{"paper_id":"49591310-a1e5-487d-8acf-895cfcc50164","body_text":"© 2010. Al Ameen Charitable Fund Trust, Bangalore  322  \nAJMS                     A l  A me e n  J  M e d  S c i  (2 010 )3 (4 ):3 2 2 -3 3 1  \n(An US National Library of Medicine enlisted journal)                                     I S S N  0 9 7 4 - 1 1 4 3  \n  \nO RI G I NAL  ART I CL E  \n \nInfestation Of Endometrium By Mycobacterium Tuberculosis \nBacilli-Cause Of Reproductive Failure \n \nRajib Gon Chowdhury 1, Suman Kalyan Paine 2, Basudev Bhattacharjee 2 \nand Siddhartha Chatterjee 1* \n \n1Calcutta Fertility Mission, Pushpanjali Apartment, 102C Ballygunge Place,  \nKolkata- 700019 India and 2 Department of Biochemistry,Institute of Post-\nGraduation Medical Education & Research (IPGMER), SSKM Hospital,244,AJC \nBose Road, Kolkata-700 020 \n \nAbstract:  Tuberculosis is known to be one of the major disea ses, causing infertility in India. \nThe prevalence of tuberculosis causing infertility is different in different countries. \nEstablished tubercle infection may cause irreversib le infertility, on many occasions as it may \naffect all the reproductive organs. It also produce s lower pregnancy rate in Assisted \nReproductive Technology program as well. Tubercular  bacillary infestation of the \nendometrium that is mere presence of Mycobacterium Tuberculosis (MTB) bacilli on the \nendometrial surface has been found to affect fertil ity as well. In this study, we have detected \nMycobacterium Tuberculosis (MTB) infestation of endometrium, causing implanta tion failure \nor early embryonic rejection. In many cases of unex plained infertility, tubercular bacilli \ninfestation of endometrium, has come out to be the root cause of infertility. Association of \ntubercular bacillary infestation and endometriosis is another cause of concern. Recurrent \nabortions and ectopic pregnancy may be precipitated  by the same genital pathology as well. \nThe presence of very small number of bacilli, which  escapes detection by AFB smear, culture \nor histology, may be detected by polymerase chain r eaction techniques. These bacillary \ninfections bring an inflammatory change in the endo metrium and produces harmful cytokines \nwhich are responsible for implantation failure of micro abortion.  \nKeywords:  MTB infestation, PCR study, unexplained infertilit y, recurrent pregnancy loss, \nendometriosis \n \nIntroduction \nInfertility is the commonest symptom associated wit h genital tuberculosis [1, 2] and \nit seems to be an important under-diagnosed factor in infertility [3]. So for any \nmeasure to cure infertility, tuberculosis must be e xcluded. The exact incidence of \nfemale genital tuberculosis is not known partly bec ause more often the disease \nremains silent and in addition, there are lack of r eliable confirmatory investigations. \nThe first reported case of gynaecological tuberculo sis was described by Morgagni in \n1744. On making a post-mortem examination on a woma n aged 20 years, he found \nthe uterus and both tubes filled with caseous mater ial. Interest in the field of \ngynaecological tuberculosis began to build up in th e early years of this century.  The \nmost important early contributions were given by Be rkeley (1903), Daniel (1925), \nGreenberg (1924), Jameson (1935), Murphy (1903, 190 4), Norris (1921) and \nWilliams (1894) [4]. Genital tuberculosis (TB) in f emales, is found in 0.75 to 1% of \ngynaecological admissions in India with considerabl e variation from place to place. \nThe disease is responsible for 5% of all female pel vic infections and occurs in 10%  \n\nAl Ameen J Med. Sci, Volume 3, No.4, 2010                                                         Gon Chowdhury R. et al  \n© 2010. Al Ameen Charitable Fund Trust, Bangalore  323  \n \ncases of pulmonary tuberculosis. Although most of t he affected belong to \nreproductive age- group, the disease has been reported in post-menopausal females as \nwell. Lately, an increase in