RECURRENT PREGNANCY LOSS & Clinical Lab. RECURRENT PREGNANCY LOSS & Clinical Lab. Dr.Mir Majid Mossalaeie (DCLS) Parseh Path. & Genetics Lab.

Occurrence of 3 or more clinically recognized consecutive or nonconsecutive pregnancy losses before 26 weeks from last menstrual period Occurrence of 3 or more clinically recognized consecutive or nonconsecutive pregnancy losses before 26 weeks from last menstrual period Primary- No previous full term pregnancy Primary- No previous full term pregnancy Secondary- At least one successful pregnancy Secondary- At least one successful pregnancy Recurrent Miscarriage-Definition

Incidence 15-20% of all pregnancies 15-20% of all pregnancies % in first pregnancy % in first pregnancy % after first abortion % after first abortion 38 % after two abortions 38 % after two abortions 55% after three abortions 55% after three abortions

Anatomic(Sporadic)12%-16% Anatomic(Sporadic)12%-16% Endocrine17%-20% Endocrine17%-20% Luteal phase deficiency Luteal phase deficiency Uncontrolled DM Uncontrolled DM PCOS PCOS Immunological10%-16% Immunological10%-16% Anti phospholipid syndrome Anti phospholipid syndrome Environmental Environmental Alcohol, Smoking Alcohol, Smoking Genetic factors 3.5-5% Genetic factors 3.5-5% Explained Un-explained 50% Recurrent Miscarriage Etiology

Single Gene Disorders in RM Second and 3 rd trimester losses Alpha Thalassemia Myotonic dystrophy X linked Dominant disorder –Incontinentia Pigmenti –Chondrodysplasia punctata –Focal dermal hypoplasia of Goltz –Rett Syndrome –Aicardi Syndrome 5

Single Gene Disorders in RM Hereditary thrombophilia –First and later trimester losses –Microthrombosis in placenta ;Impaired uteroplacental circulation Factor V Leiden gene mutation Evidence based Prothrombin G 20210A mutation inc. risk Protein C,S deficiency Antithrombin III No significant association MTHFR C677T mutation Combination of any of above-Increased risk 6

Role of Infections

Venn diagram of the responsibilities of Reproductive Failure EGG 80% SPERM 10% UTERUS 10%

Doubtful causes of RSA TORCH infections TORCH infections Endocrine and metabolic disease Endocrine and metabolic disease Untreated adrenal hyperplasia, hypothyroidism & diabetes mellitus. Untreated adrenal hyperplasia, hypothyroidism & diabetes mellitus. Exogenous causes Exogenous causes Environmental factors, alcohol, street drugs, anesthesia gases etc Environmental factors, alcohol, street drugs, anesthesia gases etc

Its time to say goodbye to TORCH tests……. Cochrane Review has categorically proven in multiple meta-analysis that none of the “TORC H” group of infections are responsible for RECURRENT SPONT ANEOUS ABORTIONS TORCH TESTS

So which infections, if any are responsible for RSA? Female Viral infections ? ? Viral infections ? ? Coxasackie B Coxasackie B Parovo-virus B Parovo-virus B Bacterial infections Bacterial infections Bacterial Vaginosis Bacterial Vaginosis Tuberculosis Tuberculosis Chlamydia trachomatis Chlamydia trachomatis Male factors: Semen infections can cause anueploidy and be the reason of RSA Semen infections can cause anueploidy and be the reason of RSA

The influence of Chlamydia trachomatis infection on RSA The influence of Chlamydia trachomatis infection on RSA Specific anti-chlamydial antibodies in 3 groups of women IgA class IgA class – 7.9% (p=0.082) in group 1 (RSA group), – 4.5% (p=0.236) in group 2 (1 abortion) – 0% in group 3 ( no abortions) IgG class in 21.1% (p=0.024), 36.4% (p=0.000) and in 4.4%, respectively. IgG class in 21.1% (p=0.024), 36.4% (p=0.000) and in 4.4%, respectively.CONCLUSIONS: C.t. infection is an important causative agent in RSA C.t. infection is an important causative agent in RSA Anti-Chlamydial antobodies included in screening tests Anti-Chlamydial antobodies included in screening tests Wilkowska-Trojniel M. Adv Med Sci. 2009;54(1):86-90 Wilkowska-Trojniel M. Adv Med Sci. 2009;54(1):86-90 Kavalier F, BMJ Jul 16;331(7509): Kavalier F, BMJ Jul 16;331(7509):121-2.

