex• vitamin D+calcium• pentoxifylline• omega-3-polyunsaturated fatty acids

Cochrane Review

Showell MG, Mackenzie-Proctor R, Jordan V, Hart RJ. Antioxidants forfemale subfertility. Cochrane Database of Systematic Reviews 2017, Issue7. Art. No.: CD007807.

• Antioxidants may be associated with an increased livebirth rate ad clinical pregnancy rate

• Expected livebirth rate 20% with antioxidants 26-43%• Expected clinical pregnancy rate 22% with antioxidants 27-

33%

Very low-quality evidence

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Melatonin Trial – Monash University

• Four groups:– Placebo– 2mg Melatonin (4mg/d)– 4mg Melatonin (8mg/d)– 8mg Melatonin (16mg/d)

• Twice a day dosage from Day 2 until oocyte retrieval

Dr Shavi Fernando, Professor Euan Wallace,Professor Luk Rombauts

The Ritchie Centre, Hudson Institute of Medical Research,Monash University and Monash IVF

160 Randomised

150 included foranalysis

10 Withdrewbefore trial

medication began6 pregnant

2 cancelled IVF1 could not comply

1 used excludedadjuvants

PlaceboN=36

2mgMelatonin

N=38

4mgMelatonin

N=36

8mgMelatonin

N=40

783 identified fromMonash IVF database Sep

2014-Sep 20161st cycle IVF/ICSI

Age and BMI within range

412Ineligible

211Declined

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No significant difference in birth outcomes

Placebo

N=36

2mg

N=38

4mg

N=36

8mg

N=40

P value AnyMelatoni

n

N=114

P value

Clinical pregnancyper cycle started(N %)

6

(16.7)

11

(28.9)

6

(16.7)

9

(22.5)

0.52 26

(22.8)

0.43

Live birth per cyclestarted (N %)

6

(16.7)

11

(28.9)

6

(16.7)

9

(22.5)

0.52 26

(22.8)

0.43

Clinical pregnancyper embryotransfer (N %)

6

(22.2)

11

(40.7)

6

(30.0)

9

(33.3)

0.54 26

(35.1)

0.21

Miscarriages (N %) 2

(5.6)

3

(7.9)

0

(0.0)

0

(0.0)

0.13 3

(2.6)

0.61

Conclusion

Melatonin does not improve or reduce clinical pregnancyrates or live birth rates unselected IVF patients

Satisfactory Safety profile– 1 baby with absent right kidney in the 2mg group

Power calculation for future studies1500 patients need to be enrolled to reach significance

Recruitment problemsPatients declining randomisation to take active melatoninMany using adjuvants from other health providers

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Melatonin

• Melatonin not likely to benefit unselected patients

on first cycle

• Untested in selected populations at risk of elevated

peritoneal levels of oxidative stress

• No evidence of harm

What should be our current clinical practice?

• Understand the literature and explain it to patients

• Use pregnancy multivitamins

• Test for markers of oxidative stress/ causes of oxidativestress (homocysteine, MTHFR, Vit D, glucose, insulin,lipids)

• Replace supplement if indicated (vitamin D, B6 , B 12,folate, fish oil)

• Stay neutral if already on antioxidants

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Immune suppression to enhance implantation

• Glucocorticoids• Immunoglobulins• Intralipid• Anti-TNF alpha• G-CSf• Lymphocyte immune therapy

SA Robertson, M Jin, D Yu, LM Moldenhauer, M Davies, M L Hull, RJ Norman.Corticosteroid therapy in assisted reproduction – a faulty premise justifies immune

suppression at conception, Human Reproduction, 2016, 31 (10): 2164.

Professor Sarah RobertsonUniversity of Adelaide

Dr Gavin SacksIVF Australia

Professor Ashley MoffittUniversity of Cambridge

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The foetus inherits 50% of genes from the mother but50% from the father ……. which are is ‘foreign’ to thefemale body

The immune paradox of pregnancy

+

Maternal uNK cell(uterine natural killer)Maternal uNK cell(uterine natural killer)

Paternal HLA-CPaternal HLA-C

Maternal HLA-CMaternal HLA-C

Immune recognition of trophoblast:Maternal KIR binds to fetal HLA-C molecules

Placental cell

KIRHLA-C

Both KIR and HLA-C are highly polymorphic

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Keeping the balance towards immunotolerance

