report assessment immunolog] reproductive

ReproductiveImmunologyAssessmentReport

PatientJane DoeDate of Birth01/02/1983

Report Date11/04/2021Report IDDOEJ010283F-PGM1

Copyright ©2021 Pregimmune Corp. All rights reserved.

https://pregmune.com

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Consent Release

Pregmune Medical PA is pleased to deliver this Reproductive Immunology Assessment Report (PGM-1) toyou and your ordering physician via the Pregmune Platform consistent with the terms and conditions withinthe Consent and Agreement previously executed by you, which you can find within your Patient Portalaccount. The remote consultation represented by this report is complex and should be reviewed by you inconjunction with your physician. In connection with this Report you acknowledge receipt of PregmuneMedical’s Notice of Privacy Practices (shown in your Patient Portal account) which describes the howinformation about you is used and disclosed by Pregmune Medical and consulting medical providers whoare supported by business associates under contract to deliver services on their behalf consistent withHIPAA and the terms and conditions within the Consent and Agreement.

Patient History & Demographics

Results Summary

Parental Compatibility

Female Assessment

Male Assessment

Additional Reading

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4

6

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2Jane Doe DOEJ010283F-PGM1 11/04/2021

Reproductive Immunology Assessment Report

38

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https://pregmune.com/

Patient History & Demographics

Name: Jane Doe

Patient Demographics Partner Demographics

Patient Fertility & Medical History Summary

Patient ID:

DOB: 01/02/1983

Age: 38

Height: 5'5"

Weight: 125 lbs.

BMI: 20.8

Patient ID:

Name: John Doe

DOB: 01/10/1977

Age: 43

Height: 6'0"

Weight: 210 lbs.

BMI: 28.5

Address: 21200 Pinelakes boulevard

City/State/Zip: Jersey City, NJ 7305 Email: [email protected]

Phone: 605-523-9823

3Jane Doe DOEJ010283F-PGM1 11/04/2021


Type of Infertility: PrimaryNumber of Pregnancies: 4RPL: YesStillbirths: 0Live Births: 0Miscarriages: 5Failed IVF Transfers: 5Any medical/autoimmune conditions? Prediabetic, dysmenorrhea, endometriosis, fibroidAny family medical/autoimmune conditions?Is she on fish oil or any medications? NoKnown allergies to medications:Male partner: any issues? NoEmbryos stored? NoNumber of embryos (if yes) 0Number of embryos genetically tested? 0

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Results Summary

Above Average

Live Birth Success Rate

Below Average Average Above Average

What you need to know

The predicted Live Birth Success Rate tells you the likelihood of a successful full-term deliveryassuming the implementation of all recommended follow-up care. The calculation is generated usinga customized algorithm based on a source data set of over 1000 patient observations (patient using

own egg and <45 years old).

Fetal HLA-C and maternal KIR (uterine NK cell) interaction

HLA Mismatches

HLA Homozygosity

Anti-HY Immunity

Anti-HLA Antibodies

Embryo MTHFR prediction and Risk for implantation failure

Chromosome Analysis

Thrombophilia

Autoimmunity

Thyroid

Serological

Inflammatory

Metabolic

Nutritional Analysis

Chromosome Analysis

Thrombophilia

MTHFR Analysis


Female Assessment Male Assessment


Lower Risk

Lower Risk

Lower Risk

Lower Risk

Lower Risk

Lower Risk

Normal

Medium Risk

Medium Risk

Lower Risk

Lower Risk

Medium Risk

Medium Risk

Higher Risk

Normal

Lower Risk

Lower Risk

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Jane Doe DOEJ010283F-PGM1 11/04/2021

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Higher Risk




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Results Summary (cont'd.)

Recommended Actions

Immunological/Fertility TreatmentACOG/ESHRE recommendationsHigher risk for thrombophilia (inherited): Lovenox and low dose aspirin (75 to 100mg a day).Other recommendations (published clinical trial)

Hormonal/Metabolic TreatmentThe patient is pre-diabetic and may benefit from Metformin.

Nutritional RecommendationsThe patient is in the High-risk category and may benefit from EPA/DHA supplementation of 6g /day-3 pills, 2 times a day- as her w6/w3 ratio is at 15. After 2 weeks, her intake should be reduced to 4g/day for 2 weeks and further reduced to 3g/day for another 2 weeks. The patient can buy fish oilon our supplement website; it is named Omega 3 Optimize and contains two essential omega 3 fattyacids (EPA and DHA). The patient can buy fish oil on our supplement website; it is named Omega 3Optimize and contains two essential omega 3 fatty acids (EPA and DHA).https://reproductiveimmunologysupplements.com/The patient's MTHFR activity is of about 60% which places her in the moderate-risk category forhomocysteine accumulation leading to inflammation and pregnancy complications. She may benefitfrom methylated folate supplementation. She can buy methylated folate on our supplement website;it is named Methyl Optimize and contains methylated folate in addition toessential B vitamins. https://reproductiveimmunologysupplements.com/

Further Evaluation

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Pregmune’s parental compatibility assessment determines your risk for a defective placentationby assessing the type of protein KIR (expressed on your uterine NK cells) as well as your and yourpartner’s HLA-C genotype. Further, the level of HLA homozygosity and the degree of HLA allelesharing modulates maternal immune tolerance towards the embryo thus impacting chances for a

successful pregnancy.

Fetal HLA-C and Maternal KIR (Uterine NK Cell) Interaction

Maternal HLA-C Group Paternal HLA-C GroupC1/C1 C1/C2



KIR AB

Maternal KIRHaplotype

Uterine NK cell

+KIR2DS1PaternalHLA-CC1/C2

MaternalHLA-CC1/C1

Fetal HLAC1/C150%

Fetal HLAC1/C250%

Lower Risk

0 1 2

Maternal KIR A Content Score

6

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Fetal HLA-C and Maternal KIR (Uterine NK Cell) Interaction (cont'd.)

HLA Mismatches

Class II Alleles4 of 6 Total Class II Mismatches0 of 2 Supertype Mismatches

The patient is at lower risk for failure to generate immune tolerance towards paternal antigen foundon the embryo due to her specific maternal KIR-fetal HLA-C interaction. All looks good! No further

recommendations.

What this means for you


There is no lack of HLA class II allele mismatching which does not impact the ability of the maternalimmune system to generate tolerance for paternal antigens present on the embryo. All looks good!

No further recommendations.

Lower Risk

DQ Alpha DQ Beta HLA-DRB1 DRB3/4/5

0 of 22 of 21 of 21 of 2



Uterine natural killer cells (uNK) secrete a unique repertoire of cytokines and growth factors thatregulate blood vessel growth and development (also known as spiral artery remodeling) leadingto a healthy placentation that supports embryo growth. This secretion is modulated based on thetype of interaction (activating or inhibitory) taking place between your KIR receptor (present onthe surface of uNK) and the HLA-C of your embryo (inherited from both partners). This test aims topredict the risk you may have for a placental dysfunction that can lead to miscarriage and pregnancy

complications. Read more


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A certain level of difference between the mother's and father's HLA alleles (inherited by the embryoand defined as a mismatch) is necessary to actively generate immune tolerance of the embryo. Thus,couples with low number or no mismatched alleles for HLA genes may be more prone to infertility,

repeated implantation failure, and recurrent pregnancy loss. Read more

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HLA Homozygosity

Homozygosity of Class II Alleles

Anti-HY Immunity

HY Immunity


Homozygosity of HLA class II alleles leads to a limitation in the repertoire of paternal class II antigensthat can be presented to the maternal immune system. Thus, your immune system does not generatetolerance for paternal antigens found on the embryo, in the most optimum way. The more alleles

diversity there is, the better it is.


The patient is at lower risk for failure to generate immune tolerance towards paternal antigen foundon the embryo as there is no significant homozygosity of class II alleles. All looks good! No further

recommendations.


The patient is at lower risk for triggering the development of anti-HY responses. All looks good! Nofurther recommendations.


Healthy women, pregnant with a boy, may generate an immune response against HY antigens (malespecific minor histocompatibility antigen). Women with a first born boy and carrying HY restrictingHLA allele (HYrHLA alleles) have significantly reduced chances for a subsequent live birth.

Read more

Lower Risk

Lower Risk



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Your immune system (B cells) can target paternally derived HLA molecules, present on the embryo.HLA antibodies are commonly found in pregnant women and paternal-specific anti-HLA antibodiesare considered harmless during most pregnancies. Nevertheless, they have been associated withearly miscarriages, obstetrical complications and secondary infertility. They may denote a particularability from the maternal immune system to develop an aberrant immune response to paternally

derived antigens. Read more


The patient is seronegative for anti HLA antibodies. No further recommendation.

Anti-HLA Antibodies

HLA Class 1 Ab HD HLA Class 2 Ab HD

Lower Risk

>10K MFI4-10K MFI1.5-4K MFI0-1.5K MFI Anti-HLA Ab partner specificAnti-HLA Ab

Anti-HLA Ab typeAnti-HLA Ab titer



aHLA-Ab class I aHLA-Ab class II

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Your MTHFR genotype (both partners) can help us predict your risk of having embryos carrying twocopies of the T allele. This genotype has been associated with a failed implantation in patientsexperiencing reccurent pregnancy losses and/or recurrent implantation failures. Read more



The couple has 0% probability to have an embryo carrying MTHFR T/T mutation, which places themat Lower Risk for failed embryo transfer.

MTHFR 677 MTHFR 677Carrier father: MTHFR C/C Carrier mother: MTHFR C/T

Embryo MTHFR Prediction and Risk for Implantation Failure

Lower Risk

Carrier father Carrier motherMTHFR C/C MTHFR C/T

50% Non carrierHomozygous MTHFR C/C

50% carrierHeterozygous MTHFR C/T



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Female Assessment

Pregmune’s female assessment is an extensive panel of tests that determines if your thyroidfunction, hormonal and inflammation levels are within optimal range. It determines your risk forautoimmunity (production of antibodies potentially attacking your own body or your baby), as wellas your risk for blood clot formation. The panel also determines whether your diet is balanced or ifit may trigger inflammation by looking at your free fatty acid, folic acid and Vitamin D levels.

Female Chromosome Analysis

Low Risk46,XXNormal female karyotype

A karyotype (or chromosome analysis) is a test that evaluates the number and the structure of yourchromosomes (genetic blueprint) to detect any abnormalities.

Cytogenetic analysis of PHA stimulated cultures has revealed a FEMALE karyotype with anapparently normal GTG banding pattern in all cells observed. This result does not exclude thepossibility of subtle rearrangements below the resolution of cytogenetics or congenital anomalies

due to other etiologies.



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MTHFR, DNA Analysis

DNA Analysis

MTHFR (%)

30 36 65

677C/677T1298A/1298C

677T 1298C

677C 1298A

MTHFR gene

Medium Risk



MTHFR (5, 10 methylenetetrahydrofolate reductase) is one of the key enzymes involved in folicacid metabolism. A normal activity of this protein is crucial to maintain optimal levels of circulatingfolate and methionine and is key to prevent the accumulation of homocysteine responsible forinflammation and pregnancy complications. This test aims to detect genetic mutation that lower

MTHFR activity and trigger inflammation. Read more

60

12

*Disclaimer: Please note that this is not a complete thrombophilia assessment. Our limited thrombophilia panel analyzes four genes that areassociated with an increased risk for developing thromboembolism (blood clot formation) and experiencing reccurent pregnancy losses.Individuals who have inherited a pathogenic variant for one of these genes have a predisposition to excessive blood clot formation andmiscarriages.

Female Thrombophilia*

You have a MTHFR activity of about 60% which places you in the moderate-risk category forhomocysteine accumulation leading to inflammation and pregnancy complications.

You may benefit from methylated folate supplementation. You can buy methylated folate on oursupplement website; it is named Methyl Optimize and contains methylated folate in addition to

essential B vitamins.

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Female Thrombophilia (cont'd.)

Lower RiskHomocysteine

Homocysteine (umal/L)

14.5

Hyperhomocysteinemia (high homocysteine levels) is observed in approximately 5% of the generalpopulation and is associated with increased risk for many inflammatory disorders as well as adverse

pregnancy outcome.

The patient's homocysteine level is NORMAL

5.1

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DNA Analysis




PAI-1 4G/5G PolymorphismHeterozygous 4G/5G

Medium Risk

Promoter region of PAI-1 gene

The main function of plasminogen activator inhibitor type 1 (PAI-1) is to decrease fibrinolysis (processthat prevents blood clot formation). PAI-1 gene polymorphism (variation of the gene) has been shownto modulate the expression of PAI-1. This test aims to determine if you carry a specific allele that may

increase PAI-1 activity thus increasing your risk for blood clot and miscarriage. Read more

The patient is heterozygous 4G/5G for the PAI-1 genotype which places her in the lower riskcategory for thrombosis risk but she is at increased risk for miscarriage by +46%.

Your PAI-1 activity is within normal range but you are at higher risk for miscarriage. This shouldwarrant a close monitoring during pregnancy.

5G allele

4G allele

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DNA Analysis




Factor IINegative G/G

The prothrombin factor II 20210 G→A mutation has been identified as the second most commonindependent risk factor for blood clot and is associated with many disorders including thrombophiliaand pregnancy complications. This test checks if you carry the mutation responsible for increasing

your risk of blood clot and pregnancy complications.will help reduce your risk for blood clot. Read more

The patient is homozygous G/G for the Prothrombin Factor II 20210 nucleotide which places her atthe lower risk for thrombosis and does not increase her risk for another miscarriage. All is good! No

further recommendation.

Lower Risk

G allele

G allele

Prothrombin Factor II,nucleotide position 20210

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DNA Analysis




Factor VNegative G/G

Lower Risk

Factor V gene,nucleotide position 1691

Factor V (Leiden) mutation 1691 is a common point mutation G→A that increases you risk for bloodclot formation and accounts for many cases of reccurent pregnancy losses. This test checks if you

carry the mutation responsible for increasing these risks. Read more

The patient is homozygous G/G for the Factor V 1691 nucleotide which places her at the lower riskcategory for thrombosis risk and does not increase her risk for another miscarriage. All is good! No

further recommendation.

G allele

G allele

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The patient is not at higher risk for blood clot formation. All is good! No further recommendation.

INR, PT and aPTT are part of a prothrombin test aiming to determine your risk for blood clot orexcessive bleeding.

Lower Risk

1.0

10.1

26.0

INR, PT, aPTT

INR (ratio)

PT (sec)

aPTT (sec)

0.91.2

9.1 12.0

24 33

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HLA Autoimmune Disease Predisposition

Female Autoimmunity

Human Leukocyte Antigen (HLA) genes are a cluster of genes involved in initiating an immuneresponse. Some HLA alleles/haplotypes (group of genes) are associated with a predisposition to thedevelopment of various autoimmune conditions that can negatively impact your fertility. This testassesses your HLA system which is crucial to determine key mechanisms that may be impact your

fertility. Read more

You harbor DRB1*09:01. You harbor DRB1*11 which is part of the DR5 serotype. You harbor B*51. You harbor B*57 andC*07. This may predispose you to the following conditions: rheumatoid arthritis, lupus, Hashimoto’s thyroiditis, primaryantiphospholipid syndrome, thrombotic thrombocytopenic purpura, Behcet’s disease, psoriasis, and psoriatic arthritis.

Medium Risk

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Female Autoimmunity (cont'd.)

Lower RiskAntiphospholipid Antibodies (APAs)

Anticardiolipin IgG (GPL U/mL)

1420 80

<9

Anticardiolipin IgM (MPL U/mL) <9

12 20 80

Anticardiolipin IgA (APL U/mL) <9

12 20 80

Anti Beta-2 Glycoprotein I IgG (GPI IgG units) <9

20

Anti Beta-2 Glycoprotein I IgM (GPI IgM units) <9

32

Anti Beta-2 Glycoprotein I IgA (GPI IgAQ units) <9

25

Antiphosphatidylserine IgG (GPS IgG) 3

30

Antiphosphatidylserine IgM (MPS IgM) 9

22

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Antiphospholipid Antibodies (cont’d.)

