RECURRENT PREGNANCY LOSS is defined as loss of three or more consecutive pregnancy¹. According to ASRM (American Society for Reproductive Medicine), Recurrent pregnancy loss is considered as two or more miscarriages². Clinical investigation may be initiated after two pregnancy losses.The occurrence of spontaneous miscarriage in India is reported to be around 6.37%³, recurrent miscarriage is around 1 to 2%⁴. The risk of miscarriage in subsequent pregnancy is 30% after 2 losses compared to 33% after 3 losses⁵. The prevalence of Hyperhomocysteinemia in recurrent pregnancy loss in India is 38%⁶.

Recurrent pregnancy loss (RPL) is a very traumatic condition both physically and mentally not just to female but also to the family. Its definition has always been controversial. According to RCOG, RPL is defined as three consecutive pregnancy losses, before 24 weeks gestation¹. According to the American Society for Reproductive Medicine (ASRM), it is defined as two or more clinical pregnancy losses (documented by ultrasonography or histopathology examination), and non- visualized pregnancy losses are not included².

RPL is an important health issue in reproductive females, because it affects 2% -5%of couple ^{7, 8,}The incidence of RPL varies widely from one report to other due to differences in the definition among international societies. Primary RPL refers to multiple losses in a woman with no previous viable infants, whereas secondary RPL refers to multiple losses in a woman who had previous pregnancy beyond 20 gestational weeks. Tertiary RPL refers to multiple pregnancy losses between normal pregnancies^9,10

Homocysteine is non-essential sulfur containing amino acid that is biosynthesized from methionine.With the help of Vitamin B12and Folicacid it is brokendowntomethionine by the enzyme Methylene-tetrahydrofolatereductase (MTHER). Homocysteine is also converted to cysteine through trans-sulphuration pathway with the help of pyridoxal 5 phosphate (active form of Vitamin B6). Hereditary defect in MTHER, hereditary lack of cystathionine-B- synthase, Homocysteine metabolism errorand dietary deficiency of folic acid, vitamin B6 and B12 cases hyperhomocysteinemia.

HYPERHOMOCYSTEINEMIA in pregnancy is associated with various placental pathology, like preeclampsia, IUGR, abruptio placenta, RPL, still births and also associated with neural tube defects¹¹ Exact pathophysiology is not known, studies have shown that the free radical produced as the result of homocysteine causes oxidative damageto the endothelium directly and high homocysteine have thrombotic impact on coagulation cascaderesultingin occlusivearterial and venous disease. Also homocysteine affects placental implantation by inhibition of trophoblast function and cell death.

This Casecontrolstudy was conducted in VanivilashospitalattachedtoBMCRI\, Bangalore. Study period was February2021toJune2022(18months)

InclusionCriteria:

GROUPA-CASES

1. Pregnant women with 2 or more spontaneous consecutive pregnancy losses at the booking visit.
2. Pregnantwomeninreproductiveage group(18to 39years)
3. Pregnantwomenwillingtogiveconsentforthe
4. Nootherknown

GROUP-BCASES

1. Matchedcontrolswithatleastonepreviouslivepregnancy

ExclusionCriteria:

1. Patientnotwillingtogiveinformed

1. Womenwithnonconsecutivepregnancy

1. Patientwithknownuterine
2. Women with known co morbidity like immunological diseases,antiphospholipid antibody syndrome , endocrinology disorder ( eg Hypothyroidism) .
3. Womenwithhistoryofmedical terminationof

1. Pregnant women with history of folic acid or vitamin B6 supplementation inthe last 6month.

SampleSize Calculation:

BasedonpreviousstudiesinINDIAhomocysteinelevelsof10.5istakenascutoff12,13.

n=2σ²x(zα+zβ)/d² n=sample size

σ^–arangeodSDfrompreviousstudyIe4.26 zα- standard deviation at 95% CI = 1.95

zβ–POWEROFSTUDY=0.84

d–Precision=1 n=45.3675

Substituting the values above, gives a sample size of 45.3675 which is rounded off to 50.

Minimalsamplesizeis50.

Sampletakenis100ineachgroup

After obtaining approval and clearance from the institutional ethics committee, the data is be collected using a prepared Performa by means of personal interview of the patient after taking informed consent and after applying inclusion and exclusion criteria.

1. Thorough history was taken of present pregnancy and previous pregnancy: Pattern, trimester, and characteristics of prior pregnancy losses.

History of sub fertility or in fertility. Menstrual history.

Prioror current gynecologic orobstetric infections.

Signs or symptoms of thyroid, glucose to lerance and hyper and rogenicd is orders (including polycystic ovarian syndrome).