the trend of this dise ase has been reported, which may be \npartly due to an overall rise in tuberculosis cases . The other contributory factors may \nbe HIV infection, as tuberculosis is the most commo n HIV-related opportunistic \ninfection in India [5]. However, the average world- wide incidence of female genital \ntuberculosis in infertile population has been repor ted as 5-10% [6, 7], with the range \nvarying between <1% in USA and about 10% in India [ 8]. Incidence at our clinic is \n9.6%. Incidence of infertility in genital tuberculo sis may vary between 40–75.6% [9, \n10]. Infertility in genital tuberculosis may be due  to various causes. The average \nincidence of genital TB in infertility clinics worl dwide is 5-10%, and varies from \n0.69% in Australia to 17.4% in India  [11]. Tuberculosis may cause minimal damage \nto the tube leading to ectopic pregnancy. Extensive  damage may lead to complete \ntubal occlusion [12]. Peri-tubal adhesions and tubo -ovarian mass have been found in \n47.2% of cases [13]. Various grades of intrauterine adhesion (Asherman’s syndrome) \nor non-receptive endometrium have been reported in association with genital \ntuberculosis [14]. Although pulmonary TB (PTB) rema ins the commonest and the \nmost infectious type of TB, extra-pulmonary TB (EPTB) is becoming more prevalent \nespecially in young women throughout the world [15] . Female genital TB (FGTB), \nbeing non-infectious, has been neglected by healthc are providers, but is an important \ncause of both significant morbidity and short- and long-term sequelae for the affected \nwomen [15, 16]. The incidence of active tuberculosi s in infected individuals is only \n10% [17]. Depending on the virulence of organism an d immune response generated \nby the host, the disease remains either active or b ecomes asymptomatic with latent \ninfection persisting for many years [18]. Latent in fected individuals contain dormant, \nyet viable bacilli, which may re-activate when the host response becomes low, and as \na consequence, the disease may become active again.  During the process of \nreactivation, the bacilli induce immune modulation within the local tissues, which \nmimics the process seen during infection. There is release of harmful cytokines like \nIL2, TNF α and INF γ. The final effect will depend upon how strongly th e host tissue \n(ovarian tissue and endometrium) can resist this tr auma [19]. If unable to resist, \nimmuno-modulatory impact will affect adversely, the  endometrial receptivity. Once \nthe adverse impact has been established on the deli cate function of this reproductive \norgan, the consequences may continue to persist; in fection subsequently remains \ndormant or even cured. So for any measure to cure i nfertility, tuberculosis must be \nexcluded. Since physical symptoms are usually not p resent definitively, the disease \nremains undiagnosed or specific investigations are not undertaken to rule out the \nproblem (Bateman et al, 1986) [20]. It is a pauci-b acillary form of disease of which \ncultures and smears are often negative (Baum et al,  2001) [21]. The primary focus is \nrarely found outside the genital tract (Sutherland 1985) [22]. Routine screening tests \nfor pulmonary tuberculosis like X-ray chest, tuberc ulin test and sputum examination \nare usually negative (Schaetzing, 1986) [23]. With the above mentioned background \nin mind this study was conducted employing a multip lex PCR, developed in our \nlaboratory to evaluate its effectivity in diagnosing genital tuberculosis among patients \nattending infertility clinic and how far it can be implicated for infertility. \n \n\nAl Ameen J Med. Sci, Volume 3, No.4, 2010                                                         Gon Chowdhury R. et al  \n© 2010. Al Ameen Charitable Fund Trust, Bangalore  324  \n \nMaterials and Methods \n517 cases having C/O unexplained infertility, Recur rent Spontaneous Abortions \n(RSA) Ectopic Pregnancy and Mild Endometriosis