Antiphosphatidylserine IgA (APS IgA) 1

19

Lupus Anti Coagulant dPT (sec) 28.70

47.6

Lupus Anti Coagulant dPT Confirm Ratio (MPL U/mL) 1.24

1.34

Lupus Anti Coagulant Thrombin Time (sec) 17.50

23

Lupus Anti Coagulant PTT-LA (sec) 32.50

51.9

Lupus Anti Coagulant DRVVT (sec) 34.20

47.0

Antiphospholipid antibody syndrome (APS) is an autoantibody-mediated disorder whereantiphospholipid antibodies are produced by the immune system against itself. This can lead toblood clot, multiple miscarriages and other pregnancy complications. This test aims to detect the

presence of these antibodies in your blood. Read more

The patient is negative for all APAs tested.

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Lower RiskAntinuclear Antibodies

Sjogrens Ab SS-A igG (Ro) (AI)

Sjogrens Ab SS-B igG (La) (AI)

<0.2

0.9

<0.2

0.9

ANA-DS.DNA (IU/mL) 1

5

Anti-Jo1 Ab (AI) <0.2

0.9

ANA RNP (AI) 0.2

0.9

ANA SM (AI) <0.2

0.9

ANA SCL (AI) <0.2

0.9

Antichromatin (AI) <0.2

0.9

21

9

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Antinuclear Antibodies (cont’d.)

ANA CENT (AI) <0.2

0.9

Antinuclear antibodies are autoantibodies targeting “normal” proteins within the nucleus of yourcells. Their presence in small amount is not worriesome and may not be associated to autoimmunedisease. But, when present in high concentration, they can reveal disease such as lupus. This test

will tell you if you are positive for the ANA listed below. Read more

The patient is NEGATIVE for all ANAs tested.

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Anti-CCP Antibodies and RheumatoidArthritis Factor Lower Risk

Anti-CCP Antibodies IgG/IgA (IU/mL)

Rheumatoid Factor Levels (IU/mL)

4

<10

20 40 60

13.9

Anti-citrullinated protein antibodies (anti-CCP) and rheumatoid factor (RF) are two tests used todetect rheumatoid Arthritis (RA) and other collagen vascular disease. Read more

The patient is NEGATIVE for both Rheumatoid Factor and Anti-CCP antibodies. All looks good! Nofurther recommendation.

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The thyroid is a butterfly-shaped gland located at the base of your throat. It is a key regulator of yourhormones production and its optimal function is crucial during pregnancy. It also helps to keep yourmetabolism in check, regulates your heart, body temperature and digestive system. Read more

Female Thyroid

Thyroid Function Lower Risk

Anti-Thyroglobulin Antibody (IU/mL)

0.9

<1

Thyroid Peroxidase Antibody (IU/mL) 9

34

TSH (ug/dL) 1.08

0.45 4.5

TSH Receptor Antibody (ug/dL) <1.10

1.75

Total T4 (ug/dL) 8.40

4.5 12

Total T3 (ng/dL) 123

71 180

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Patient 's white blood cell count and platelet count are within normal range. All looks good! Nofurther recommendation.

Female Thyroid (cont'd.)

Female Serological

Cell Blood Count (CBC)

White Blood Cells (WBC) (x10E3/uL) 7.9

Lower Risk

Your white blood cell (WBC) are part of your immune system and help fight infections. Platelets areessential for normal blood clotting. Determining your levels will help detect some health issues.

3.4 10.8

Platelets (x10E3/uL) 406

150 450


25

The patient's hormones are all within normal level and no antibodies have been detected. All looksgood! No further recommendations.

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The patient has normal levels of total immunoglobulins. All looks good! No further recommendation.

Immunoglobulins are proteins produced by specific immune cells (plasma cells) and play a key rolein the body’s immune system. They are produced in response to bacteria, viruses and

non-selfantigens. This test can help us determine if you have an allergic reactions, among otherthings.

Female Inflammatory

Total Immunoglobulin Lower Risk

Immunoglobulin G Levels (mg/dL) 1359

586 1602

87 352

Immunoglobulin A Levels (mg/dL) 179

Immunoglobulin M Levels (mg/dL) 64

26 217

Immunoglobulin E Levels (IU/mL) 217

6 495

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Female Inflammatory (cont'd.)

Complement C4 Activity (mg/dL)

Complement Activity Lower Risk

28

Complement C3 Activity (mg/dL) 172

The complement is a system of proteins that play a key role in inflammation. Complement activationhas been linked to pregnancy complications such as pre-eclampsia. Read more

The patient's C3 and C4 complement levels are within normal range.

12 38

82 167

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The patient has a TH2 bias. The patient may discuss her results with her healthcare provider as herTh2 profile may reflect an underlying issue.

The TH1:TH2 intracellular cytokine ratio characterizes the balance between pro-inflammatory IFNg Thelper producing cells and anti-inflammatory IL-4 T helper producing cells. Read more

Th1/Th2 Medium Risk

Th1/Th2 (mg/dL) 7.00

7.8 19.5 22.5 28

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The patient's NK cytotoxic activity (NKa) levels are within normal levels. All looks good! No furtherrecommendation.

Natural killer (NK) cells are a population of immune cells, found at high levels in patients withautoimmune disease. NK cell activity has been associated with the pathogenesis of reccurent

pregnancy loss. Read more

Natural Killer Cell Activity (NKa) Lower Risk

NK Cytotoxic Activity Assay (%) 12.5

12.1 37.4

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Regulatory T cells (Treg cells) are key immune cells promoting maternal tolerance towards theembryo. During pregnancy, these cells are recruited from your blood to your uterus to promote

embryo implantation and the progression of your pregnancy. Read more

The patient's Treg cell levels are within normal levels.

7.51.6

1.8Regulatory T Cell Levels

Regulatory T Cells (Treg cells) Lower Risk

30

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Female Metabolic

SHBG (nmol/L) 26.4

24.6 122

Free Testosterone (pg/mL) 3.2

4.2

Testosterone (Premenopausal) (ng/dL) 17

8 60

Insulin (uIU/mL) 15.2

2.6 24.9

DHEA-Sulfate (ug/dL) 94.0

57.3 279.2

17 Hydroxyprogesterone (ng/dL) 18

15 290

Fasting Glucose (mg/dL) 94.0

65 99

AMH (ng/mL) 0.264

Insulin Resistance and Polycystic OvarianSyndrome (PCOS) Assessment Medium Risk

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Based on her hemoglobin A1c levels, the patient is pre-diabetic. The patient may discuss theseresults with her healthcare provider as she may benefit from therapies to restore her hormonal levels

within normal range.

Polycystic ovarian syndrome (PCOS) is a common endocrine condition, triggered by high levels ofandrogens and resistance to insulin. Women with PCOS may experience infertility and are at

increased risk for pregnancy complications such as gestational diabetes and hypertension. This testscreen for many hormones whose dysregulations may cause PCOS. Read more

Female Metabolic (cont'd.)

Insulin Resistance and Polycystic OvarianSyndrome (PCOS) Assessment (cont’d.)

Homocysteine

Hemoglobin A1C

5.1

5.9

5.7 6.4

14.5

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Vitamin D is a fat-soluble vitamin belonging to the steroid hormone family. Expressed in the ovaryand uterus, this vitamin regulates many factors including the steroid hormones estrogen and

progesterone, the immune function and reduces oxydative stress. Read more

20 30 100

25 Hydroxy Vitamin D levels (ng/mL) 28.1

Medium RiskVitamin D

Female Nutritional

33

The patient has insufficient Vitamin D levels, which does require supplementation. You can buyVitamin D on our supplement website; it is named Vitamin D-Optimize.

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Folic acid is a B vitamin, found naturally in food. A compromised folate status in pregnant women isassociated with recurrent miscarriages and fetal neural tube defects. Read more

Folic Acid levels (ng/mL) 17.6

3

Lower RiskFolic Acid

Female Nutritional (cont’d.)

34

The patient has sufficient folic acid levels.

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Both ω3 and ω6 fatty acids are essential components of your cells but ω3 only (EPA and DHA) canhelp reduce maternal inflammation and oxidative stress. This can have benefits on your egg qualityand can reduce placental inflammation. This test determines your fatty acid levels and will let you

know if you are at increased risk for inflammation and pregnancy losses. Read more

W6/W3 (ratio)Propensity to stimulate pro (ω6) or anti (ω3) inflammatory pathways

5 10.8

15.00

2.7 10.9

Competition between pro (ω6) and anti (ω3) inflammatory precursorPotential for Inflammation (mmol/L) 13.46

6 14

Long-term intake of EPA+DHA3.60Omega 3 Index (mmol/L)

Fatty Acid Higher Risk

35

The patient is in the High-risk category and may benefit from EPA/DHA supplementation of 6g /day-3 pills, 2 times a day- as her w6/w3 ratio is at 15. After 2 weeks, her intake should be reduced to 4g/day for 2 weeks and further reduced to 3g/day for another 2 weeks. The patient can buy fish oil onour supplement website; it is named Omega 3 Optimize and contains two essential omega 3 fatty

acids (EPA and DHA).

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The patient has a ω6 bias with higher ω6 metabolites levels and lower ω3 metabolites production.The patient has a ω6 profile (pro-inflammatory) and is prone to inflammation.

8 130

EPA (nmol/mL) 47

45 365

DHA (nmol/mL) 162

DHA & EPA levels Higher Risk

36

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Leptin is produced by adipocytes (fat cells) and play an important role in reproduction. High leptin levels have beenshown to disrupt folliculogenesis (maturation of follicle leading to the production of a fertilizable egg). This translates

into “poor” embryo quality and ultimately higher risks for pregnancy failure through IVF. Read more

4.6 24

Leptin Levels (ng/mL) 70.1

Leptin Levels Higher Risk

37

The patient's leptin levels are high.

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Cytogenetic analysis of PHA stimulated cultures has revealed a MALE karyotype with an apparentlynormal GTG banding pattern in all cells observed. This result does not exclude the possibility ofsubtle rearrangements below the resolution of cytogenetics or congenital anomalies due to other

etiologies.

Low Risk46,XYNormal male karyotype

Pregmune’s male assessment is an extensive panel of tests that determines if your partner’s se-menquality is within optimal range. It determines if he is at risk for blood clot formation. By looking at hisfree fatty acid as well as his MTHFR genotype, the panel determines whether his diet is balanced or

if it may trigger inflammation.

Male Assessment

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Male Chromosome Analysis




DNA Analysis

MTHFR (%)

30 36 65

MTHFR gene



MTHFR (5, 10 methylenetetrahydrofolate reductase) is one of the key enzymes involved in folicacid metabolism. A normal activity of this protein is crucial to maintain optimal levels of circulatingfolate and methionine and is key to prevent the accumulation of homocysteine responsible forinflammation and pregnancy complications. This test aims to detect genetic mutation that lower

MTHFR activity and trigger inflammation.

92

Lower RiskMTHFR 677

Male MTHFR

39

677C 1298A

677C 1298C

677C/677C1298A/1298C

The patient has a MTHFR activity of about 92% which places him in the low-risk category forhomocysteine accumulation leading to inflammation and pregnancy complications.

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INR, PT, aPTT

INR (ratio)

PT (sec)

aPTT (sec)

The patient is at not at higher risk for blood clot formation. All is good! No further recommendation.

INR, PT and aPTT are part of a prothrombin test aiming to determine your risk for blood clot orexcessive bleeding. Read more

1.0

10.6

28

0.91.2

9.1 12.0

24 33

Lower Risk

40

*Disclaimer: Please note that this is not a complete thrombophilia assessment. Our limited thrombophilia panel analyzes 3 parameters: theprothrombine time (time range for blood to clot), its ratio INR (used for easier comparison between laboratories) and the activated partialthromboplastin time (another measure of time range for blood to clot with a narrower reference range).

Male Thrombophilia*

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W6/W3 (ratio)Propensity to stimulate pro (ω6) or anti (ω3) inflammatory pathways

Competition between pro (ω6) and anti (ω3) inflammatory precursorPotential for Inflammation (mmol/L)

Long-term intake of EPA+DHAOmega 3 Index (mmol/L)

Fatty Acid

Both ω3 and ω6 fatty acids are essential components of your cells but ω3 only (EPA and DHA) canhelp reduce inflammation and oxidative stress. This can have benefits on your semen quality bypreventing sperm DNA damage. This test determines your fatty acid levels and will let you know if

you are at increased risk for inflammation and poor semen quality. Read more

16.90

18.00

3.30

AA/DHA (ratio)Propensity to stimulate pro (ω6) or anti (ω3) inflammatory pathways

7.60

Higher Risk

Male Nutritional Analysis

3.3 6.4

22.8

6.7 12

6.3 14.8

41

The patient is in the High-risk category and may benefit from EPA/DHA supplementation of 6g /day -3 pills, 2 times a day-as her w6/w3 ratio is at 16.9. After 2 weeks, his intake should be reduced to 4 g/day for 2 weeks and further reduced to3g/day for another 2 weeks. The patient can buy fish oil on our supplement website; it is named Omega 3 Optimize and

contains two essential omega 3 fatty acids (EPA and DHA).

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8 130

EPA (nmol/mL)

45 365

DHA (nmol/mL)

DHA & EPA levels

The patient has a ω6 bias with higher ω6 metabolites levels and lower ω3 metabolites production.The patient has a ω6 profile (pro-inflammatory) and is prone to inflammation which may impact his

semen quality.

Male Nutritional Analysis (cont’d.)

Higher Risk

121

51

42

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Leptin Levels (ng/mL)

Leptin Levels

Leptin is produced by adipocytes (fat cells) and play an important role in reproduction. High leptinlevels have been shown to disrupt folliculogenesis (maturation of follicle leading to the production ofa fertilizable egg). This translates into “poor” embryo quality and ultimately higher risks for pregnancy

failure through IVF. Read more

Male Nutritional Analysis (cont’d.)

0.3 13.4

30.1

Higher Risk

43

The patient's leptin levels are high.

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44

Report Review

Disclaimer

Patient Name: Jane Doe

Partner Name: John Doe

Report ID: DOEJ010283F-PGM1

Provider:

NPI:

Physician’s Signature Date

DOB: 01/02/1983

DOB: 01/10/1977

Dr. Andrea Vidali

1265014583

Please note that this remote consultation is to be considered of high complexity and will be billedaccordingly. This consultation is to be considered complete with the creation of this document and ofthe forwarding of this document to the ordering physician. This document required about 75 minutesto be created and completed. Please also note that this document constitutes the entire content of

the patient’s medical record.

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Additional Reading

Parental compatibility

Fetal HLA-C and maternal KIR (uterine NK cell) interaction

Physiology:

Uterine natural killer cells (uNK) secrete a unique repertoire of cytokines and growth factors that regulate blood vessel growth and development (also known as spiral artery remodeling) leading to a healthy placentation that supports embryo growth1. Killer immunoglobulin-like receptors (KIRs) present on the surface of uNK interact with HLA-C on the trophoblast to promote (activating KIR) or suppress (inhibiting KIR) this vascular remodeling2.

HLA-C genotype of both the father and the mother has a significant impact on the risk for defective placentation which can manifest as recurrent miscarriage, intrauterine growth restriction and/or preeclampsia3.

Published clinical data:

Clinical studies found that the combination of maternal KIR-AA and fetal HLA-C2, but not fetal HLA-C1, lead to increased risk of recurrent losses4, preeclampsia3 and low birth weight5.

Indeed, maternal KIR AA frequency is increased in pregnancies ending with a loss or affected pre-eclampsia compared with control pregnancies when the fetus has more C2 genes than the mother or when fetal C2 is inherited paternally4.

Further, a study comparing HLA-C and KIR haplotypes of both partners in couples with three or more spontaneous miscarriages (RPL) versus couples with no fertility issues showed a significant association of the lack of activating KIR in the affected woman (KIR AA genotype) and an increased HLA-C2 group frequency in the RPL couples6.

Pregmune clinical data:

In our practice, a retrospective analysis based on 761 patients has shown that pregnancy success rate is significantly reduced (*** means p-value=.005) in patient with KIRAA haplotype as compared to patients with KIRAB haplotype with KIR2DS1 as seen in Figure 1.

Although not significant (p value=.1), the same trend was observed when comparing KIR AA to KIR AB without KIR2DS1 haplotype.

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Figure 1: Pregnancy success rate in our patient population based on the maternal KIR haplotype: KIR AA, KIR AB + KIR2DS1 or KIR AB – KIR2DS1.

This observation is true in patients with KIRAA haplotype specifically when the fetus has more C2 than the mother (*** means p-value=.005).