Personal or familial thrombotic history.

Features associated with the antiphospholipid syndrome (thrombosis, false positive test for syphilis). Other autoimmune disorders.

Use of any Medications.

Environmental exposures, illicit and common drug use particularly caffeine, alcohol, cigarettes, and in utero diethylstilbestrol exposure).

1. Gestationalagewasbasedontheparticipants’lastnormalmenstrual
2. Thorough clinical examination including General physical examination, vital, systemic examination.
3. Group A cases, patient who come to the hospital for first Antenatal care visit with history of 2 or more consecutive pregnancy loss with no history of folic acid consumption in the last 6months were taken as study and patients with at least one successful pregnancy were taken as control.
4. Both cases and control group previous records were examined for laboratory investigation to rule out associated co-morbidities like uterine anomaly on ultrasound

, complete blood count for platelets, coagulation profile, thyroid profile, blood glucose, renal and liver function test, urine albumin, urine routine and microscopy and serology test for Human immune virus, Hepatitis B, syphilis, TORCH infection.

1. Cases and control were advised on first visit to come next morning on empty stomach with fasting for minimum 8hrs and was advised to take the test.
2. A venous blood sample by venipuncture to be used to assess Fasting homocysteine level. Total homocysteine concentration was measured by enzymatic photometric method, after centrifugation and storing. Patient sample Id was taken and confirmed with laboratory.
3. Hyperhomocysteinemiaisdefinedashomocysteinelevelmorethan95

In Indian studies, Yajniketal and Kumar etal have taken cut of fas10.5micromol.

STATISTICALANALYSIS

Data will be entered in the excel spread sheet. Descriptive statistics will be done which provides the mean, standard deviation and percentages. Inferential statisticslike chi-square and unpaired t test will be used for qualitative data and quantitative data respectively using SPSS (statistical Package for Social Sciences) version 20. (IBM SPASS statistics [IBM corp. released 2011]. Any other necessary tests will be dealt at the time of analysis based on data distribution

Atotalof200subjectswereincludedinfinal analysis. Among the study population, 100 (50%) participants were in Cases (RPL) and remaining 100(50%) participants were in control group.

TABLE1:Analysisoffrequencydistributionamongstudypopulation(N=200)

Among the case group,the mean age was 25.66 years (SD+/- 4.699 ) and it was 23.56 years(SD+/-3.557 ) in control group. The mean age was higher among patients with RPL.P VALVE IS <0.0001 which implies that there is significant age distribution among case and control group.

TABLE2: Comparison of age among study population (N=100)

SOCIOECONOMICCLASSES: Kuppuswamy classification was used to assess socioeconomic class of both groups. The SES was comparable in both the groups. In the lower socioeconomic status, 66 (66%) participants in cases and 54(54%) participants were in control group. Out of 200 participants 120(60%) belonged to lower class of Kuppuswamy classification.

TABLE3: Comparison of socioeconomic classes among cases and control:

TABLE4 : Meanhomocysteinelevelin case and control groups. (N=200)

Themeanhomocysteineincasegroupis8.43andcontrolgroupis5.77.Theabove table show

p<0.0001whichmeansthatthereisstatisticallysignificantdifferenceinmeanhomocysteine levels in cases and control groups.

TABLE5:              Number of           cases      and        control  with hyper homo cysteinemia

Thetableshows17%ofcasesand5%ofcontrol hadhyperhomocysteinemia.Thereis significant statistical difference in number of participant with increased homocysteine levels in case and control groups.

TABLE6:NumberofPrimaryandsecondaryabortershavinghyperhomocysteinemiaamongCases.

This table shows homocysteinemia is more common in primary aborters. HyperhomocysteinemiawasthricemorecommoninPrimaryaborterswhencompared to secondary aborters.

Multiple mechanisms are said to play a role in the etiology of spontaneous and recurrent abortions like immunological disorders, Genetic disorders, Endocrine disorders, Psychological disorders, inherited disorders are responsible for embryoloss. Studies reveal the pathogenic of inherited thrombophilia in RPL. Of these factors, moderate hyperhomocysteinemia has also been found to be a risk factor for RPL¹⁴

Severalpathophysiologicalhypothesesincludingimpairedcellproliferation,increased oxidative stress, apoptosis, reduced extra-embryonic vascular development are responsible for recurrent pregnancy loss in hyperhomocysteinemia¹⁵.It is not clear whether hyper homo cysteinemia is a causative factor or marker of RPL. Lowering homocysteine concentration by B-vitamin supplementation has shown to have a positive effect in several casereports¹⁶withspontaneouspregnanciesoccurringaftera few months of treatment in patients who had previously experienced early spontaneous abortion between 4 qt 12 weeks. Increasing evidences are available for the relationship between hyper homo cysteinemia and MTHFR C677T gene polymorphism and unexplained recurrent pregnancy loss^17,18.