Hattori Y, Nakanishi T. Am J Reprod Immunol Oct;58(4): Uterine cervical inflammatory cytokines, interleukin-6 and -8, as predictors of RSA Uterine cervical inflammatory cytokines, interleukin-6 and -8, as predictors of RSA Both IL-6 and IL-8 in cervical mucus were significantly higher in patients who miscarried subsequently than in those who had a live birth. Both IL-6 and IL-8 in cervical mucus were significantly higher in patients who miscarried subsequently than in those who had a live birth.

Other rare viruses Coxsackie B virus (CBV) & RSA Parvovirus B19

Is it time to look at the sperm?

? No DNA Repair Fertilisation Failure Abnormal offspring Normal offspring DNA Repair Fertilisation Partial DNA Repair Fertilisation Consequences of fertilisation by sperm with nuclear DNA damage

SEMEN CULTURE Male accessory gland infection with E coli / Staph aureus Male accessory gland infection with E coli / Staph aureus Bacteria ride on the sperm tails into uterine cavity Bacteria ride on the sperm tails into uterine cavity Produce low grade endometritis Produce low grade endometritis

Possible role of male factors in recurrent pregnancy loss Amongst male partners of women with RSA 3 (4%) had varicocele, 23 (30.6%) had infection, 1 (1.3%) immunological and 1 (1.3%) had genetic abnormality Amongst male partners of women with RSA 3 (4%) had varicocele, 23 (30.6%) had infection, 1 (1.3%) immunological and 1 (1.3%) had genetic abnormality Sperm motility, viability and sperm function tests were significantly lower in the RPL group as compared to the control group (P = 0.000) Sperm motility, viability and sperm function tests were significantly lower in the RPL group as compared to the control group (P = 0.000) Male factor might be a contributing factor towards RPL Male factor might be a contributing factor towards RPL Both the partners should be evaluated Both the partners should be evaluated Infection treated in both Infection treated in both Saxena P, Misro MM et al. Indian J Physiol Pharmacol Jul-Sep;52(3):274-82

Conclusion Problems of Research in RSA The cause of individual abortion may be different The cause of individual abortion may be different More than one factor may exist More than one factor may exist Thorough investigation often fails to reveal a cause Thorough investigation often fails to reveal a cause Infections must be ruled out Infections must be ruled out Fertil Steril Mar 1;93(4): Epub 2009 Mar 31

Conclusions TORCH group DOES NOT cause RSA TORCH group DOES NOT cause RSA Infections in both partners need to be evaluated in cases of RSA Infections in both partners need to be evaluated in cases of RSA Therefore the genetic counseling of couples should include thorough medical examination and evaluation for infections Therefore the genetic counseling of couples should include thorough medical examination and evaluation for infections

Antiphospholipid Antibody Syndrome and Recurrent Pregnancy Loss 21

Autoimmune etiology Secondary to autoimmune disease such as SLE, Polyarteritis nodosa, etc Secondary to autoimmune disease such as SLE, Polyarteritis nodosa, etc Primary Antiphospholipid Syndrome (PAPS) refers to the association of adverse pregnancy outcome and presence of antiphospholipid antibodies Primary Antiphospholipid Syndrome (PAPS) refers to the association of adverse pregnancy outcome and presence of antiphospholipid antibodies 22

Which antibodies ? A number of antibodies have been studied A number of antibodies have been studied The antibodies with the greatest significance and association with obstetric events are The antibodies with the greatest significance and association with obstetric events are Lupus anticoagulant (LA) Lupus anticoagulant (LA) Anticardiolipin antibodies (ACL IgG and ACL IgM) Anticardiolipin antibodies (ACL IgG and ACL IgM) Others have such as β 2 glycoprotein-I, antiphosp hatidylserine antibodies, annexin, etc may not be obstetrically significant Others have such as β 2 glycoprotein-I, antiphosp hatidylserine antibodies, annexin, etc may not be obstetrically significant 23

Incidence About 1% of couples have recurrent miscarriages About 1% of couples have recurrent miscarriages Antiphospholipid antibodies are found in about 2% of a Caucasian population. Not studied in a general Asian / In dian population Antiphospholipid antibodies are found in about 2% of a Caucasian population. Not studied in a general Asian / In dian population 5 – 20% of women with recurrent miscarriages have anti phospholipid antibodies 5 – 20% of women with recurrent miscarriages have anti phospholipid antibodies MacLean AS et al, BJOG 1994 Rai RS et al, Hum Reproduction 1995 Balasch J et al, Hum Reproduction