The success rate in KIRAA patient is reduced by 35% as compared to KIR AB-2DS1 patients and by 23% as compared to KIRAB+2DS1 patients with less C2 than the fetus.

References:

1. Moffett-King A. Natural killer cells and pregnancy. Nat Rev Immunol (2002) 2(9):656–63.2. Chazara O, Xiong S, Moffett A. Maternal KIR and fetal HLA-C: a fine balance. J Leukoc Biol. 2011

Oct;90(4):703-16. doi: 10.1189/jlb.0511227. Epub 2011 Aug 26. Review.3. Hiby SE,Walker JJ, O’Shaughnessy KM, Redman CW, Carrington M, Trowsdale J, et al.

Combinations of maternal KIR and fetal HLA-C genes influence the risk of preeclampsia andreproductive success. J Exp Med (2004) 200(8):957–65.

4. Hiby SE, Apps R, Sharkey AM, Farrell LE, Gardner L, Mulder A, Claas FH, Walker JJ, Redman CW,Morgan L, Tower C, Regan L, Moore GE, Carrington M, Moffett A. Maternal activating KIRs protectagainst human reproductive failure mediated by fetal HLA-C2. J Clin Invest. 2010 Nov;120(11):4102-10.

5. Hiby SE, Apps R, Chazara O, Farrell LE, Magnus P, Trogstad L, Gjessing HK, Carrington M, MoffettA. Maternal KIR in combination with paternal HLA-C2 regulate human birth weight. J Immunol. 2014Jun 1;192(11):5069-73.

6. Hiby SE, Regan L, Lo W, Farrell L, Carrington M, Moffett A. Association of maternal killer-cellimmunoglobulin-like receptors and parental HLA-C genotypes with recurrent miscarriage. HumReprod. 2008 Apr;23(4):972-6.

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HLA mismatches

Physiology:

Human Leukocyte Antigen (HLA) genes [commonly referred to as the major histocompatibility complex (MHC) genes], are a cluster of genes present on the chromosome 6 and are involved in antigen presentation to T cells to initiate an immune response.

In most cases, this immune reaction leads to the destruction of cells displaying "non-self" peptides.

There are two groups of HLA molecules, the class I includes HLA-A, -B, -C and the class II that includes HLA-DR, -DQ, and -DP.

HLA molecules play a key role in organ transplantation and are associated with many diseases including auto-immune disorders1-2.

While a mismatch in Human Leukocyte Antigen (HLA) between a donor and a recipient in organ transplantation may lead to a graft rejection, most often due to the formation of antibodies3, a certain level of difference between the mother's and father's HLA alleles4 (inherited by the embryo and defined as a mismatch) is necessary to actively generate immune tolerance of the embryo5. Thus, couples with significant matched alleles for HLA genes may be more prone to infertility, repeated implantation failure, and recurrent pregnancy loss6.


Although still controversial, the influence of HLA sharing on pregnancy outcome has been shown in clinical studies. Prospective studies of pregnancy outcome in an inbred population of European origins named “Hutterites” previously demonstrated increased fetal loss rates among couples with matching HLA4-6.

References:

1. Doherty PC,Zinkernagel RM. A biological role for the major histocompatibility antigens. Lancet(1975) 1(7922):1406–9.

2. Complete sequence and gene map of a human major histocompatibility complex. The MHCsequencing consortium. Nature (1999) 401(6756):921–3.

3. Jucaud V. The Immunogenicity of HLA Class II Mismatches: The Predicted Presentation of NonselfAllo-HLA-Derived Peptide by the HLA-DR Phenotype of the Recipient Is Associated with theFormation of DSA. J Immunol Res. 2017; 2017:2748614.

4. Ober C. Studies of HLA, fertility and mate choice in a human isolate. Hum Reprod Update. 1999Mar-Apr;5(2):103-7. Review.

5. Papúchová H, Meissner TB, Li Q, Strominger JL, Tilburgs T. The Dual Role of HLA-C in Toleranceand Immunity at the Maternal-Fetal Interface. Front Immunol. 2019 Dec 9;10: 2730.

6. Ober C, Hyslop T, Elias S, Weitkamp LR, Hauck WW. Human leukocyte antigen matching and fetalloss: results of a 10-year prospective study. Hum Reprod. 1998 Jan;13(1):33-8.

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Anti HY-immunity

Physiology:

During a first pregnancy with a male fetus, the maternal immune system could be activated by allogenic fetal cells possessing male-specific minor histocompatibility inherited antigens (HYrHLA allele) that are encoded by genes localized on the Y chromosome. In some women, this can lead to an acute immune reaction leading to the production of HY antibodies by B cells. These antibodies can last for several years in the maternal serum1. This may lead to secondary recurrent miscarriage (with male or female embryos) and to giving birth to boys with a low birth weight2-3.


Although present in 30% of women, anti HY antibodies have been linked to secondary recurrent miscarriage in subsequent pregnancy and other pregnancy complications such as stillbirth, placental abruption or fetal growth retardation, all these events being the results of an inflammatory environment.

A large cohort study including women with unexplained secondary RPL showed that these patients were more prone to miscarriage during their subsequent pregnancy when having a firstborn boy compared to a firstborn girl (46 % versus 24 % respectively)4. In these patients with a first-born son, the presence of H-Y restricting HLA class II alleles (HLA-DRB1*15, HLA- DQB1*05:01/02 and HLA–DRB3*03:01) has been correlated with lower chance of live birth and a low male/female ratio among the subsequent births (increased loss rate of male embryos)5.

Patients with no copy of HY restricting alleles have a subsequent live birth rate of 73%, which dropped to 58% with 1 allele copy and 49% with two allele copies.

Pregmune clinical data:

In our practice, a retrospective analysis based on 828 patients has shown that miscarriage rate is significantly increased (** means p-value=.01) in patient with at least one HY restricting allele and undergoing an embryo transfer (Figure 2).

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Figure 1: Miscarriage rate in our patient population based on the presence of HY restricting alleles in female patients.

References:

1. Christiansen OB, Steffensen R, Nielsen HS. Anti-HY responses in pregnancy disorders. Am JReprod Immunol. 2011 Jul;66 Suppl 1:93-100.

2. Kolte AM, Steffensen R, Christiansen OB, Nielsen HS. Maternal HY-restricting HLA class II allelesare associated with poor long-term outcome in recurrent pregnancy loss after a boy. Am J ReprodImmunol. 2016 Nov;76(5):400-405.

3. Nielsen HS, Steffensen R, Varming K, Van Halteren AG, Spierings E, Ryder LP, Goulmy E,Christiansen OB. Association of HY-restricting HLA class II alleles with pregnancy outcome inpatients with recurrent miscarriage subsequent to a firstborn boy. Hum Mol Genet. 2009 May1;18(9):1684-91.

4. Christiansen OB, Steffensen R, Nielsen HS. The impact of anti-HY responses on outcome in currentand subsequent pregnancies of patients with recurrent pregnancy losses. J Reprod Immunol. 2010May;85(1): 9-14.

5. Nielsen HS, Steffensen R, Varming K, Van Halteren AG, Spierings E, Ryder LP, Goulmy E,Christiansen OB. Association of HY-restricting HLA class II alleles with pregnancy outcome inpatients with recurrent miscarriage subsequent to a firstborn boy. Hum Mol Genet. 2009 May 1;18(9): 1684-91.

Anti-HLA Antibodies

Physiology:

Pregnancy is marked by profound changes of the maternal immune system that allow the semi-allogeneic fetus to implant and grow within the uterus.

Among immune cells, B lymphocytes (B cells) are key players in the maternal immune adaptation towards fetal implantation.

0

5

10

15

20

25

30

35

Loss

Perc

enta

ge (%

)

Miscarriage rate based on presence/absence of HY restricting allele

Absence of HY restricting allele Presence of HY restricting allele

**, p=.007

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Some B cell subsets are capable of producing antibodies that target components of the embryo/fetus encoded by the paternal genetics – most notably paternally derived HLA molecules.

Anti-HLA antibodies are present in one third of healthy successful pregnancies1. Nevertheless, the presence of partner-specific anti-HLA antibodies (particularly those that fix complement) can be harmful to pregnancy maintenance and can induce miscarriage, or later complications such as preeclampsia, intrauterine growth restriction, or stillbirth.


In patient with a history of secondary recurrent pregnancy losses (SRPL: patient experiencing several losses after the birth of a first child), the frequency of anti HLA antibodies was found to be higher in those who had obstetrical complications compared to those with uncomplicated prior birth2. Further, when detected in early pregnancy in SRPL patients, the chances for a live birth are decreased.

References:

1. L. Regan, P.R. Braude, D.P. Hill. A prospective study of the incidence, time of appearance andsignificance of anti-paternal lymphocytotoxic antibodies in human pregnancy. HumanReproduction, Volume 6, Issue 2, February 1991, Pages 294–298.

2. Henriette Svarre Nielsen 1, Marian D Witvliet, Rudi Steffensen, Geert W Haasnoot, Els Goulmy, OleBjarne Christiansen, Frans Claas. The presence of HLA-antibodies in recurrent miscarriage patientsis associated with a reduced chance of a live birth. Reprod Immunology.2010 Dec;87(1-2):67-73. doi: 10.1016/j.jri.2010.05.006

Embryo MTHFR prediction and risk for implantation failure

Physiology:

MTHFR (5, 10 methylenetetrahydrofolate reductase) is one of the key enzymes involved in folic acid metabolism and is directly responsible for homocysteine conversion to methionine (Figure 1). Folate and methionine are essential for nucleic acid synthesis (component of your DNA) and are required for methylation reactions.

The gene methylenetetrahydrofolate reductase (MTHFR) shows a high prevalence of polymorphism, folate metabolism can be impaired with MTHFR 677C/T and MTHFR 1298A/C polymorphism, two variants in the MTHFR gene that are commonly found in the population regardless of ethnicity1.

MTHFR 677C>T polymorphism is associated with a 70% decrease of MTHFR enzymatic activity in homozygous (MTHFR T/T), while the heterozygous (C/T) genotype has a 35% decreased activity. MTHFR 1298A>C polymorphism results in a slight decrease in MTHFR activity 2-4.

Normal MTHFR activity is crucial to maintain normal levels of circulating folate and methionine and is key to prevent the accumulation of homocysteine.

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Hyperhomocysteinemia is observed in approximately 5% of the general population and is associated with increased risk for many inflammatory disorders including auto immune disorders (Rheumatoid Arthritis, Diabetes, Multiple Sclerosis, Systemic Lupus erythematosus, Grave disease), birth defects and adverse pregnancy outcomes (pre-eclampsia, placental abruption, spontaneous abortion, low birth weight), as well as vascular and neurodegenerative diseases. Homocysteine is at the epicenter of oxidative stress and DNA methylation errors.

Figure 1: The folate cycle and the methionine cycle are two metabolic pathways existing independently. In the folate cycle, folic acid is imported into cells and reduced to tetrahydrofolate (THF). THF is converted to 5, 10-methylene-THF (me-THF). Me-THF is then reduced to 5 methyltetrahydrofolate (mTHF) by methylenetetrahydrofolate reductase (MTHFR). 5-mTHF is demethylated to complete the folate cycle by donating a carbon into the methionine cycle through the methylation of homocysteine (hCYS) by methionine synthase and its cofactor vitamin B12 (B12).


While MTHFR missenses are largely present in the general population that is overall healthy and fertile5, it is true that women experiencing fertility issues are more likely to carry at least one of these mutations 6-7.

Numerous studies showed that a compromised folate status in pregnant women is associated with recurrent spontaneous abortion (miscarriage)8-10 and fetal neural tube defects11.

A study12 found an association between the embryo genotype (MTHFR 677 TT) and a failed implantation in patients experiencing reccurent pregnancy losses and/or recurrent implantation failures. The study showed a higher proportion of embryos carrying 2 copies of the missense

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(677TT) in the failed implantation group (25%) as compared to embryos with successful implantation (5%).

References:

1. Chango, A. et al. The effect of 677C—>T and 1298A—>C mutations on plasma homocysteine and5,10-methylenetetrahydrofolate reductase activity in healthy subjects. Br J Nutr 83, 593-596(2000).

2. van der Put, N. M. et al. A second common mutation in the methylenetetrahydrofolate reductasegene: an additional risk factor for neural-tube defects? Am J Hum Genet 62, 1044-1051,doi:10.1086/301825 (1998).

3. Weisberg, I., Tran, P., Christensen, B., Sibani, S. & Rozen, R. A second genetic polymorphism inmethylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. MolGenet Metab 64, 169-172, doi:10.1006/mgme.1998.2714 (1998).

4. Ogino S, Wilson RB. Genotype and haplotype distributions of MTHFR677C>T and 1298A>C singlenucleotide polymorphisms: a meta-analysis. J Hum Genet. 2003;48(1):1-7.

5. Peng F, Labelle LA, Rainey BJ, et al. Single nucleotide polymorphisms in themethylenetetrahydrofolate reductase gene are common in US Caucasian and Hispanic Americanpopulations. Int J Mol Med 2001; 8(5):509–11.

6. Clément A, Menezo Y, Cohen M, Cornet D, Clément P. 5-Methyltetrahydrofolate reduces bloodhomocysteine level significantly in C677T methyltetrahydrofolate reductase single-nucleotidepolymorphism carriers consulting for infertility. J Gynecol Obstet Hum Reprod. 2019 Aug 22.

7. Zeng S, Wang X, Wang Y, Xu Z, Zhang J, Liu W, Qian L, Chen X, Wei J, Yang X, Gong Z, Yan Y.MTHFR C677T polymorphism is associated with follicle-stimulating hormone levels and controlledovarian hyperstimulation response: a retrospective study from the clinical database. Fertil Steril.2019 May;111(5):982-990.e2.

8. Gupta S, Agarwal A, Banerjee J, Alvarez JG. The role of oxidative stress in spontaneous abortionand recurrent pregnancy loss: a systematic review. Obstet Gynecol Surv. 2007 May; 62(5):335-47;quiz 353-4. Review. PubMed PMID: 17425812.

9. Xu Y, Ban Y, Ran L, Yu Y, Zhai S, Sun Z, Zhang J, Zhang M, Hong T, Liu R, Ren L, Hu L. Relationshipbetween unexplained recurrent pregnancy loss and 5,10-methylenetetrahydrofolate reductase)polymorphisms. Fertil Steril. 2019 Mar;111(3):597-603.

10. Pereza N, Ostojić S, Kapović M, Peterlin B. Systematic review and meta-analysis of geneticassociation studies in idiopathic recurrent spontaneous abortion. Fertil Steril. 2017 Jan;107(1):150-159.e2.

11. Cai CQ, Fang YL, Shu JB, Zhao LS, Zhang RP, Cao LR, Wang YZ, Zhi XF, Cui HL, Shi OY, Liu W.Association of neural tube defects with maternal alterations and genetic polymorphisms in one-carbon metabolic pathway. Ital J Pediatr. 2019 Mar 14;45(1):37.

12. Brenner B, Aharon A. Thrombophilia and adverse pregnancy outcome. Clin Perinatol. 2007 Dec;34(4):527-41, v. Review. PubMed PMID: 18063103.

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Female Assessment

Thrombophilia

a. MTHFR

Physiology:

MTHFR (5, 10 methylenetetrahydrofolate reductase) is one of the key enzymes involved in folic acid metabolism and is directly responsible for homocysteine conversion to methionine (Figure 1). Folate and methionine are essential for nucleic acid synthesis (component of your DNA) and are required for methylation reactions.

The gene methylenetetrahydrofolate reductase (MTHFR) shows a high prevalence of polymorphism, folate metabolism can be impaired with MTHFR 677C/T and MTHFR 1298A/C polymorphism, two variants in the MTHFR gene that are commonly found in the population regardless of ethnicity1.

MTHFR 677C>T polymorphism is associated with a 70% decrease of MTHFR enzymatic activity in homozygous (MTHFR T/T), while the heterozygous (C/T) genotype has a 35% decreased activity. MTHFR 1298A>C polymorphism results in a slight decrease in MTHFR activity 2-4.

Normal MTHFR activity is crucial to maintain normal levels of circulating folate and methionine and is key to prevent the accumulation of homocysteine.