In this study we evaluated homocysteine levels in women showing RPL. During pregnancy, the level sofplasma homo cysteinetendt of all due to increase dmethionine requirement by fetus, hemodilution and increased renal clearance of homocysteine. Hence the value tends tof all from 15 μmol to10.5 μmol¹³. Hence values more than10.5 taken as hyperhomo cysteinemia. In our study out of 100 cases with RPL17cases had hyper homo cysteinemia which is statistically significant.

Table7:Comparisonofpvaluebetweenvariousstudies

The women with RPL show significant higher mean homocysteine concentrations than controls, correlating with Nisha et al study.

Table 8: Comparison of number of primary and secondary aborters having hyperhomocysteinemia

In our study, hyperhomocysteinemia is more common in primary aborters while Comans et al and Wouters et al found Hyper homo cysteinemia more common in secondary aborters. The mechanism could be an intrinsic genetic metabolic disorder in primary aborters rather than dietary and environmental factors that could have a role in secondary aborters.

Hyperhomocysteinemia also found to be a risk factor for recurrent pregnancy loss as per this study. Daily supplementation with vitamin B12 and folic acid- reduce homocysteine concentration. Regarding MTHFR mutation, treatment is dietary intervention and supplementation with folic acid and vitamin B group^20,21.

Hence we can infer from present study that there is a statistically significant association of Hyper homo cysteinemia with complications likes early pregnancy loss. Many unknown causes remain regarding the impact of hyper homo cysteinemia on pregnancy. Large scale of study and control groups needed to define relation between homocysteine, folic acid and pregnancy loss.

Ourstudycomprises100recurrentpregnancylosscases and100controls. Outof100 cases, 17 patients show increased homocysteine levels. Homocysteine group shows P value of 0.001, which is significant at 1% levels.Homocysteine levels more than 10.5 μmol/dl is a risk factor for pregnancy loss.

This study shows there is positive correlation of socioeconomic class with hyperhomocysteinemia. Pregnant women belonging to Lower class of Kuppuswamy class had higher serum homocysteine level indirectly indicating poor nutrition among this pregnant.

1. American College of Obstetricians and Gynecologists' Committee on Practice Bulletins—Gynecology. ACOG Practice Bulletin No. 200: Early Pregnancy Loss. ObstetGynecol. 2018;132(5):e197-e207.
2. PracticeCommitteeoftheAmericanSocietyforReproductive

Definitionsofinfertilityandrecurrentpregnancyloss:acommittee opinion. FertilSteril. 2013;99(1):63.

3. PallikadavathS,StonesRW.MiscarriageinIndia:apopulation-based

FertilSteril[Internet].2005;84(2):516–8.

4. Antepartum hemorrhage in edited by HiralaKonar Text book of Obstetrics Eighthedition JaypeeBrother Medical Publishers, revised in 2015 p.282-301.
5. ACOGpracticebulletin.

Managementofrecurrent pregnancyloss.Number 24,February2001.(Replaces Technical Bulletin Number 212, September 1995).American College of Obstetricians and Gynecologists.IntJGynaecolObstet. 2002;78(2):179-190.

6. Bhatia N, B. H. Hyperhomocysteinemia in Recurrent pregnancy loss. Int J ReprodContraceptObstetGynecol [Internet]. 2017;6(7):2919.
7. PracticeCommitteeoftheAmericanSocietyforReproductive Evaluation and treatment of recurrent pregnancy loss: a committee opinion. FertilSteril. 2012;98(5):1103–1111.
8. RoyalCollegeofObstetriciansandGynaecologists,ScientificAdvisoryCommittee, Guideline No 17. The Investigation and treatment of couples withrecurrentmiscarriage,2011.Availablefrom:http://www.rcog.org.uk/womens-health/clinical-guidance/investigation-and-treatmentcouples-recurrent- miscarriage-green-top-. Accessed January 10, 2017.
9. KolteAM,Bernardi LA, ChristiansenOB,et al; ESHRESpecialInterest Group, EarlyPregnancy. Terminologyfor pregnancyloss prior to viability: a consensus statementfromtheESHREearlypregnancyspecialinterestgroup.HumReprod. 2015;30(3):495–498.
10. Silver RM, Branch DW, Goldenberg R, Iams JD, Klebanoff MA. Nomenclature for pregnancyoutcomes: time for a change. Obstet Gynecol. 2011;118(6):1402–