Statistical Distribution Prevalence of antiphospholipid antibodies in various categories of women was studied Prevalence of antiphospholipid antibodies in various categories of women was studied Women with 3 or more early fetal losses Women with normal pregnancy outcome Women who have not been pregnant (includes women not desiring pregnancy and infertile women) 16%7%3% Parke AL et al, Arch Rheumat

Antiphospholipid Antibodies Phospholipid molecules are normal components of all cell membranes. Some also have glue like properties and allo w cells to fuse. Antibodies to phospholipid molecules can, therefore, cause problems. Specifically, they can damage the inside of the blood vessel wall. This allows blood cells to stick to the site of the injury and cause blood clots. Phospholipid molecules are normal components of all cell membranes. Some also have glue like properties and allo w cells to fuse. Antibodies to phospholipid molecules can, therefore, cause problems. Specifically, they can damage the inside of the blood vessel wall. This allows blood cells to stick to the site of the injury and cause blood clots.

Antiphospholipid antibodies can also cause blood vessels to constrict, causing decreased blood flow throughout the circulatory system. Antiphospholipid antibodies can also cause blood vessels to constrict, causing decreased blood flow throughout the circulatory system. The combination of blood clots and constricted blood vessels may impair blood supply to the fetus and placenta resulting in complete fetal demise or growth retardation.

Diagnosis of PAPS Based on clinical and laboratory criteria Based on clinical and laboratory criteria One obstetric or thrombotic criteria and one laboratory criteria should be present to diagnose PAPS One obstetric or thrombotic criteria and one laboratory criteria should be present to diagnose PAPS Other autoimmune disease has to be ruled out to make the diagnosis of PAPS Other autoimmune disease has to be ruled out to make the diagnosis of PAPS Wilson A et al, International Consensus statement on APS, Arthritis Rheumatol

Obstetric Criteria Three or more consecutive spontaneous abortion before the 10 th week of gestation Three or more consecutive spontaneous abortion before the 10 th week of gestation One or more unexplained fetal death at or beyond the 10 th week of gestation One or more unexplained fetal death at or beyond the 10 th week of gestation Severe preeclampsia or placental insufficiency (IUGR) necessitating birth before the 34 th week of gestation Severe preeclampsia or placental insufficiency (IUGR) necessitating birth before the 34 th week of gestation 29

Vascular Thrombosis Criteria Unexplained venous thrombosis Unexplained venous thrombosis Unexplained arterial thrombosis Unexplained arterial thrombosis Small vessel thrombosis in any tissue or organ, without significant evidence of inflammation of the vessel wall Small vessel thrombosis in any tissue or organ, without significant evidence of inflammation of the vessel wall 30

Laboratory Criteria Anticardiolipin antibody IgG or IgM isotype in medium to high titers by standardized ELISA assay Anticardiolipin antibody IgG or IgM isotype in medium to high titers by standardized ELISA assay Lupus anticoagulant present Lupus anticoagulant present A positive test has to be repeated on at least one more occasion 12 weeks apart to fulfill the laboratory criteria A positive test has to be repeated on at least one more occasion 12 weeks apart to fulfill the laboratory criteria 31

Lupus anticoagulant testing Screen with demonstration of prolonged phospholipid dependent coagulation screening test (eg: activated partial thromboplastin time, kaolin clotting time, diluted Russell’s viper venom time, dilute prothrombin time) Screen with demonstration of prolonged phospholipid dependent coagulation screening test (eg: activated partial thromboplastin time, kaolin clotting time, diluted Russell’s viper venom time, dilute prothrombin time) Failure to correct the prolonged screening test by mixing with normal platelet-poor plasma Failure to correct the prolonged screening test by mixing with normal platelet-poor plasma Shortening or correcting the prolonged screening test by addition of excess phospholipids Shortening or correcting the prolonged screening test by addition of excess phospholipids Exclusion of other coagulopathies if clinically indicated Exclusion of other coagulopathies if clinically indicated 32

Laboratory Diagnosis of Lupus Anti coagulants test: test: 3 group : 3 group : Screening test Screening test Tests for Rule out other coagulopathies Tests for Rule out other coagulopathies Confirmatory test Confirmatory test