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Figure 1: The folate cycle and the methionine cycle are two metabolic pathways existing independently. In the folate cycle, folic acid is imported into cells and reduced to tetrahydrofolate (THF). THF is converted to 5, 10-methylene-THF (me-THF). Me-THF is then reduced to 5 methyltetrahydrofolate (mTHF) by methylenetetrahydrofolate reductase (MTHFR). 5-mTHF is demethylated to complete the folate cycle by donating a carbon into the methionine cycle through the methylation of homocysteine (hCYS) by methionine synthase and its cofactor vitamin B12 (B12).


While MTHFR missenses are largely present in the general population that is overall healthy and fertile5, it is true that women experiencing fertility issues are more likely to carry at least one of these mutations 6-7.


Some of these studies compared the relation between the maternal MTHFR polymorphism (a key factor in homocysteine regulation) and plasma homocysteine levels in pregnant women. Elevated serum homocysteine concentration is an important problem causing birth defects12, pre-eclampsia13-15, placental abruption13, low birth weight and other maternal or fetal complications16.

b. PAI-1

Physiology:

The main function of plasminogen activator inhibitor type 1 (PAI-1) is to decrease fibrinolysis (process that prevents blood clot formation), which leads to fibrin accumulation and vascular thrombosis17. An elevated plasma PAI-1 concentration has been identified as a risk factor for the development of myocardial infarction18.

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Further, during implantation and placentation, PAI-1 inhibits the extra cellular matrix (ECM) degradation and remodeling of the endometrium, which inhibits the invasion of trophoblast19-20. This suggests that PAI-1 expression may be a contributing factor in the etiology of recurrent pregnancy loss.

Indeed, the resulting shallow implantation may be associated with PAI-1 polymorphism and pregnancy losses or pre-eclampsia.

PAI-1 gene polymorphism has been shown to modulate the expression of PAI-1. Individuals homozygous for 4G (4G/4G) have the highest plasma PAI-1 level, heterozygous individuals (4G/5G) display an intermediate level while 5G homozygous individuals (5G/5G) have the lowest PAI-1 level21-22.


Many published studies comparing PAI-1 genotype between fertile controls and women experiencing reccurent pregnancy losses (RPL) and/or reccurent implantation failure (RIF) showed an increased incidence of the PAI-1 4G/4G allele in infertile patients. Indeed, PAI-1 4G/4G is associated with increased risks of RIF (+350%), RPL (+120%) and RIF with RPL (+170%)25.

The presence of one allele 4G (heterozygous patient) was shown to increase a patient risk for miscarriage by 46%23 while being homozygous (4G/4G) increases a patient risk for a miscarriage by 89%-100%23-24.

Coagulation and fibrinolytic cascades are key component in the process leading to pre-eclampsia. Therefore, increased PAI-1 levels may promote spiral arterial or intervillous thrombosis that reduces placental perfusion and may trigger pre-eclampsia27.

Indeed, in a review including 58 meta-analysis, PAI-1 4G/5G polymorphism was shown to be a significant contributor to the pathogenesis of PE28.

c. Factor V Leiden

Physiology:

Factor V (Leiden) mutation 1691 is a common point mutation G→A that causes a resistance of Factor V protein degradation by the activated protein C (APC).

This mutation is associated with increased risk of venous thrombosis (increased risk of developing blood clots) and accounts for many cases of reccurent pregnancy losses29-30.

While the mutation is present in 5% of the Caucasian women population31, most people with factor V Leiden will not develop abnormal clots.

Nevertheless, this mutation is responsible for 20-25% of the inherited venous thrombosis cases32.

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Heterozygous individuals (G/A) have 3-8 times more risk of developing thrombosis than the normal population33 and homozygous individuals (A/A) have 50 to 100 times greater risk for thrombosis34.

Women carrying the factor V Leiden mutation, who are pregnant or on estrogen therapy, have a 30 times higher risk for venous thrombosis35.


Studies comparing Factor V genotype between fertile controls and women experiencing reccurent pregnancy losses (RPL) showed an increased incidence of the 1691 A allele in infertile patients. The A allele frequency of Factor V Leiden was shown to be significantly higher in RPL patients (7.5%) as compared to fertile controls (1.88%)36.

Depending on the studies, the A allele frequency varies from 8 to 32% in RPL patients as compared to 4 to 10% in the fertile control population37-38.

Further, the presence of the A allele was found to place a patient with RPL at a four times higher risk for another miscarriage39 while a meta-analysis estimates that this risk doubles in RPL patients40.

Hypercoagulability due to thrombophilic factors is a key event leading to a decreased placental perfusion which induces symptoms of pre-eclampsia.

A meta-analysis including thirty-one studies with 7,522 patients showed that the factor V Leiden polymorphism (1691 G→A) is associated with a 2-fold increased risk for all and severe preeclampsia41. Another meta-analysis comparing healthy pregnancy to pre-eclamptic pregnancy showed that factor V Leiden polymorphism (1691 G→A) is associated with a +87% increased risk for pre-eclampsia42.

Risk for pre-eclampsia has been estimated to double in carrier patients for the allele 1691 A allele40.

d. Prothrombin Factor II

Physiology:

The prothrombin factor II 20210 G→A mutation has been identified as the second most common independent risk factor for venous thrombosis43 and is associated with many disorders including thrombophilia, pregnancy complications or cardiovascular diseases.

The mutation is associated with increased plasma levels of prothrombin and is present in 1-3 % of the Caucasian women population31.

The 20210 A allele of the prothrombin gene is associated with an approximately 3-fold increased risk of venous thrombosis44-48.

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Many studies have found that carrier for the prothrombin factor II 20210 G→A mutation are at significantly higher risk for early recurrent pregnancy loss and sporadic later loss (risk doubles)49-

50. Pregnancy-associated thrombosis has been estimated to occur in 1 in 17 heterozygous carrierand in 1 in 6 homozygous carrier for the prothrombin factor II 20210 G→A51-52.

A meta-analysis of 11 studies including a total of 856 women with pre-eclampsia and 1244 controls (healthy pregnancies) showed that carrier of the prothrombin factor II 20210 G→A are at increased risk for pre-eclampsia (+80%)40.

References:

1. Chango, A. et al. The effect of 677C—>T and 1298A—>C mutations on plasma homocysteine and5,10-methylenetetrahydrofolate reductase activity in healthy subjects. Br J Nutr 83, 593-596(2000).

2. van der Put, N. M. et al. A second common mutation in the methylenetetrahydrofolate reductasegene: an additional risk factor for neural-tube defects? Am J Hum Genet 62, 1044-1051,doi:10.1086/301825 (1998).

3. Weisberg, I., Tran, P., Christensen, B., Sibani, S. & Rozen, R. A second genetic polymorphism inmethylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. MolGenet Metab 64, 169-172, doi:10.1006/mgme.1998.2714 (1998).

4. Ogino S, Wilson RB. Genotype and haplotype distributions of MTHFR677C>T and 1298A>C singlenucleotide polymorphisms: a meta-analysis. J Hum Genet. 2003;48(1):1-7.

5. Peng F, Labelle LA, Rainey BJ, et al. Single nucleotide polymorphisms in themethylenetetrahydrofolate reductase gene are common in US Caucasian and Hispanic Americanpopulations. Int J Mol Med 2001; 8(5):509–11.

6. Clément A, Menezo Y, Cohen M, Cornet D, Clément P. 5-Methyltetrahydrofolate reduces bloodhomocysteine level significantly in C677T methyltetrahydrofolate reductase single-nucleotidepolymorphism carriers consulting for infertility. J Gynecol Obstet Hum Reprod. 2019 Aug 22.

7. Zeng S, Wang X, Wang Y, Xu Z, Zhang J, Liu W, Qian L, Chen X, Wei J, Yang X, Gong Z, Yan Y.MTHFR C677T polymorphism is associated with follicle-stimulating hormone levels and controlledovarian hyperstimulation response: a retrospective study from the clinical database. Fertil Steril.2019 May;111(5):982-990.e2.





12. Zhang Y, He X, Xiong X, Chuan J, Zhong L, Chen G, Yu D. The association between maternalmethylenetetrahydrofolate reductase C677T and A1298C polymorphism and birth defects andadverse pregnancy outcomes. Prenat Diagn. 2019 Jan;39(1):3-9.

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13. Chaudhry SH, Taljaard M, MacFarlane AJ, Gaudet LM, Smith GN, Rodger M, Rennicks White R,Walker MC, Wen SW. The role of maternal homocysteine concentration in placenta-mediatedcomplications: findings from the Ottawa and Kingston birth cohort. BMC Pregnancy Childbirth.2019 Feb 19;19(1):75.


15. Den Heijer M, Koster T, Blom HJ, Bos GMJ, Briët E, Reitsma PH, Vandenbroucke JP, Rosendaal FR.Hyperhomocysteinemia as a risk factor for deep-vein thrombosis. N Engl J Med. 334, 1996, 759-762.

16. Enciso M, Sarasa J, Xanthopoulou L, Bristow S, Bowles M, Fragouli E, Delhanty J, Wells D.Polymorphisms in the MTHFR gene influence embryo viability and the incidence of aneuploidy.Hum Genet. 2016 May;135(5):555-68.

17. Kuhli C, Luchtenberg M, Scharrer I, Hattenbach LO. Massive subhyaloidal hemorrhage associatedwith severe PAI-1 deficiency. Graefes Arch Clin Exp Ophthalmol. 2005;243(10):963-966.

18. Sartori MT, Danesin C, Saggiorato G, Tormene D, Simioni P, Spiezia L, Patrassi GM, Girolami A. ThePAI-1 gene 4G/5G polymorphism and deep vein thrombosis in patients with inheritedthrombophilia. Clin Appl Thromb Hemost. 2003 Oct;9(4):299-307.

19. Floridon C, Nielsen O, Holund B, et al. Does plasminogen activator inhibitor-1 (PAI-1) controltrophoblast invasion? A study of 1558 Reproductive Sciences 24(11) fetal and maternal tissue inintrauterine, tubal and molar pregnancies. Placenta. 2000;21(8):754-762.

20. Ye Y, Vattai A, Zhang X, Zhu J, Thaler CJ, Mahner S, Jeschke U, von Schönfeldt V. Role ofPlasminogen Activator Inhibitor Type 1 in Pathologies of Female Reproductive Diseases. Int J MolSci. 2017 Jul 29;18(8).

21. Festa A, D’Agostino R Jr, Rich SS, Jenny NS, Tracy RP, Haffner SM. Promoter (4G/5G) plasminogenactivator inhibitor-1 genotype and plasminogen activator inhibitor-1 levels in blacks, Hispanics, andnon-Hispanic whites: the Insulin Resistance Atherosclerosis Study. Circulation. 2003;107(19):2422-2427.

22. Grubic N, Stegnar M, Peternel P, Kaider A, Binder BR. A novel G/A and the 4G/5G polymorphismwithin the promoter of the plasminogen activator inhibitor-1 gene in patients with deep veinthrombosis. Thromb Res. 1996 Dec 15;84(6):431-43.

23. Seguí R, Estellés A, Mira Y, España F, Villa P, Falcó C, Vayá A, Grancha S, Ferrando F, Aznar J. PAI-1promoter 4G/5G genotype as an additional risk factor for venous thrombosis in subjects withgenetic thrombophilic defects. Br J Haematol. 2000 Oct;111(1):122-8.

24. Huang Z, Tang W, Liang Z, Chen Q, Li M, Li Y, Lao S, Pan H, Huang L, Huang M, Hu X, Zhao J.Plasminogen Activator Inhibitor-1 Polymorphism Confers a Genetic Contribution to the Risk ofRecurrent Spontaneous Abortion: An Updated Meta-Analysis. Reprod Sci. 2017 Nov;24(11):1551-1560.

25. Li X, Liu Y, Zhang R, Tan J, Chen L, Liu Y. Meta-analysis of the association between plasminogenactivator inhibitor-1 4G/5G polymorphism and recurrent pregnancy loss. Med Sci Monit. 2015 Apr11;21: 1051-6.29- Salazar Garcia MD, Sung N, Mullenix TM, Dambaeva S, Beaman K, Gilman-Sachs A, Kwak-KimJ. Plasminogen Activator Inhibitor-1 4G/5G Polymorphism is Associated with Reproductive Failure:Metabolic, Hormonal, and Immune Profiles. Am J Reprod Immunol. 2016 Jul;76(1):70-81.

26. Williams PJ, Broughton Pipkin F (2011) The genetics of pre-eclampsia and other hypertensivedisorders of pregnancy. Best Pract Res Clin Obstet Gynaecol 25: 405–17.

27. Belo L, Santos-Silva A, Rumley A, Lowe G, Pereira-Leite L, et al. (2002) Elevated tissueplasminogen activator as a potential marker of endothelial dysfunction in pre-eclampsia:correlation with proteinuria. BJOG 109: 1250–5.

28. Giannakou K, Evangelou E, Papatheodorou SI. Genetic and non-genetic risk factors for pre-eclampsia: umbrella review of systematic reviews and meta-analyses of observational studies.Ultrasound Obstet Gynecol. 2018 Jun;51(6):720-730.

29. Dawood F, Mountford R, Ferquharson R, Quenby S. Genetic polymorphisms on the factor V gene inwomen with recurrent miscarriage an acquired APCR. Hum Reprod 2007; 22:2546-53. 7.

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30. Mierla D, Szmal C, Neagos D, Cretu R, Stoian V, Jardan D. Association of prothrombin (A20210G)and factor V Leiden (A506G) with recurrent pregnancy loss. J Clin Med 2012; 7:222-26.

31. Zöller B, García de Frutos P, Hillarp A, Dahlbäck B. Thrombophilia as a multigenic disease.Haematologica. 1999 Jan;84(1):59-70. Review.

32. Ridker PM, Hennekens CH, Lindpaintner K, Stampfer MJ, Eisenberg PR, Miletich JP . Mutation inthe gene coding for coagulation factor V and the risk of myocardial infarction, stroke, and venousthrombosis in apparently healthy men. N Engl J Med 1995; 332: 912–917

33. Horne MK 3rd, McCloskey DJ. Factor V Leiden as a common genetic risk factor for venousthromboembolism. J Nurs Scholarsh. 2006;38(1):19-25. Review.

34. Juul K, Tybjaerg-Hansen A, Schnohr P, Nordestgaard BG. Factor V Leiden and the risk for venousthromboembolism in the adult Danish population. Ann Intern Med. 2004 Mar 2;140(5):330-7.

35. Calderwood CJ, Greer IA. The role of factor V Leiden in maternal health and the outcome ofpregnancy. Curr Drug Targets. 2005 Aug;6(5):567-76. Review

36. Nahas R, Saliba W, Elias A, Elias M. The prevalence of thrombophilia in women with recurrent fetalloss and outcome of anticoagulation therapy for the prevention of miscarriages. Clin Appl ThrombHemost 2018; 24:122-8. 25.

37. D’Uva M, Di Micco P, Strina I, Placido GD. Recurrent pregnancy loss and thrombophilia. Journal ofClinical Medical Research 2010; 2:18-22.

38. Rey E, Kahn SR, David M, Shrier I. Thrombophilic disorders and foetal loss: a meta-analysis. Lancet2003; 361:901–8.

39. Jusić A, Balić D, Avdić A, Pođanin M, Balić A. The association of factor V G1961A (factor V Leiden),prothrombin G20210A, MTHFR C677T and PAI-1 4G/5G polymorphisms with recurrent pregnancyloss in Bosnian women. Med Glas (Zenica). 2018 Aug 1;15(2):158-163.

40. Tempfer CB, Riener EK, Hefler LA, Keck C. Genetic thrombophilia has pleiotropic effects inpregnancy. Per Med. 2004 Dec;1(1):105-114.

41. Lin J, August P. Genetic thrombophilias and preeclampsia: a meta-analysis. Obstet Gynecol. 2005Jan;105(1):182-92.

42. Fong FM, Sahemey MK, Hamedi G, Eyitayo R, Yates D, Kuan V, Thangaratinam S, Walton RT.Maternal genotype and severe preeclampsia: a HuGE review. Am J Epidemiol. 2014 Aug15;180(4):335-45.

43. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3’-untranslated regions of the prothrombin gene is associated with elevated plasma prothrombinlevels and an increase in venous thrombosis. Blood 1996; 88:3698-703.

44. Rosendaal FR, Doggen CJ, Zivelin A, et al. Geographic distribution of the 20210 G to Aprothrombin variant. Thromb Haemostas 1998; 79:706-8.