Immunotolerant cells Inflammatory Immune Cells

Th1/Th17 T cells

M1 phagocytic macrophages

Cytokines : TNFα, INFγ

T2 /Treg cells

Cytokines: TGFβ, IL-10

M2 repair macrophages

Cytotoxic (CD56dim) NK cells

******

****

****

uNK (CD56bright) cells

Immune suppression may alter the balance

Immunotolerant cells Inflammatory Immune Cells

Th1/Th17 T cells

M1 phagocytic macrophages

Cytokines : TNFα, INFγ

T2 /Treg cells

Cytokines: TGFβ, IL-10

M2 repair macrophages

Cytotoxic (CD56dim) NK cells

******

****

****

uNK (CD56bright) cells Immunesuppression

Mayhinder

Mayhelp

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Cochrane Review

No Significant difference in live birth rates (OR 1.21, 95% CI 0.67 to 2.19).

Boomsma CM, Keay SD, Macklon NS. Peri-implantation glucocorticoid administration forassisted reproductive technology cycles. Cochrane Database of Systematic Reviews 2012

Live birth rate per couple in unselected IVF : Glucocorticoids versus placebo orno glucocorticoids

Negative effects of glucocorticoid use

• Small risk of cleft lip and palate (Carmichael and Shaw 1999)

• Miscarriage, preterm birth, hypertension (Gur et al 2004, Laskin

et al 1997)

• Maternal side effects –weight gain, mood

• Negative impact on sugar and insulin levels

• Negative impact on underlying infections (mycoplasma)

• May exacerbate endometriosis (D’Hooghe et al 1995)

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Is there a subgroup that benefits from steroids?

Difficulties identifying a subgroup

• Aneuploidy confounds RCTS

• Heterogeneity of population who have implantation

failure and recurrent miscarriage

• Many factors contribute to a pro-inflammatory

endometrial immune profile

• Poor ability to test for immune dysfunction

Aneuploidy confounds the pictureImmunotolerant cells

T2 /Treg cells

Cytokines: TGFβ, IL-10

M2 repair macrophages

uNK (CD56bright) cells

Ogasawara et al 2010

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SecretoryProliferative

%CD56+cells

Day of menstrual cycle

Testing Uterine Natural Killer (uNK) in endometrium

NK cells in blood and uterus

Blood NK cells

CD56dim, CD16+

High cytotoxic activity

Low cytokine production

Uterine NK cells

CD56bright, CD16-

Low cytolytic activity

Cytokine producers(MIP-1α, GM-CSF)

Moffett 2002 Nature Reviews Immunology

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Conditions associated with inflammatory immune profiles

Inflammatory Immune Cells

Th1/Th17 T cells

M1 phagocytic macrophages

Cytokines : TNFα, INFγ

Cytotoxic (CD56dim) NK cells

******

****

****

Immune system disease

Diabetes and glucose intolerance

Endometriosis

Infection

Hydrosalpinx

Obesity

Smoking

Vitamin D and other deficiencies

Can randomise on the basis of NK cells toassess the efficacy of steroids?

LBR % LBR RR (CI) P-value

Prednisolone 12/20 60% 1.5 (0.79–2.86) NS

Placebo 8/20 40%

Tang et al 2013 –feasibility study –RCT in Recurrent miscarriage160 women with high uNK cells (>5%) and ≥ 3 consecutivemiscarriages40 randomised when pregnant to prednisone (8 weeks) or placebo

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Corticosteroid trials in the subgroup of womenwith elevated NK cells undertaking IVF

Systematic review: Interventions to improve reproductive outcomes in women withelevated natural killer cells undergoing assisted reproduction techniquesPolanski et al 2014, Hum Reprod, 29,1:65-75

RCT of 112 womenNK cell activation marker CD69+ on >1% lymphocytes20mg prednisolone or placebo from Day1

Conclusion:Prednisone seemed to confer a significant benefit on ART outcomesBut as only 1 small study with a low quality score the authors could not supportthe use of prednisolone

What should be our current clinical practice?

• Understand the literature and explain it to patients

• Immune suppression should not empirical– Try to eliminate aneuploidy as a confounding factor

– Test for autoimmune conditions (ANAs, APLS, others)

– Exclude and treat other non-immune inflammatoryconditions

• Be aware of the caveats of NK cell testing

• Be aware of the negatives of glucocorticoid use

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At the end of the day……..We are trying to help couples have a family

and ‘Add ons’ could be treatments of the future-need to be safe-need to be honest about benefits-need to be intended to help the couple(not the clinician or the clinics bottom line)-need to be well informed-need to keep assessing and trialing