Pitfalls in diagnosis of PAPS Usually an over diagnosed syndrome Usually an over diagnosed syndrome Not meeting clinical and the strict laboratory criteria Not meeting clinical and the strict laboratory criteria Not repeating the laboratory test at 12 weeks Not repeating the laboratory test at 12 weeks Non standardized ELISA for ACL antibodies Non standardized ELISA for ACL antibodies Interlaboratory variations for phospholipid dependent coagulation tests used for screening for lupus anticoagulant Interlaboratory variations for phospholipid dependent coagulation tests used for screening for lupus anticoagulant 35

False results in PAPS Improperly collected and processed samples Improperly collected and processed samples Temporal and trimester wise fluctuations Temporal and trimester wise fluctuations VDRL positive patients who may or may not have syphilis VDRL positive patients who may or may not have syphilis General infections and inflammations General infections and inflammations Coagulopathies and anticoagulant medication users (including aspirin, heparin) Coagulopathies and anticoagulant medication users (including aspirin, heparin) 36

Goals for treating PAPS Avoid early pregnancy loss Avoid early pregnancy loss Normalize placental and fetal circulations to prevent early birth from obstetric complications such as preeclampsia and growth restriction Normalize placental and fetal circulations to prevent early birth from obstetric complications such as preeclampsia and growth restriction Prevent maternal vascular thrombosis in pregnancy and postpartum Prevent maternal vascular thrombosis in pregnancy and postpartum 37

Women with PAPS without a history of thrombotic events (most women with RPL) Women with PAPS with history of thrombotic events (past or present) Prophylactic therapies such as aspirin, heparin in pregnancy and 6 to 8 weeks postpartum Full anticoagulation with heparin (or warfarin) in pregnancy and postpartum 38

Aspirin alone v/s Aspirin + Heparin Recent metaanalysis shows that the combi nation of Aspirin + Heparin is better than Aspirin alone in achieving live births in women with recurrent pregnancy loss and antiphospholipid antibodies Recent metaanalysis shows that the combi nation of Aspirin + Heparin is better than Aspirin alone in achieving live births in women with recurrent pregnancy loss and antiphospholipid antibodies Mak A et al, Rheumatology (Oxford)

Is Heparin + Aspirin really better? The metaanalysis was based on data from five trials involving 334 patients across non uniform care platforms The metaanalysis was based on data from five trials involving 334 patients across non uniform care platforms Overall live birth rates were and 55.83% in the combination and aspirin alone groups Overall live birth rates were and 55.83% in the combination and aspirin alone groups RR 1.301; 95% CI 1.040, RR 1.301; 95% CI 1.040, Number needed to treat is 5.6 Number needed to treat is 5.6 There is no placebo group for comparison There is no placebo group for comparison Another metaanalysis showed that LMW heparin + Asprin does not significantly improve birth rates. The benefits is present only with unfractionated heparin Another metaanalysis showed that LMW heparin + Asprin does not significantly improve birth rates. The benefits is present only with unfractionated heparin Zikas PD et al, Obstet Gynecol

Clinical Tips for using Heparin There is controversy as to whether LMW Heparin is effective in preventing recurrent pregnancy loss There is controversy as to whether LMW Heparin is effective in preventing recurrent pregnancy loss Consider costs, convenience and compliance before initiating therapy Consider costs, convenience and compliance before initiating therapy Therapy should be started when fetal cardiac activity is demonstrated and continued throughout pregnancy and postpartum Therapy should be started when fetal cardiac activity is demonstrated and continued throughout pregnancy and postpartum Heparin in prophylactic doses needs to be stopped for about 24 hours around the time of labor and delivery Heparin in prophylactic doses needs to be stopped for about 24 hours around the time of labor and delivery 41

Heparin in prophylactic doses can not be monitored and does not require monitoring by coagulation parameters Heparin in prophylactic doses can not be monitored and does not require monitoring by coagulation parameters Do a platelet count at 3 days, 1 week and bimonthly when the patient is on heparin Do a platelet count at 3 days, 1 week and bimonthly when the patient is on heparin Standard doses Standard doses Unfractionated heparin – 5000 units sc bd Unfractionated heparin – 5000 units sc bd Enoxaparin – 40 mg sc daily or in two doses Enoxaparin – 40 mg sc daily or in two doses Clinical Tips for using Heparin 42