45. Arruda VR, Annichino Bizzacchi JM, Goncalves MS, Costa FF. Prevalence of the prothrombin genevariant (nt20210A) in venous thrombosis and arterial disease. Thromb Haemostas 1997; 78:1430-3.

46. Brown K, Luddington R, Williamson D, Baker P, Baglin T. Risk of venous thromboembolismassociated with a G to A transition at position 20210 in the 3’-untranslated region of theprothrombin gene. Br J Haematol 1997; 98:907-9.

47. Ferraresi P, Marchetti G, Legnani C, et al. The heterozygous 20210 G/A prothrombin genotype isassociated with early venous thrombosis in inherited thrombophilias and is not increased infrequency in artery disease. Arterioscler Thromb Vasc Biol 1997; 17:2418-22.

48. Hillarp A, Zöller B, Svensson PJ, Dahlbäck B. The 20210 A allele of the prothrombin gene is acommon risk factor among Swedish outpatients with verified deep venous thrombosis. ThrombHaemostas 1997; 78:990-2.

49. Rey E, Kahn SR, David M, Shrier I. Thrombophilic disorders and fetal loss: a meta-analysis. Lancet.2003 Mar 15;361(9361):901-8.

50. Gao H, Tao FB: Prothrombin G20210A mutation is associated with recurrent pregnancy loss: Asystematic review and meta-analysis update. Thromb Res, 2015; 135: 339–46

51. Gerhardt A, Scharf RE, Beckmann MW, Struve S, Bender HG, Pillny M, Sandmann W, Zotz RB.Prothrombin and factor V mutations in women with a history of thrombosis during pregnancy andthe puerperium. N Engl J Med. 2000 Feb 10;342(6):374-80.

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52. Samama MM, Rached RA, Horellou MH, Aquilanti S, Mathieux VG, Plu-Bureau G, Elalamy I, ConardJ. Pregnancy-associated venous thromboembolism (VTE) in combined heterozygous factor VLeiden (FVL) and prothrombin (FII) 20210 A mutation and in heterozygous FII single gene mutationalone. Br J Haematol. 2003 Oct;123(2):327-34.

Autoimmunity

a. HLA autoimmune disease predisposition

Our analysis indicates HLA alleles and haplotypes that have been shown to predispose to certain autoimmune conditions. The presence of one or more predisposing HLA alleles/haplotypes is not diagnostic of the existence of an autoimmune condition. However, when combined with other analysis in this report, the presence of specific alleles/haplotypes can provide valuable insight into the state of the patient’s immune system that could be contributing to failure to initiate or maintain pregnancy. HLA alleles and haplotypes that contribute to autoimmunity may also directly lead to an inability to appropriately establish maternal immune tolerance to an embryo or fetus.

b. Antiphospholipid antibodies (APA)

Physiology:

Antiphospholipid antibody syndrome (APS) is an autoantibody-mediated disorder where antiphospholipid antibodies are produced by the immune system against itself.

The presence of antiphospholipid antibodies may trigger a thrombophilic disorder that causes excessive clotting and can lead to venous thromboembolism, stroke, multiple miscarriages and other pregnancy complications.

APS is considered primary if it occurs in a patient with no underlying disease and secondary if it is related to an underlying pathology such as systemic lupus erythematous (SLE).

Among the different antiphospholipid antibodies, anti-beta 2 glycoprotein I (anti-β2-GP1 ) antibodies is the best to support the diagnosis of APS1. Indeed, many studies supports the fact that anti-β2-GP1 are more specific for APS than anti-cardiolipin antibodies1-3.

All three isotypes of anti-β2-GP1 (IgG, IgM, and IgA) have been associated with thrombosis3-5. The presence of one or both β2-GP1 IgG and IgM antibodies is an independent risk factor for thrombosis and pregnancy complications3.


APS is the most frequent acquired risk factor for recurrent pregnancy loss. Disrupting the placental function and impairing the maternal–fetal blood exchange, it also increases the risk for pregnancy complications such as stillbirth, intrauterine death, preeclampsia (PE), premature birth, and fetal growth restriction.

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In RPL women, 26.4% of reccurent miscarriages were associated with the presence of anti-phospholipid antibodies6 while pregnancy complications were found in up to 20% of APS pregnancies7.

In a large meta-analysis including over 200 000 participants, the risk for spontaneous abortion in women with APS increased by a factor 2.58. Further, in a meta-analysis, moderate to high levels of anti-cardiolipin antibodies (aCL) were associated with higher risk of PE9. When anti-phospholipid antibodies are high and persistent, there is a higher risk for preterm birth and fetal growth restriction10-11.

Ultimately, elevated titers for aCL and anti-β2GPI antibodies were associated with a 3- to 5-fold increased odd of stillbirth12.

Altogether, these studies showed that detection, close monitoring and adequate care should be given to pregnant women with APS to allow a successful pregnancy.

References:

1. Harris EN, Pierangeli SS, Gharavi AE. Diagnosis of the antiphospholipid syndrome: A proposal foruse of laboratory tests. Lupus. 1998; 7(Suppl 2): S144-S148.

2. Carreras LO, Forastiero RR, Martinuzzo ME. Which are the best biological markers of theantiphospholipid syndrome? J Autoimmun. 2000 Sep; 15(2):163-172.

3. Levine JS, Branch DW, Rauch J. The antiphospholipid syndrome. N Engl J Med. 2002 Mar 7;346(10):752-763.

3. Reddel SW, Krilis SA. Testing for and clinical significance of anticardiolipin antibodies. Clin DiagnLab Immunol. 1999 Nov; 6(6):775-782.

4. Brey RL, Abbott RD, Curb JD, et al. beta (2)-Glycoprotein 1-dependent anticardiolipin antibodiesand risk of ischemic stroke and myocardial infarction: The Honolulu Heart Program. Stroke. 2001Aug; 32(8):1701-1706.

5. Greco TP, Amos MD, Conti-Kelly AM, Naranjo JD, Ijdo JW. Testing for the antiphospholipidsyndrome: Importance of IgA anti-beta 2-glycoprotein I. Lupus. 2000; 9(1):33-41.

6. Rai RS, Regan L, Clifford K, et al. Antiphospholipid antibodies and beta 2-glycoprotein-I in 500women with recurrent miscarriage: results of a comprehensive screening approach. Hum Reprod1995;10(8):2001–2005.

7. Cervera R, Serrano R, Pons-Estel GJ, et al; Euro-Phospholipid Project Group (European Forum onAntiphospholipid Antibodies). Morbidity and mortality in the antiphospholipid syndrome during a10-year period: a multicentre prospective study of 1000 patients. Ann Rheum Dis 2015;74(6):1011–1018.

8. Liu L, Sun D. Pregnancy outcomes in patients with primary antiphospholipid syndrome: Asystematic review and meta-analysis. Medicine (Baltimore). 2019 May;98(20):e15733.

9. do Prado AD, Piovesan DM, Staub HL, Horta BL. Association of anticardiolipin antibodies withpreeclampsia: a systematic review and meta-analysis. Obstet Gynecol 2010;116(6):1433–1443.

10. Clark EAS, Silver RM, Branch DW. Do antiphospholipid antibodies cause preeclampsia and HELLPsyndrome? Curr Rheumatol Rep 2007;9(3):219–225 54.

11. Yamada H, Atsumi T, Kobashi G, et al. Antiphospholipid antibodies increase the risk of pregnancy-induced hypertension and adverse pregnancy outcomes. J Reprod Immunol 2009;79(2):188–195.

12. Silver RM, Parker CB, Reddy UM, et al. Antiphospholipid antibodies in stillbirth. Obstet Gynecol2013;122(3):641–657.

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c. Antinuclear antibodies (ANA)

Physiology:

Antinuclear antibodies are autoantibodies targeting “normal” proteins within the nucleus of your cells.

The presence of ANA antibodies in small amount may not be a sign for autoimmune diseases1 and can fluctuate over time2.

Nevertheless, large titers can indicate an autoimmune disease such as lupus, scleroderma, juvenile arthritis, autoimmune hepatitis, autoimmune thyroid disease or Sjögren's syndrome.


ANA may play a role in early pregnancy as well as in reccurent pregnancy losses (RPL) by negatively impacting embryo development3. ANA have been detected more frequently in women with RPL than in control women4. They are also associated with poor outcomes of IVF/ICSI cycles5. ANA positivity may predict a subsequent miscarriage6 in women with RPL.

Indeed, ANA+ RPL women had a higher number of miscarriages and lower number of successful subsequent pregnancies than ANA− women7.

It is important to note that miscarriages occurred in women positive for ANA+ before pregnancy and who remained ANA+ in the first trimester.

Pregnancy in RA patients is associated with higher risk for complications such as IUGR (intra uterine growth retardation) and premature rupture of membranes with rates that are 1.5–2 times higher than in the general obstetric population8.

References:

1. Giannouli E, Chatzidimitriou D, Gerou S, Gavrilaki E, Settas L, Diza E. Frequency and specificity ofantibodies against nuclear and cytoplas-mic antigens in healthy individuals by classic and newmethods. Clin Rheumatol. 2013; 32:1541–1546. 5.

2. Faria AC, Barcellos KS, Andrade LE. Longitudinal fluctuations of anti-bodies to extractable nuclearantigens in systemic lupus erythemato-sus. J Rheumatol. 2005; 32:1267–1272.

3. Kaider BD. Cavalcante MB, da Silva Costa F, Araujo EJr, Barini R. Risk factors as-sociated with anew pregnancy loss and perinatal outcomes in cases of recurrent miscarriage treated withlymphocyte immunotherapy. J Matern Fetal Neonatal Med. 2014; 28:1092–1096.

4. Ticconi C, Rotondi F, Veglia M, et al. Antinuclear autoantibod-ies in women with recurrentpregnancy loss. Am J Reprod Immunol. 2010; 64:304–392.

5. Zhu Q, Wu L, Xu B, et al. A retrospective study on IVF/ICSI outcome in patients with anti- nuclearantibodies: the effects of prednisone plus low- dose aspirin adjuvant treatment. Reprod BiolEndocrinol. 2013; 11:98.

6. Cavalcante MB, da Silva Costa F, Araujo EJr, Barini R. Risk factors as-sociated with a newpregnancy loss and perinatal outcomes in cases of recurrent miscarriage treated with lymphocyteimmunotherapy. J Matern Fetal Neonatal Med. 2014; 28:1092–1096.

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7. Sakthiswary R, Rajalingam S, Norazman MR, Hussien H. Antinuclear antibodies predict a highernumber of pregnancy loss in unexplained recurrent pregnancy loss. Clin Ter. 2015; 166: e98–e101.

8. Chakravarty EF, Nelson L, Krishnan E. Obstetric hospitalizations in the United States for womenwith systemic lupus erythematosus and rheumatoid arthritis. Arthritis Rheum. 2006; 54:899–907.

d. Anti CCP and RF

Physiology:

Anti-citrullinated protein antibodies (anti-CCP) and rheumatoid factor (RF) are two tests used to detect rheumatoid Arthritis (RA). Elevated RF levels are found in collagen vascular diseases such as SLE, RA, scleroderma, Sjogren’s Syndrome or thyroid disease. RF is not very specific for RA diagnosis and can also be detected in patients with other rheumatic disorders, infections, as well as in apparently healthy patients1. Anti-CCP antibodies are more specific than RF for RA2.

References:

1. Eggeland T, Munthe E (1983) The role of the laboratory in rheumatology. Rheumatoid factors ClinRheum Dis 9:135–160.

2. Dos Anjos LM, Pereira IA, Orsi E, Seaman AP, Burlingame RW, Morato EF (2009) A comparativestudy of IgG second- and thirdgeneration anti-cyclic citrullinated peptide (CCP) ELISAs and theircombination with IgA third-generation CCP ELISA for the diagnosis of rheumatoid arthritis. ClinRheumatol 28:153–158.

Thyroid

Physiology:

The thyroid gland, located at the base of your neck, is part of your endocrine system.

Thyroid dysfunction is the second most common endocrine disorder after diabetes that affects women of reproductive age1, among whom 5–15% test positive for thyroid autoantibodies.

It is caused by genetic factors and environmental conditions, both of which are yet to be fully understood.

Hypothyroidism is the most common thyroid alteration observed during pregnancy with the most frequent cause being autoimmune thyroiditis (Hashimoto's thyroiditis).


Untreated hypothyroidism can lead to fertility issues with a wide range of adverse outcomes: miscarriage, preterm delivery, gestational hypertension or reduced cognitive function in the offspring2. Thyroid autoimmunity with the production of anti-thyroid antibodies (anti-TPO antibodies) may directly attack the fetoplacental unit thus leading to embryo losses or obstetrical complications.

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The miscarriage rate in women with subclinical or clinical hypothyroidism at the time of conception, is significantly increased ranging from 60% up to 71.4%3. On the other hand, when adequately treated, no losses were seen and over 90% of patients delivered at term regardless of the severity of the hypothyroidism.

In women inefficiently treated for hypothyroidism4, fetal loss rate was 29% compared with 6% in patients whose serum TSH values were restored within the reference range after therapy. Even in least severe case of hypothyroidism with no anti-thyroid antibodies and low TSH levels (between 2.5 and 5 mUI/mL), there is an increased risk of miscarriages that almost doubles as compared with control patients when treatment is not administrated5.

Lastly, in a large study including 25 756 women with a singleton pregnancy6, pregnancies in women with subclinical hypothyroidism were three times more likely to be complicated by placental abruption and preterm birth as compared to pregnant women with normal thyroid function. These complications were estimated to increase by 60% for every doubling of the serum TSH concentration.

All together, these data showed that screening, monitoring and adequate treatment to restore optimal thyroid function during pregnancy is crucial to minimize risks and lead to term pregnancies with no obstetrical complication in patients affected with hypothyroidism.

References:

1. W. Teng, Z. Shan, K. Patil-Sisodia, D.S. Cooper. Hypothyroidism in pregnancy. Lancet DiabetesEndocrinol, 1 (2013), pp. 228-237.

2. Milanesi A & Brent GA. Management of hypothyroidism in pregnancy. Current Opinion inEndocrinology, Diabetes, and Obesity 2011 18 304–309.

3. Abalovich M, Gutierrez S, Alcaraz G, Maccallini G, Garcia A & Levalle O. Overt and subclinicalhypothyroidism complicating pregnancy. Thyroid 2002 12 63–68. (doi:10.1089/105072502753451986) 23 Hallengren B, Lantz M, Andreasson B & Grennert L. Pregnant women on thyroxinesubstitution are often dysregulated in early pregnancy. Thyroid 2009 19 391–394.

4. Hallengren B, Lantz M, Andreasson B & Grennert L. Pregnant women on thyroxine substitution areoften dysregulated in early pregnancy. Thyroid 2009 19 391–394.

5. Negro R, Schwartz A, Gismondi R, Tinelli A, Mangieri T & Stagnaro-Green A. Increased pregnancyloss rate in thyroid antibody negative women with TSH levels between 2.5 and 5.0 in the firsttrimester of pregnancy. Journal of Clinical Endocrinology and Metabolism 2010 95 E44–E48.

6. Casey BM, Dashe JS, Wells CE, McIntire DD, Byrd W, Leveno KJ & Cunningham FG. Subclinicalhypothyroidism and pregnancy outcomes. Obstetrics and Gynecology 2005 105 239–245.

7. Benhadi N, Wiersinga WM, Reitsma JB, Vrijkotte TG & Bonsel GJ. Higher maternal TSH levels inpregnancy are associated with increased risk for miscarriage, fetal or neonatal death. EuropeanJournal of Endocrinology 2009 160 985–991.

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Inflammatory

a. Complement activity

Physiology:

The complement is a system of serum proteins that comprises an important effector arm of the innate immune system and is associated with macrophage activation and inflammation.

Three different pathways activate the complement system:

• the classical pathway is activated by antigen–antibody complexes• the alternative pathway activated by microbial surfaces• the lectin pathways are activated by microbial surfaces.

These pathways converge and their activation results in the generation of C3a, C4a, and C5a anaphylatoxins (potent inflammatory molecules) and the membrane attack complex MAC (C5b, 6,7,8, and 9).