were  investigated by multiplex \nPCR screening. Out of 517 cases, in 49 cases for th e detection of AFB smear by ZN \nstain or culture of menstrual blood in LJ media, fo r the detection of tubercle bacilli \nwas performed, along with TB PCR study. In rest of the cases, only TB PCR study \nwas undertaken. Patients were advised to attend the clinic on d2 of period. Menstrual \nflow was usually considerable on 2nd day of menstru al cycle and that confirms \nproper menstrual flow. A sterile Cusco’s speculum w as introduced in the vagina \ncarefully, avoiding contamination/contact to skin. Menstrual blood was collected by \na 1cc/2cc sterile syringe and transferred to a ster ile vial containing normal saline. \nUtmost care was taken to avoid contamination with s kin. On many occasions, the \nskin may contain commensals mycobacteria. \n \nSelection of Cases: Studies were undertaken in 517 cases. These cases w ere selected \nfrom infertile patients in the following group:  \nGroup A:  246 patients, who were apparently unexplained. The se cases were either \nprimarily normal, with tubal, ovulatory or seminal factor or after correction of the \nabove factors,  they were sub-normal. The usual wai ting period for pregnancy to \noccur often about one year, following they were declared normal. \nGroup B:  218 patients with GI to II endometriosis, without a ny anatomical distortion \nof the pelvic organs, where good tubo-ovarian relat ion along with free Pouch of \nDouglus (POD) was maintained. Here also, the waiting period for pregnancy to occur \nwas about one year, with or with out ovulation induction.  \nGroup C:  46 patients with more than one spontaneous abortion (RSA) \nGroup D:  7 cases with previous Ectopic Pregnancy \n \nExclusion Criteria: Patients with previous history of anti-tubercular d rug (ATD) \nintake were excluded from this study. For Group A, that is Unexplained Infertility, if \nthere was ovulatory factor, previous correction of the thyroid status and control of \nprolactin level, followed by ovulation induction wi th oral agents like Clomifene or \nLetrozole are included in the study. Any case which  required ovulation induction \nwith Gonadotrophin, or cases where Clomifene or Let rozole failed in inducing \novulation induction, were excluded. Cases with endo metriosis, having pelvic \nadhesion, distorting tubo-ovarian relation, or abno rmal Pouch of Douglas, were \nexcluded from this study. For recurrent spontaneous  abortions, where no \nendocrinological, anatomical or immunological cause s were found, were included in \nthis study. Previous ectopic pregnancies, where def inite tubal defects were detected, \nwere excluded from the study. \n \n \nCollection of Samples: After taking informed consent, the patients were ad vised to \nattend the clinic on 2nd day of menstrual cycle. Me nstrual flow is usually \nconsiderable on 2 nd day of menstrual cycle. In 49 of these cases, endom etrial biopsy \nsamples were collected by curetting the endometrial cavity by a small curette with or \nwithout dilating the cervix under deep sedation or anaesthesia, in the pre-menstrual \nphase of the same cycle. Menstrual fluid specimens were collected in three parts.  \n \n\nAl Ameen J Med. Sci, Volume 3, No.4, 2010                                                         Gon Chowdhury R. et al  \n© 2010. Al Ameen Charitable Fund Trust, Bangalore  325  \n \nEndometrial specimens were aliqueted into three portions - one kept in normal saline \nfor microscopy and culture, one in formal saline fo r histopathology and one in lysis \nbuffer(50mmol/l KCl,10 mmol/lTrisHCl pH 8.3, 1.5 mm ol/l MgCl 2,  0.1% NonidetP-\n40, 0.5%Tween-20) for extraction of DNA s for subse quent  multiplex PCR study. \nSimilarly, three menstrual fluid specimens were tre ated like endometrial tissues. All \nthe clinical samples were screened with conventiona l microbiological tests such as \nZiehl–Neelsen acid fast staining for recording smea