Full Anticoagulation : Practical Preconception : Warfarin Preconception : Warfarin Switch to Heparin when fetal cardiac activity is demonstrated Switch to Heparin when fetal cardiac activity is demonstrated Warfarin should be considered in the second trimester Warfarin should be considered in the second trimester Switch back to Heparin at 34 to 36 weeks Switch back to Heparin at 34 to 36 weeks After delivery : Warfarin After delivery : Warfarin 43

What not to do for PAPS Steroid therapy should be avoided for PAPS because it significantly increases morbidity (hypertension, diabetes, preterm births) without any demonstrable benefit Steroid therapy should be avoided for PAPS because it significantly increases morbidity (hypertension, diabetes, preterm births) without any demonstrable benefit Immunoglobulin therapy is experimental and not for clinical use at present Immunoglobulin therapy is experimental and not for clinical use at present 44

RECOMMENDED INVESTIGATIONS Grade A (FOR ALL PATIENTS) Grade A (FOR ALL PATIENTS)  Hysterosalpingography/ Hysteroscopy  APTT/ dRVVT/ Lupus anticoagulant  IgG & IgM anticardiolipin antibodies  TSH / Prolactin / Testosterone / HbA1C/ 2 hrs Post Prandial INSULIN  Karyotyping of both parents &  If possible abortus

RECOMMENDED INVESTIGATIONS Grade B (FOR SELECTED PATIENTS) Grade B (FOR SELECTED PATIENTS)  ANDROGENS, LH, FSH IN PATIENTS WITH IRREGULAR MENSTRUATION  SERUM PROGESTERONE  EB & TB PCR, CULTURE  SERUM HOMOCYSTEINE LEVELS / THROMBOP HILIA SCREEN  HVS / WET PREP & pH / KOH Whiff test  SEMEN CULTURE / TB PCR

ANTI PHOSPHOLIPID SYNDROME LOW DOSE ASPIRIN pre preg clinic LOW DOSE ASPIRIN pre preg clinic HEPARIN after ultrasound viability HEPARIN after ultrasound viability Low molecular weight heparin Low molecular weight heparin Intravenous immunoglobulins Intravenous immunoglobulins Corticosteroids only used in aps associated with sle Corticosteroids only used in aps associated with sle Warfarin if previous arterial thrombosis in second & third trimester Warfarin if previous arterial thrombosis in second & third trimester

INFECTVE CAUSES Screening and treatment of bacterial vaginosis Screening and treatment of bacterial vaginosis Screening and treatment of occult genital tuberculo sis Screening and treatment of occult genital tuberculo sis Chlamydia screening & treatment Chlamydia screening & treatment

ENDOCRINAL FACTORS Polycystic ovaries ? metformin Polycystic ovaries ? metformin Luteal phase defects progesterone / Duphaston Luteal phase defects progesterone / Duphaston Thyroid replacement therapy Thyroid replacement therapy Optimising HbA1c levels Optimising HbA1c levels Correct hyperprolactinaemia Correct hyperprolactinaemia

THROMBOPHILIA SCREEN POSITIVE LOW MOLECULAR WEIGHT HEPARIN LOW MOLECULAR WEIGHT HEPARIN UNFRACTIONATED HEPARIN UNFRACTIONATED HEPARIN (From 6 weeks to 36 weeks of pregnancy)

51HAEMATOLOGICAL Folic acid, vitamin b6, vitamin b12 in hyperhomocystinaemia Folic acid, vitamin b6, vitamin b12 in hyperhomocystinaemia Low dose aspirin Low dose aspirin Heparin or LMWH Heparin or LMWH Full dose heparin in case of DVT Full dose heparin in case of DVT WARFARIN if arterial thrombosis WARFARIN if arterial thrombosis

the chance of a viable birth the chance of a viable birth even after four prior losses : 60% even after four prior losses : 60% cytogenetic etiology : 20%-80% cytogenetic etiology : 20%-80% corrected anatomic anomalies : 60%-90% corrected anatomic anomalies : 60%-90% corrected endocrinologic abnormalities : higher than 90% corrected endocrinologic abnormalities : higher than 90% women receiving therapy for antiphospholipid antibodies : 70% - 90% women receiving therapy for antiphospholipid antibodies : 70% - 90%

THANK YOU for your valuable time

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