The complement and coagulation pathways are closely interacting1-2 with C5a inducing procoagulant activity1 and reducing fibrinolysis. C3a and C5a also activate endothelial cells and platelets3, inducing increased levels of adhesion factors and procoagulant activity4-6.

Complement activation has dramatic effects on placenta causing inflammation and placental injury7, two main culprits of fetal loss in APS8 in addition to triggering pre-eclampsia9.


Various studies explored complement activation in the circulation of women with preeclampsia and found higher complement activity as compared to healthy control pregnancies10-12.

Similarly, clinical studies support the role of complement activity as seen by lower levels of C3 and C4 complement levels in obstetrical complications occurring in patient with antiphospholipid syndrome13-16 or Systemic Lupus Erythematosus 17.

Indeed, a correlation between elevated levels of complement activation fragments (Bb and C3a) and preterm birth has been reported18-19.

Interestingly, some proteins of the complement activation (factor B and H) were found to be useful biomarkers to predict preterm birth as early as 15 weeks of pregnancy 20.

References:

1. Ritis K, Doumas M, Mastellos D, Micheli A, Giaglis S, Magotti P, et al. A novel C5a receptor-tissuefactor crosstalk in neutrophils links innate immunity to coagulation pathways. J Immunol.(2006) 177:4794–802.

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2. Foley JH, Conway EM. Cross talk pathways between coagulation and inflammation. Circ Res.(2016) 118:1392–408.

3. Subramaniam S, Jurk K, Hobohm L, Jäckel S, Saffarzadeh M, Schwierczek K, et al. contributions ofcomplement factors to platelet activation and fibrin formation in venous thrombusdevelopment. Blood. (2017) 129:2291–302.

4. Facciabene A, De Sanctis F, Pierini S, Reis ES, Balint K, Facciponte J, et al. Local endothelialcomplement activation reverses endothelial quiescence, enabling t-cell homing, and tumor controlduring t-cell immunotherapy. Oncoimmunology. (2017) 6: e1326442.

5. Noone DG, Riedl M, Pluthero FG, Bowman ML, Liszewski MK, Lu L, et al. Von Willebrand factorregulates complement on endothelial cells. Kidney Int. (2016) 90:123–34.10.1016/j.kint.2016.03.023.

6. Riedl M, Noone DG, Khan MA, Pluthero FG, Kahr WHA, Palaniyar N, et al. Complement activationinduces neutrophil adhesion and neutrophil-platelet aggregate formation on vascular endothelialcells. Kidney Int Rep. (2017) 2:66–75.

7. Redecha P, Franzke CW, Ruf W, Mackman N, Girardi G. Neutrophil activation by the tissuefactor/Factor VIIa/PAR2 axis mediates fetal death in a mouse model of antiphospholipidsyndrome. J Clin Invest. (2008) 118:3453–61.

8. Chaturvedi S, Brodsky RA, McCrae KR. Complement in the Pathophysiology of theAntiphospholipid Syndrome. Front Immunol. 2019; 10:449. Published 2019 Mar 14.

9. Pierik E, Prins JR, van Goor H, et al. Dysregulation of Complement Activation and PlacentalDysfunction: A Potential Target to Treat Preeclampsia? Front Immunol. 2020;10: 3098. Published2020 Jan 15. doi:10.3389/fimmu.2019.03098.

10. Ye Y, Kong Y, Zhang Y. Complement split products C3a/C5a and receptors: are they regulated bycirculating angiotensin II type 1 receptor autoantibody in severe preeclampsia? Gynecol ObstetInvest.

11. Boij R, Svensson J, Nilsson-Ekdahl K, Sandholm K, Lindahl TL, Palonek E, et al. Biomarkers ofcoagulation, inflammation, and angiogenesis are independently associated with preeclampsia. AmJ Reprod Immunol. (2012) 68:258–70.

12. Derzsy Z, Prohászka Z, Rigó J, Füst G, Molvarec A. Activation of the complement system in normalpregnancy and preeclampsia. Mol Immunol. (2010) 47:1500–6.

13. Reggia R, Ziglioli T, Andreoli L, Bellisai F, Iuliano A, Gerosa M, et al. Primary anti-phospholipidsyndrome: any role for serum complement levels in predicting pregnancycomplications? Rheumatology. (2012) 51:2186–90.

14. Oku K, Atsumi T, Bohgaki M, Amengual O, Kataoka H, Horita T, et al. Complement activation inpatients with primary antiphospholipid syndrome. Ann Rheum Dis. (2009) 68:1030–5.

15. Breen KA, Seed P, Parmar K, Moore GW, Stuart-Smith SE, Hunt BJ. Complement activation inpatients with isolated antiphospholipid antibodies or primary antiphospholipid syndrome. ThrombHaemost. (2012) 107:423–9.

16. De Carolis S, Botta A, Santucci S, Salvi S, Moresi S, Di Pasquo E, et al. Complementemia andobstetric outcome in pregnancy with antiphospholipid syndrome. Lupus. (2012) 21:776–8.

17. Hazeltine M, Rauch J, Danoff D, Esdaile JM, Tannenbaum H. Antiphospholipid antibodies insystemic lupus erythematosus: evidence of an association with positive Coombs’ andhypocomplementemia. J Rheumatol. (1988) 15:80–6.

18. Lynch AM, Gibbs RS, Murphy JR et al (2008) Complement activation fragment bb in earlypregnancy and spontaneous preterm birth. Am J Obstet Gynecol 199: 354.e1–354.e8

19. Lynch AM, Gibbs RS, Murphy JR et al (2011) Early elevations of the complement activationfragment C3a and adverse pregnancy outcomes. Obstet Gynecol 117:75–83.

20. Lynch AM, Wagner BD, Deterding RR et al (2016) The relationship of circulating proteins in earlypregnancy with preterm birth. Am J Obstet Gynecol 214:517.e1–517.e8.

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b. Th1/Th2

Physiology:

CD4+ T cells, CD8+ T cells, NKT cells, and NK cells comprise four of the major effector cell types of the immune system. These cells can respond to the presence of foreign antigens, including paternally derived antigens present on conceptuses, and elicit either immunogenic or tolerogenic responses.

Naïve CD4+ T cells can differentiate into one of several lineages upon antigen priming by antigen presenting cells (APCs) depending on the nature of the APCs involved and the profile of soluble molecules secreted by the APCs during priming. These lineages include Th1, Th2, Th17, and Treg cells (regulatory T cells) which are distinguished by unique cytokine expression profiles. Cells expressing these cytokines (IFNg for Th1, IL-4 for Th2, IL-17 for Th17, and IL-10 for Treg) can be identified by flow cytometry and ratios of these cells can be determined to characterize the CD4+ T cell lineage profile for an individual. Analogous lineages also exist for CD8+ T cells, NKT cells, and NK cells which can be similarly characterized. Levels of TNFa positive cells can also be used as a general marker of cellular activation.

Relative balances of these lineages within each of these cell types can be used to help characterize the nature of any underlying immune conditions. For example, some autoimmune conditions such as rheumatoid arthritis are Th1-dominant, whereas other autoimmune conditions such as systemic lupus erythematosus are Th2-dominant. Other conditions including endometriosis, PCOS, and atopy are also characterized by differential intracellular cytokine profiles. These profiles can be present many years prior to the full clinical manifestation and diagnosis of an underlying autoimmune/inflammatory immune condition and therefore, in combination with other genetic and cellular data, can be used to characterize preclinical/asymptomatic conditions that may affect the immune response to foreign antigens.

Changes in intracellular cytokine profiles can also be used to track the maternal immune response to pregnancy, assess the efficacy of immune treatment, and determine if any failures of pregnancy are immunological in nature.


Altered intracellular cytokine profiles have been found in the peripheral blood of women with a history of recurrent implantation failure or recurrent miscarriage. These include an elevated ratio of Th1 to Th2 cells, elevated levels of TNFa positive cells, decreased levels of IL-10 positive cells, and increased levels of Th17 cells1-2.

The normal (non-pathological) response to pregnancy is characterized by a tolerogenic response to paternal antigens present on the conceptus. This tolerogenic response is characterized by specific changes to the intracellular cytokine profile of various immune cell types. These tolerogenic responses include a shift to Th2 dominance (a decrease in the Th1/Th2 ratio) and an increase in levels of IL-10 positive cells3-4.

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Pathological immune responses to a conceptus involve a failure to properly develop immunological tolerance to paternally derived antigens. Rather, an immunogenic response is elicited which can lead to cellular and/or humoral (antibody-mediated) responses. Such pathological immune responses to a conceptus can result in various clinical manifestations including implantation failure, spontaneous abortion, preeclampsia, intrauterine growth restriction (IUGR), and stillbirth.

Defective development of immunological tolerance during pregnancy is characterized in the peripheral blood in part by a failure in a shift to Th2 dominance, a failure in the increase in IL-10 positive cells and increases in levels of IL-17 positive cells5-6.

References:

1. Yockey LJ, Iwasaki A. Interferons and Proinflammatory Cytokines in Pregnancy and FetalDevelopment. Immunity. 2018 Sep 18;49(3):397-412. doi: 10.1016/j.immuni.2018.07.017. PMID:30231982; PMCID: PMC6152841.

2. Raghupathy R, Kalinka J. Cytokine imbalance in pregnancy complications and its modulation. FrontBiosci. 2008 Jan 1;13:985-94. doi: 10.2741/2737. PMID: 17981605.

3. Saito S, Nakashima A, Shima T, Ito M. Th1/Th2/Th17 and regulatory T-cell paradigm in pregnancy.Am J Reprod Immunol. 2010 Jun;63(6):601-10. doi: 10.1111/j.1600-0897.2010.00852.x. Epub 2010Apr 23. PMID: 20455873.

4. Ali S, Majid S, Niamat Ali M, Taing S. Evaluation of T cell cytokines and their role in recurrentmiscarriage. Int Immunopharmacol. 2020 Mar 3;82:106347. doi: 10.1016/j.intimp.2020.106347. Epubahead of print. PMID: 32143004.

5. Sykes L, MacIntyre DA, Yap XJ, Teoh TG, Bennett PR. The Th1:th2 dichotomy of pregnancy andpreterm labour. Mediators Inflamm. 2012;2012:967629. doi: 10.1155/2012/967629. Epub 2012 Jun7. PMID: 22719180; PMCID: PMC3376783.

6. Raghupathy R. Pregnancy: success and failure within the Th1/Th2/Th3 paradigm. Semin Immunol.2001 Aug;13(4):219-27. doi: 10.1006/smim.2001.0316. PMID: 11437629.

c. Natural Killer cell activity (NKa)

Physiology:

Natural killer (NK) cells are a population of cytotoxic innate lymphocytes1. Natural killer cell activity has been found elevated in patients with autoimmune diseases such as Graves’ disease, Hashimoto’s thyroiditis as compared to a heathy control population2.

NK cells may be directly involved in these diseases through their potential autoreactivity or through their interaction with dendritic cells, macrophages or T lymphocytes, thereby inducing excessive inflammation or favoring the adaptive autoimmune response3.

NK cells constitute the dominant population of lymphocytes in decidua during early pregnancy4. Although endometrial NK cells are phenotypically different from circulating NK cells (with a weak cytotoxicity and immunoregulatory activities)5, it is speculated that peripheral blood NK cells are strongly related to NK cell populations found in the decidua6.

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NK cell activity has been associated with the pathogenesis of reccurent pregnancy loss7-8, even when the embryo was chromosomally normal9.

A clinical study has shown that women with high NK cell activity pre-conception have a higher risk for pregnancy loss (3.5- fold higher risk) than women with normal levels7. More recent studies showed a strong correlation between high NK cell activity and recurrent spontaneous abortion10-12 and suggested that NK cell activity can be used as a predictive biomarker.

IVIG can induce a down-regulation of NK cell activity among women with reccurent pregnancy losses13 which may help improve a patient’s pregnancy outcome.

Nevertheless, the significance of pre-conceptional NK activity as a predictive value remains debated as a study showed no correlation between NK activity and risk for a subsequent loss14. This study could be bias as it is considering clinical losses only as miscarriages (excluding all chemical losses).

References:

1. Moffett, A., & Shreeve, N. (2015). First do no harm: Uterine natural killer (NK) cells in assistedreproduction. Human Reproduction, 30(7), 1519–1525.

2. Hidaka Y, Amino N, Iwatani Y, Kaneda T, Nasu M, Mitsuda N, Tanizawa O, Miyai K. Increase inperipheral natural killer cell activity in patients with autoimmune thyroid disease. Autoimmunity.1992;11(4):239-46.

3. Fogel LA, Yokoyama WM, French AR. Natural killer cells in human autoimmunedisorders. ArthritisRes Ther. 2013 Jul 11;15(4):216. doi: 10.1186/ar4232. Review.

4. Ntrivalas, E. I., Kwak-Kim, J. Y. H., Gilman-Sachs, A., Chung-Bang, H., Ng, S. C., Beaman, K. D., …Beer, A. E. (2001). Status of peripheral blood natural killer cells in women with recurrentspontaneous abortions and infertility of unknown aetiology. Human Reproduction, 16(5), 855–861.

5. Koopman, L. A., Kopcow, H. D., Rybalov, B., Boyson, J. E., Orange, J. S., Schatz, F, Strominger, J. L.(2003). Human decidual natural killer cells are a unique NK cell subset with immunomodulatorypotential. Journal of Experimental Medicine, 198(8), 1201–1212.

6. Male, V., Hughes, T., McClory, S., Colucci, F., Caligiuri, M. A., & Moffett, A. (2010). Immature NK cells,capable of producing IL-22, are present in human uterine mucosa. The Journal of Immunology,185(7), 3913–3918.

7. Aoki K, Kajiura S, Matsumoto Y, et al. Preconceptional natural-killer cell activity as a predictor ofmiscarriage. Lancet (London, England). 1995; 345:1340–1342.

8. Higuchi, K., Aoki, K., Kimbara, T., Hosoi, N., Yamamoto, T., & Okada, H. (1995). Suppression ofnatural killer cell activity by monocytes following immunotherapy for recurrent spontaneousaborters. American Journal of Reproductive Immunology, 33(3), 221–227.

9. Kwak-Kim, J., & Gilman-Sachs, A. (2008). Clinical implication of natural killer cells and reproduction.American Journal of Reproductive Immunology, 59(5), 388–400.

10. Perricone C, De Carolis C, Giacomelli R, et al. High levels of NK cells in the peripheral blood ofpatients affected with anti-phospholipid syndrome and recurrent spontaneous abortion: a potentialnew hypothesis. Rheumatology (Oxford, England). 2007; 46:1574–1578.

11. Seshadri S, Sunkara SK. Natural killer cells in female infertility and recurrent miscarriage: asystematic review and meta-analysis. Hum Reprod Update. 2014; 20:429–438.

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12. Miyaji M, Deguchi M, Tanimura K, Sasagawa Y, Morizane M, Ebina Y, Yamada H. Clinical factorsassociated with pregnancy outcome in women with recurrent pregnancy loss. Gynecol Endocrinol.2019 Apr 23:1-6.

13. Ahmadi M, Ghaebi M, Abdolmohammadi-Vahid S, Abbaspour-Aghdam S, Hamdi K, Abdollahi-FardS, Danaii S, Mosapour P, Koushaeian L, Dolati S, Rikhtegar R, Oskouei FD, Aghebati-Maleki L, NouriM, Yousefi M. NK cell frequency and cytotoxicity in correlation to pregnancy outcome andresponse to IVIG therapy among women with recurrent pregnancy loss. J Cell Physiol. 2019Jun;234(6):9428-9437.

14. Katano K, Suzuki S, Ozaki Y, Suzumori N, Kitaori T, Sugiura-Ogasawara M. Peripheral natural killercell activity as a predictor of recurrent pregnancy loss: a large cohort study. Fertil Steril. 2013Dec;100(6):1629-34.

d. Regulatory t cell (Treg cells)

Physiology and published clinical data:

Treg cells play a critical role in regulating tolerance to the semi-allogenic fetus presenting paternal alloantigens. These cells may prevent fetal rejection by the maternal immune system1. Treg cells may promote fetal survival by inhibiting effector T cells and secreting anti-inflammatory factors such as IL-10 and TGF-β2-3.

These cells are enriched in the fetal-maternal interface during early pregnancy4 They were shown to migrate from the peripheral blood to the decidua in pregnant subjects5. Thus, they might be recruited and expanded by the recognition to fetal antigens1.

References

1. Aluvihare VR, Kallikourdis M, Betz AG. Regulatory T cells mediate maternal tolerance to the fetus.Nat Immunol. 2004 Mar;5(3):266-71. doi: 10.1038/ni1037. Epub 2004 Feb 1. PMID: 14758358.

2. Robertson SA, Ingman WV, O'Leary S, Sharkey DJ, Tremellen KP. Transforming growth factorbeta--a mediator of immune deviation in seminal plasma. J Reprod Immunol. 2002 Oct-Nov;57(1-2):109-28. doi: 10.1016/s0165-0378(02)00015-3. PMID: 12385837.

3. Yin Y, Han X, Shi Q, Zhao Y, He Y. Adoptive transfer of CD4+CD25+ regulatory T cells forprevention and treatment of spontaneous abortion. Eur J Obstet Gynecol Reprod Biol. 2012Apr;161(2):177-81. doi: 10.1016/j.ejogrb.2011.12.023. Epub 2012 Jan 18. PMID: 22261465.

4. Mjösberg J, Berg G, Jenmalm MC, Ernerudh J. FOXP3+ regulatory T cells and T helper 1, T helper2, and T helper 17 cells in human early pregnancy decidua. Biol Reprod. 2010 Apr;82(4):698-705.doi: 10.1095/biolreprod.109.081208. Epub 2009 Dec 16. PMID: 20018909.

5. Tilburgs T, Roelen DL, van der Mast BJ, de Groot-Swings GM, Kleijburg C, Scherjon SA, Claas FH.Evidence for a selective migration of fetus-specific CD4+CD25bright regulatory T cells from theperipheral blood to the decidua in human pregnancy. J Immunol. 2008 Apr 15;180(8):5737-45. doi:10.4049/jimmunol.180.8.5737. PMID: 18390759.

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Metabolic

a. Insulin resistance and PCOS assessment

Physiology and Published clinical data:

Polycystic ovarian syndrome (PCOS), a multifactorial syndrome, is one of the most prevalent endocrine diseases affecting women of reproductive age1.

PCOS is currently diagnosed according to the Rotterdam criterion2 determining the disease based on the presence of two out of three of the following symptoms3-4

• polycystic ovarian morphology• clinical (hirsutism, acne)5 or biochemical hyperandrogenism (serum hormone

measurement)• oligo/amenorrhea

Although not included in the diagnostic criteria, insulin resistance may be central to the etiology of the syndrome6-7 and this may be exacerbated by obesity. Women with PCOS commonly present with infertility and are at increased risk for pregnancy complications such as gestational diabetes and hypertension8. Several studies have shown a strong association between AMH levels and PCOS. Serum AMH is a good diagnostic marker for PCOS as high AMH levels (cut off >3.19ng/mL) were correlated with oligo/amenorrhea and the appearance of polycystic ovaries on ultrasound9-10.

References:

1. March,W.A.; Moore, V.M.;Willson, K.J.; Phillips, D.I.; Norman, R.J.; Davies, M.J. The prevalence ofpolycystic ovary syndrome in a community sample assessed under contrasting diagnostic criteria.Hum. Reprod. 2009, 25, 544–551.

2. Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Revised 2003 consensuson diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS).Hum. Reprod. 2004, 19, 41–47.

3. ACOG Practice Bulletin No. 194: Polycystic Ovary Syndrome. Obstet Gynecol. 2018 Jun;131(6): e157-e171.

4. Bustin, S.A.; Benes, V.; Garson, J.A.; Hellemans, J.; Huggett, J.; Kubista, M.; Mueller, R.; Nolan, T.;Pfa_, M.W.; Shipley, G.L.; et al. The MIQE guidelines: Minimum information for publication ofquantitative real-time

a. PCR experiments. Clin. Chem. 2009, 55, 611–622.5. Lobo RA, Goebelsmann U, Horton R. Evidence for the importance of peripheral tissue events in the

development of hirsutism in polycystic ovary syndrome. J Clin Endocrinola. Metab 1983; 57: 393–7. (Level II-2)

6. Dunaif A. Insulin resistance and the polycystic ovary syndrome: mechanism and implications forpathogenesis. Endocr Rev 1997; 18:774–800. (Level III).

7. Wang J, Wu D, Guo H, Li M. Hyperandrogenemia and insulin resistance: The chief culprit ofpolycystic ovary syndrome. Life Sci. 2019 Oct 8; 236:116940.

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8. Boomsma CM, Eijkemans MJ, Hughes EG, Visser GH, Fauser BC, Macklon NS. A meta-analysis ofpregnancy outcomes in women with polycystic ovary syndrome. Hum Reprod Update 2006;12:673–83.

9. Abbara A, Eng PC, Phylactou M, Clarke SA, Hunjan T, Roberts R, Vimalesvaran S, Christopoulos G,Islam R, Purugganan K, Comninos AN, Trew GH, Salim R, Hramyka A, Owens L, Kelsey T, DhilloWS. Anti-Müllerian hormone (AMH) in the Diagnosis of Menstrual Disturbance Due to PolycysticOvarian Syndrome. Front Endocrinol (Lausanne). 2019 Sep 26; 10:656.

10. Ahmed N, Batarfi AA, Bajouh OS, Bakhashab S. Serum Anti-Müllerian Hormone in the Diagnosis ofPolycystic Ovary Syndrome in Association with Clinical Symptoms. Diagnostics (Basel). 2019 Oct 1;9 (4).


a. Vitamin D

Physiology:

Vitamin D is a fat-soluble vitamin belonging to the steroid hormone family.

Two main forms are essentials:

• Vitamin D2 (ergocalciferol) that you can get through your diet.• Vitamin D3 (cholecalciferol) that you synthetize after sun exposure and represent 95% of

your vitamin D production, once metabolized to generate calcitriol (the active form ofVitamin D)3.

By binding to its receptor (VDR), calcitriol (active form of vitamin D) regulates over 900 genes in human including several actions in reproductive tissues.

Vitamin D and its receptor are present in the ovaries4 where they stimulate the production of the steroid hormones estrogen and progesterone5. The endometrium (uterus) itself can synthesize the active form of Vitamin D6 and express the vitamin D receptor VDR as well4.


Low levels of vitamin D are common in pregnant women and has been shown in several epidemiological studies worldwide.

Vitamin D deficiency is associated with an increased risk of infection7, bacterial vaginosis8, pre-eclampsia9 and low serum vitamin D levels in the newborn10.

In a published study11, results showed that sufficient and adequate Vitamin D levels prior to conception may reduce the risk for another miscarriage in patients having suffered previous losses.

Vitamin D plays an important role in the modulation of the immune function12 and oxidative stress13 with dramatic impact on fertility ranging from poor oocyte quality, miscarriage to pregnancy complication including pre-eclampsia, preterm labor and stillbirth12, 14.

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The activation of the vitamin D pathway has multiple beneficial effects on the immune function leading to less inflammation15-18.

References:

1. IOM (Institute of Medicine). 2010. Dietary reference intakes for calcium and D. Washington DC: TheNational Academies Press.

2. Holick MF, Binkley NC, Bischoff−Ferrari HA, et al. Evaluation, treatment, and prevention of vitaminD deficiency: an Endocrine Society clinical practice guideline. JCEM. 2011 Jul; 96(7):1911−30.

3. Holick MF. Vitamin D deficiency. N Engl J Med. 2007 Jul 19;357(3):266-81. Review.4. Agic A, Xu H, Altgassen C, Noack F, Wolfler MM, Diedrich K, Friedrich M, Taylor RN & Hornung D.

Relative expression of 1,25-dihydroxyvitamin D3 receptor, vitamin D 1a-hydroxylase, vitamin D 24-hydroxylase, and vitamin D 25-hydroxylase in endometriosis and gynecologic cancers.Reproductive Sciences 2007 14 486–497.

5. Parikh G, Varadinova M, Suwandhi P, Araki T, Rosenwaks Z, Poretsky L & Seto-Young D. Vitamin Dregulates steroidogenesis and insulin-like growth factor binding protein-1 (IGFBP-1) production inhuman ovarian cells. Hormone and Metabolic Research 2010 42 754–757.

6. Vigano` P, Lattuada D, Mangioni S, Ermellino L, Vignali M, Caporizzo E, Panina-Bordignon P,Besozzi M & Di Blasio AM. Cycling and early pregnant endometrium as a site of regulatedexpression of the vitamin D system. Journal of Molecular Endocrinology 2006 36 415–424.

7. Gunville CF, Mourani PM, Ginde AA. The role of vitamin D in prevention and treatment of infection.Inflamm Allergy Drug Targets. 2013 Aug;12(4):239-45. Review.

8. Taheri M, Baheiraei A, Foroushani AR, Nikmanesh B, Modarres M. Treatment of vitamin Ddeficiency is an effective method in the elimination of asymptomatic bacterial vaginosis: A placebo-controlled randomized clinical trial. Indian J Med Res. 2015 Jun;141(6):799-806

9. Sablok A, Batra A, Thariani K, Batra A, Bharti R, Aggarwal AR, Kabi BC, Chellani H. Supplementationof vitamin D in pregnancy and its correlation with feto-maternal outcome. Clin Endocrinol (Oxf).2015 Oct;83(4):536-41.

10. Rodda CP, Benson JE, Vincent AJ, Whitehead CL, Polykov A, Vollenhoven B. Maternal vitamin Dsupplementation during pregnancy prevents vitamin D deficiency in the newborn: an open labelrandomized controlled trial. Clin Endocrinol (Oxf). 2015 Sep;83(3):363-8.

11. Mumford SL, Garbose RA, Kim K, Kissell K, Kuhr DL, Omosigho UR, Perkins NJ, Galai N, Silver RM,Sjaarda LA, Plowden TC, Schisterman EF. Association of preconception serum 25-hydroxyvitaminD concentrations with livebirth and pregnancy loss: a prospective cohort study. Lancet DiabetesEndocrinol. 2018 Sep;6(9):725-732.

12. Ji JL, Muyayalo KP, Zhang YH, Hu XH, Liao AH. Immunological function of vitamin D during humanpregnancy. Am J Reprod Immunol. 2017 Aug;78(2).

13. Farhangi MA,Mesgari-Abbasi M, Hajiluian G, Nameni G, Shahabi P. Adipose tissue inflammationand oxidative stress: the ameliorative effects of vitamin D. Inflammation. 2017;40(5):1688-1697.

14. Pourghassem Gargari B, Pourteymour Fard Tabrizi F, Sadien B, Asghari Jafarabadi M, Farzadi L.Vitamin D Status Is Related to Oxidative Stress but Not High-Sensitive C-Reactive Protein inWomen with Pre-Eclampsia. Gynecol Obstet Invest. 2016;81(4):308-14.

15. Adorini L, Amuchastegui S, Daniel KC. Prevention of chronic allograft rejection by Vitamin Dreceptor agonists. Immunol Lett. 2005 Aug 15;100(1):34-41. Review.

16. Cippitelli M, Santoni A. Vitamin D3: a transcriptional modulator of the interferon-gamma gene. Eur JImmunol. 1998 Oct;28(10):3017-30.

17. Helming L, Böse J, Ehrchen J, Schiebe S, Frahm T, Geffers R, Probst-Kepper M, Balling R,Lengeling A. 1alpha,25-Dihydroxyvitamin D3 is a potent suppressor of interferon gamma-mediatedmacrophage activation. Blood. 2005 Dec 15;106(13):4351-8.

18. Giarratana N, Penna G, Amuchastegui S, Mariani R, Daniel KC, Adorini L. A vitamin D analog down-regulates proinflammatory chemokine production by pancreatic islets inhibiting T cell recruitmentand type 1 diabetes development. J Immunol. 2004 Aug 15;173(4):2280-7.

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b. Folic acid

Physiology:

As a quick reminder, MTHFR induces a reaction, using 5, 10 me-THF, that will mostly produce 5-m THF (methylated folate), a substrate required to synthetize methionine from homocysteine and this is the only form of folate used in the central nervous system (CNS).




Elevated serum homocysteine concentration is an important problem causing birth defects4, pre-eclampsia5-7, placental abruption8, low birth weight and other maternal or fetal complications9.

Low folate and high homocysteine are associated with the occurrence of congenital heart disease (CHD)10-11.

References:





5. Zhang Y, He X, Xiong X, Chuan J, Zhong L, Chen G, Yu D. The association between maternalmethylenetetrahydrofolate reductase C677T and A1298C polymorphism and birth defects andadverse pregnancy outcomes. Prenat Diagn. 2019 Jan;39(1):3-9.

6. Chaudhry SH, Taljaard M, MacFarlane AJ, Gaudet LM, Smith GN, Rodger M, Rennicks White R,Walker MC, Wen SW. The role of maternal homocysteine concentration in placenta-mediatedcomplications: findings from the Ottawa and Kingston birth cohort. BMC Pregnancy Childbirth.2019 Feb 19;19(1):75.


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8. Den Heijer M, Koster T, Blom HJ, Bos GMJ, Briët E, Reitsma PH, Vandenbroucke JP, Rosendaal FR.Hyperhomocysteinemia as a risk factor for deep-vein thrombosis. N Engl J Med. 334, 1996, 759-762.

9. Enciso M, Sarasa J, Xanthopoulou L, Bristow S, Bowles M, Fragouli E, Delhanty J, Wells D.Polymorphisms in the MTHFR gene influence embryo viability and the incidence of aneuploidy.Hum Genet. 2016 May;135(5):555-68.

10. Hall J, Solehdin F: Folic acid for the prevention of congenital anomalies. Eur J Pediatr 1998;157:445-450.

11. 18- Leclerc D, Rozen R: [Molecular genetics of MTHFR: polymorphisms are not all benign]. Med Sci(Paris) 2007; 23:297-302.

c. Fatty acid

Physiology:

Both ω3 and ω6 fatty acids are essential components of phospholipids present in all tissues. They both synthesize lipids, support normal cell function as well as fetal development1. The precursors of the ω3 and ω6 pathway are both essential fatty acids (your body can not synthesize them) and must be obtained through your diet2.The Figure 1 below summarizes how ω3 (EPA and DHA) can help reduce maternal inflammation/ oxidative stress3. During pregnancy, they directly act on placenta to increase antioxidant production that will counteract the effects of reactive oxygen species (ROS). Resolvins and protectins (products of DHA and EPA metabolism) can reduce placental PGE2 (the prostaglandin associated with partition) and reduce placental inflammation. Altogether, these effects can reduce the risk of pregnancy losses or obstetrical complications.

Figure 1: Summary of omega 3 benefits

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High leptin levels have been shown to disrupt folliculogenesis (maturation of follicle leading to the production of a fertilizable egg)18.

This translates into “poor” embryo quality and ultimately higher chances for pregnancy failure19.

On the other hand, in nonoverweight PCOS women, lower leptin serum levels put them at higher risk for lower fertilization rate as the follicle maturation requires physiologic levels of leptin20. Interestingly, leptin levels are negatively associated with EPA and DHA levels21.

Further, as seen in the Figure 2, a diet rich in Fish can significantly lower leptin levels22.

Figure 2: Compared with their counterparts on vegetable diet, subjects on predominantly fish diet have strikingly lower mean adjusted (for age, alcohol, body fat, BMI, and insulin) leptin levels.

Lastly, a large meta-analysis showed that omega 3 supplementation significantly reduced leptin levels23.


In fertility, an increased ω3 intake prior to conception was shown to positively impact embryo morphology in a study on women undergoing IVF cycle4. ω3 appear to promote vascular development in the endometrium as seen by an in vitro study5.

Many other studies showed that a higher ω3 intake:

• can reduce the risk of miscarriage6.• increase uterine blood flow7.• increase the length of pregnancy and reduce preterm birth8-10.

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• reduce placental inflammation when taking during the first trimester and through thepregnancy11.

EPA and DHA are precursors to several mediators triggering anti-inflammatory12 and anti-oxidative actions13-14 and have been shown to play key roles in preventing pregnancy complications associated with excessive systemic and placental inflammation15.

In the above-mentioned review10, the authors reported that fish oil intake especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA):

• reduce by 42% the risk of early preterm births (<34 weeks)!• reduce by 11% the risk of preterm births (<37 weeks).• reduce by 25% the risk for perinatal deaths.• reduce by 11% the risk for low birth weight babies.

Knowing that labor is a pro-inflammatory process, maintaining a balance between omega 3 and omega 6 levels is crucial to allow a normal gestation length16. Further, DHA may be crucial to support fetal brain development17.

References:

1. Innis SM,Essential fatty acid transfer and fetal development. Placenta 26 (2005). S70–S75.2. Sprecher H,D.Luthria,B.Mohammed,S.Baykousheva,Reevaluation of the pathways for the

biosynthes is of polyunsaturated fatty acids,J.LipidRes.36 (1995)2471–2477.3. Leghi GE, Muhlhausler BS. The effect of n-3 LCPUFA supplementation on oxidative stress and

inflammation in the placenta and maternal plasma during pregnancy. Prostaglandins Leukot EssentFatty Acids. 2016 Oct; 113:33-39.

4. Hammiche F, Vujkovic M, Wijburg W, de Vries JH, Macklon NS, Laven JS, Steegers-Theunissen RP.Increased preconception omega-3 polyunsaturated fatty acid intake improves embryomorphology.

5. Johnsen GM, Basak S, Weedon-Fekjær MS, Staff AC, Duttaroy AK. Docosahexaenoic acidstimulates tube formation in first trimester trophoblast cells, HTR8/SVneo. Placenta. 2011Sep;32(9):626-32.

6. Di Cintio E, Parazzini F, Chatenoud L, Surace M, Benzi G, Zanconato G, La Vecchia C. Dietaryfactors and risk of spontaneous abortion. Eur J Obstet Gynecol Reprod Biol. 2001 Mar;95(1):132-6.

7. Lazzarin N, Vaquero E, Exacoustos C, Bertonotti E, Romanini ME, Arduini D. Low-dose aspirin andomega-3 fatty acids improve uterine artery blood flow velocity in women with recurrent miscarriagedue to impaired uterine perfusion. Fertil Steril. 2009 Jul;92(1):296-300.

8. Olsen SF, Sørensen JD, Secher NJ, Hedegaard M, Henriksen TB, Hansen HS, Grant A. Randomisedcontrolled trial of effect of fish-oil supplementation on pregnancy duration. Lancet. 1992 Apr25;339(8800):1003-7.

9. Kar S, Wong M, Rogozinska E, Thangaratinam S. Effects of omega-3 fatty acids in prevention ofearly preterm delivery: a systematic review and meta-analysis of randomized studies. Eur J ObstetGynecol Reprod Biol. 2016 Mar; 198:40-46.

10. Middleton P, Gomersall JC, Gould JF, Shepherd E, Olsen SF, Makrides M. Omega-3 fatty acidaddition during pregnancy. Cochrane Database Syst Rev. 2018 Nov 15;11:CD003402

11. Haghiac M, Yang XH, Presley L, Smith S, Dettelback S, Minium J, Belury MA,Catalano PM, Hauguel-de Mouzon S. Dietary Omega-3 Fatty Acid Supplementation Reduces Inflammation in ObesePregnant Women: A Randomized Double-Blind Controlled Clinical Trial. PLoS One. 2015 Sep4;10(9): e0137309.

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12. Serhan CN, Chiang N, Van Dyke TE. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat Rev Immunol. 2008 May;8(5):349-61.

13. Ambrozova G, Pekarova M, Lojek A. Effect of polyunsaturated fatty acids on the reactive oxygenand nitrogen species production by raw 264.7 macrophages. Eur J Nutr. 2010 Apr;49(3):133-9.

14. Komatsu W, Ishihara K, Murata M, Saito H, Shinohara K. Docosahexaenoic acid suppresses nitricoxide production and inducible nitric oxide synthase expression in interferon-gamma pluslipopolysaccharide-stimulated murine macrophages by inhibiting the oxidative stress. Free RadicBiol Med. 2003 Apr 15;34(8):1006-16.

15. Keelan JA, Mas E, D'Vaz N, Dunstan JA, Li S, Barden AE, Mark PJ, Waddell BJ, Prescott SL, MoriTA. Effects of maternal n-3 fatty acid supplementation on placental cytokines, pro-resolving lipidmediators and their precursors. Reproduction. 2015 Feb;149(2):171-8.

16. Zhou J, Best K, Gibson R, McPhee A, Yelland L, Quinlivan J, et al. Study protocol for a randomizedcontrolled trial evaluating the effect of prenatal omega-3 LCPUFA supplementation to reduce theincidence of preterm birth: the ORIP trial. BMJ Open 2017;7(9): e018360.

17. Shulkin M, Pimpin L, Bellinger D, Kranz S, Fawzi W, Duggan C, et al. n-3 fatty acid supplementationin mothers, preterm Infants, and term Infants and childhood psychomotor and visual development:a systematic review and meta-analysis. Journal of Nutrition 2018;148(3): 409–18.

d. Leptin levels

High leptin levels have been shown to disrupt folliculogenesis (maturation of follicle leading to the production of a fertilizable egg)1.

This translates into “poor” embryo quality and ultimately higher chances for pregnancy failure2.

On the other hand, in nonoverweight PCOS women, lower leptin serum levels put them at higher risk for lower fertilization rate as the follicle maturation requires physiologic levels of leptin3. Interestingly, leptin levels are negatively associated with EPA and DHA levels3. Further, as seen in the Figure 1, a diet rich in Fish can significantly lower leptin levels4.

Figure 1: Compared with their counterparts on vegetable diet, subjects on predominantly fish diet have strikingly lower mean adjusted (for age, alcohol, body fat, BMI, and insulin) leptin levels.

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Lastly, a large meta-analysis showed that omega 3 supplementation significantly reduced leptin levels5.

References:

1. Agarwal SK, Vogel K, Weitsman SR, Magoffin DA. Leptin antagonizes the insulin-like growth factor-Iaugmentation of steroidogenesis in granulosa and theca cells of the human ovary. J ClinEndocrinol Metab. 1999 Mar;84(3):1072-6.

2. Anifandis G, Koutselini E, Stefanidis I, Liakopoulos V, Leivaditis C, Mantzavinos T, VamvakopoulosN. Serum and follicular fluid leptin levels are correlated with human embryo quality. Reproduction.2005 Dec;130(6):917-21.

3. Winnicki M, Somers VK, Accurso V, Phillips BG, Puato M, Palatini P, Pauletto P. Fish-rich diet, leptin,and body mass. Circulation. 2002 Jul 16;106(3):289-91.

4. Santos S, Oliveira A, Pinho C, Casal S, Lopes C. Fatty acids derived from a food frequencyquestionnaire and measured in the erythrocyte membrane in relation to adiponectin and leptinconcentrations. Eur J Clin Nutr. 2014 May;68(5):555-60.

5. Garruti G, de Palo R, Rotelli MT, Nocera S, Totaro I, Nardelli C, Panzarino MA, Vacca M, SelvaggiLE, Giorgino F. Association between follicular fluid leptin and serum insulin levels in nonoverweightwomen with polycystic ovary syndrome. Biomed Res Int. 2014; 2014:980429.

6. Hariri M, Ghiasvand R, Shiranian A, Askari G, Iraj B, Salehi-Abargouei A. Does omega-3 fatty acidssupplementation affect circulating leptin levels? A systematic review and meta-analysis onrandomized controlled clinical trials. Clin Endocrinol (Oxf). 2015 Feb;82(2):221-8.

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Male Assessment

Thrombophilia

Physiology:

The prothrombin time (PT) is a test that evaluates your ability to form blood clots. The international normalized ratio or INR is a calculation based on results of a PT that is used to monitor individuals who are being treated with a blood-thinning medication.

A partial thromboplastin time (PTT) in combination with a PT test assess coagulation factors (amount and function).

Elevated plasma homocysteine have been associated with an increased risk of atherosclerosis and venous thrombosis.


Thrombophilia risk factors are common in patients with idiopathic pregnancy loss but also in pregnant women with retarded intrauterine development, such as in preeclampsia, late fetal loss and abruption placenta1.

Hyperhomocysteinemia is observed in approximately 5% of the general population and is associated with increased risk for many inflammatory disorders including auto immune disorders (Rheumatoid Arthritis, Diabetes, Multiple Sclerosis, Systemic Lupus erythematosus, Grave disease), birth defects and adverse pregnancy outcomes (pre-eclampsia, placental abruption, spontaneous abortion, low birth weight), vascular and neurodegenerative diseases.

In addition, Hyperhomocysteinemia also affects IVF outcome with significantly lower pregnancy and implantation rates whereas the miscarriage rate was higher in patients with elevated homocysteine levels2-3.

To avoid pregnancy complications, women at higher risk for thrombophilia should be placed on low-molecular-weight heparin treatment4.

References:

1. 1-Sarig G, Younis JS, Hoffman R et al: Thrombophilia is common in women with idiopathicpregnancy loss and is associated with late pregnancy wastage. Fertil Steril, 2002; 77: 342–47.

2. Pacchiarotti A, Mohamed MA, Micara G, Linari A, Tranquilli D, Espinola SB, et al. The possible roleof hyperhomocysteinemia on IVF outcome. J Assist Reprod Genet 2007;24: 459–62.

3. Gaskins AJ, Afeiche MC, Wright DL, Toth TL, Williams PL, Gillman MW, Hauser R, Chavarro JE.Dietary folate and reproductive success among women undergoing assisted reproduction. ObstetGynecol. 2014 Oct;124(4):801-9.

4. Riyazi N, Leeda M, de Vries JIP et al: Low molecular weight heparin combined with aspirin inpregnant women with thrombophilia and a history of preeclampsia or fetal growth retardation: Apreliminary study. Eur J Obstet Gynecol Reprod Biol, 1998; 80: 49–54

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Physiology:

Male infertility

Sperm DNA damage is one of the main causes of male infertility and is multifactorial as seen in the Figure1. Among these factors, poor diet and obesity cause an increase in systemic inflammation and in reactive oxygen species production (oxidative stress)1. This is associated with damage to cellular biomolecules such as DNA, in addition to damaging the acrosome reaction (activation of the spermatozoa in contact to the oocyte) and disrupting the sperm–oocyte recognition and fusion2 which ultimately lower the odd for the fertilization to occur to produce an embryo.

Figure 1: Etiological factors associated with increased human sperm damage

The significant decrease in semen parameters over the years could be, in part, attributed to a poor diet with low intake of antioxidant nutrients and an alimentation rich in sugar and saturated fatty acid3-4.

Both omega 3 (ω3) and omega 6 (ω6) polyunsaturated fatty acids are essential components of phospholipids present in all tissues including spermatozoa cell membranes5 whose potential for a successful fertilization relies on its membrane lipid composition6.

The precursors of the ω3 and ω6 pathway are both essential fatty acids (your body can not synthesize them) and must be obtained through your diet7.

Fatty acid profile in relation to sperm parameters

The intake of omega-3 polyunsaturated fatty acids is associated with a better testicular function as indicated by higher testicular volume8 and higher sperm motility, sperm concentration and normal sperm morphology 9-11. Many other similar studies showed an increased ω6/ω3 ratio in

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men’s spermatozoa with poor semen quality12 and higher ratio for Arachidonic Acid/EPA in infertile men that correlates with their serum levels13-14.

EPA and DHA supplementation benefits on male fertility: published clinical data

In infertile men with oligoasthenozoospermia15 (low sperm number and low sperm motility), EPA and DHA supplementation (1.84g per day) induced a significant improvement of sperm cell total count and sperm cell concentration. Another study16 showed that dietary supplementation (1g DHA/day in addition to oral antioxidant) in oligozoospermic men (low sperm concentration) showed:

• increase in sperm concentrations (7.4 vs. 12.5 million/ mL)• increase of the acrosome reaction (55 vs. 71%): step allowing the penetration of the

spermatozoa to fertilize the egg• significant reduction of oxidative stress with decreased in ROS levels (775 vs. 150

(1000 counts/10 second)

Further, DHA dietary supplementation at 1.5g/day17 is able by itself to induce an increase in total antioxidant capacity with a significant reduction in the percentage of spermatozoa with DNA damage.

Altogether, these studies showed the beneficial impact of omega 3 in sperm parameters. Omega 3 levels in sperm correlate with serum levels and can be modulated by dietary omega 3 (EPA+DHA) supplementation to improve male fertility.

References:

1. Kahn BE, Brannigan RE. Obesity and male infertility. Curr Opin Urol. 2017Sep;27(5):441-445.2. Agarwal A, Saleh RA, Bedaiwy MA 2003. Role of reactive oxygen species in the pathophysiology

of human reproduction. Fertility and Sterility 79, 829–843.3. Mendiola J, Torres-Cantero AM, Vioque J, Moreno-Grau JM, Ten J et al. A low intake of antioxidant

nutrients is associated with poor semen quality in patients attending fertility clinics. Fertil Steril2010; 93: 1128–33.

4. Vujkovic M, de Vries JH, Dohle GR, Bonsel GJ, Lindemans J. Associations between dietarypatterns and semen quality in men undergoing IVF/ ICSI treatment. Hum Reprod (Oxford, England)2009; 24: 1304–12.

5. Mazza M, PomponiM, Janiri L, Bria P,Mazza S. Omega- 3 fatty acids and antioxidants inneurological and psychiatric diseases: an overview. Prog Neuropsychopharmacol Biol Psychiatry2007; 31: 12–26.

6. Lenzi A, Gandini L, Maresca V, Rago R, Sgro P. Fatty acid composition of spermatozoa andimmature germ cells. Mol Hum Reprod 2000; 6: 226–31.

7. Sprecher H,D.Luthria,B.Mohammed,S.Baykousheva,Reevaluation of the pathways for thebiosynthes is of polyunsaturated fatty acids,J.LipidRes.36 (1995)2471–2477.

8. MInguez-Alarcón L, Chavarro JE, Mendiola J, Roca M, Tanrikut C, Vioque J, Jørgensen N, Torres-Cantero AM. Fatty acid intake in relation to reproductive hormones and testicular volume amongyoung healthy men. Asian J Androl. 2017 Mar-Apr;19(2):184-190.

9. Conquer JA, Martin JB, Tummon I, Watson L, Tekpetey F. Fatty acid analysis of blood serum,seminal plasma, and spermatozoa of normozoospermic vs. asthenozoospermic males. Lipids 1999;34: 793–9.

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10. Attaman JA, Toth TL, Furtado J, Campos H, Hauser R. Dietary fat and semen quality among menattending a fertility clinic. Hum Reprod 2012; 27: 1466–74.

11. Martınez-Soto JC, Landeras J & Gadea J. (2013) Spermatozoa and seminal plasma fatty acids aspredictors of cryopreservation success. Andrology 1, 365–375.

12. Aksoy Y, Aksoy H, Altinkaynak K, Aydin HR, Ozkan A. Sperm fatty acid composition in subfertilemen. Prostaglandins Leukot Essent Fatty Acids 2006; 75: 75–9.

13. Safarinejad MR, Hosseini SY, Dadkhah F, Asgari MA. Relationship of omega-3 and omega-6 fattyacids with semen characteristics, and antioxidant status of seminal plasma: a comparison betweenfertile and infertile men. Clin Nutr 2010; 29: 100–5.

14. Safarinejad MR, Hosseini SY, Dadkhah F, Asgari MA. Relationship of omega-3 and omega-6 fattyacids with semen characteristics, and antioxidant status of seminal plasma: a comparison betweenfertile and infertile men. Clin Nutr 2010; 29: 100–5.

15. Safarinejad MR. (2011) Effect of omega-3 polyunsaturated fatty acid supplementation on semenprofile and enzymatic antioxidant capacity of seminal plasma in infertile men with idiopathicoligoasthenoteratospermia: a double-blind, placebo-controlled, randomised study. Andrologia 43,38–47.

16. Comhaire FH, Christophe AB, Zalata AA, Dhooge WS, Mahmoud AM, Depuydt CE. The effects ofcombined conventional treatment, oral antioxidants and essential fatty acids on sperm biology insubfertile men. Prostaglandins Leukot Essent Fatty Acids. 2000 Sep;63(3):159-65.

17. Martínez-Soto JC, Domingo JC, Cordobilla B, Nicolás M, Fernández L, Albero P, Gadea J, LanderasJ. Dietary supplementation with docosahexaenoic acid (DHA) improves seminal antioxidant statusand decreases sperm DNA fragmentation. Syst Biol Reprod Med. 2016 Dec;62(6):387-395.

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