r-positivity, identification by \ncultural isolation and biochemical tests. Lowenstei n–Jensen medium was used for \nprimary isolation. Tests to identify M. tuberculosis complex  and NTM were: (i) \nniacin test, (ii) catalase test, (iii) heat stable catalase test, (iv) pigment production \ntest, (v) Growth at 42–44 0C, (vi) aryl sulphatase test and (vii) state-of-the -art nucleic \nacid amplification tools like a multiplex PCR techn ique. For culture, each specimen \nwas de-contaminated by N-acetyl-L-cysteine-sodium h ydroxide as per Kubica \n(Kubica et al  1963)  [24]. The processed sediment was cultured for more than six \nweeks on two Lowenstein-Jensen medium slants at 37 0C. These culture slants were \ninspected for growth every week. Positive cultures were further examined to confirm \nthe presence of M. tuberculosis.  \n \nProcessing of Positive Mycobacterial culture for ex traction of Genomic DNA: One \nloop of cultured Mycobacterium ,grown on Lowenstein-Jensen slants from menstrual \nfluid and endometrial specimens was suspended in 50 0 µl of 1X TE buffer (10 mM \nTris-HCl, 1 mM EDTA, pH 7.5). The suspension was ly sed with 0.5% SDS and \nproteins were digested with 0.5 mg of Proteinase-K/  ml for 15 minutes at 37° C \n(Syun-Ichi et al.  1993)  [25].  \n \nProcessing of endometrial specimen for extraction o f genomic DNA: A portion of \nendometrial specimen was collected in lysis buffer as described above.  Sodium \ndodecyl sulfate (SDS) and Proteinase-K were added t o final concentration of 1% and \n0.1 mg/ ml respectively and incubated at 37° C for one hour.  Subsequently, 5 mol/ L \nNaCl and 10% Cetyl trimethyl ammonium bromide (CTAB ) and 0.7 mol/ L NaCl \nwere added and kept at 65° C for 10 minutes (Herrer a et al  1996)[26]. Mycobacterial \ngenomic DNAs from all the mycobacterial strains gro wn on culture and processed \nendometrial tissues were extracted by addition of e qual volume of phenol-\nchloroform-isoamyl alcohol (25:24:1, V/V/V) solutio ns.  The aqueous phase was \ntransferred to another tube; DNAs were finally prec ipitated with 0.6 vol of isopropyl \nalcohol and DNA pellets were collected by centrifug ation.  The pellet was washed \nwith 70% ethanol, which was then dried and dissolve d in 30 µl TE buffer (pH 7.8) \nand stored at -20° C for future use. The DNA concen tration was measured from OD \nat 260 nm and run in 0.8% agarose gel with λHind III DNA molecular marker. \n \nMultiplex PCR: Samples as described were screened by multiplex PCR  system \ndeveloped in our laboratory (Bhattacharya et al  2003) [27] by modifying three \nindividual PCR (Pao et al  1990; Syun-Ichi et al.  1993; Kox et al  1994) [25, 28, 29] \nto one reaction condition. The primer pairs are sel ected by following manner (i) Two \noligonucleotide primers derived from the sequence o f gene that codes for the 65 kDa \nantigen of M. tuberculosis ; a pair of 24 base synthetic oligonucleotide (prim ers) \nbracketing a 165-bp region of a gene codes for a 65  kDa antigen (Shinnick 1987).  \n\nAl Ameen J Med. Sci, Volume 3, No.4, 2010                                                         Gon Chowdhury R. et al  \n© 2010. Al Ameen Charitable Fund Trust, Bangalore  326  \n \nBriefly, according to Pao et al  1990), from 5´ to 3´ ends CTA GGT CGG GAC GGT \nGAG GCC AGG and CAT TGC GAA GTG ATT CCT CCG GAT. (i i) In this \nreaction two genus-specific oligonucleotide primers  (forward, 5´-AAG AGG AAG \nGAG AGA GGG G-3´ and reverse, 5´-GTC GTT GAG GTT GA A CTC-3´) were \nused based on nucleotide sequence of DNA gene of M.tuberculosis  (Syun-Ichi et al.  \n1993) [25]. The amplification of the 365-bp (region  between sequence position \n1377–1741 of M. tuberculosis ) band has been observed in M..tuberculosis  and M.. \navium , but not in other species of mycobacteria.(iii)Two  oligonucleotide primers \nwithin IS 6110 insertion element, designated primer s Pt-8 (5´-GTG CGG AT GTC \nGCA GAG AT-3´) and Pt-9 (5´-CTC GAT GCC CTC ACG GTT  CA-3´), were used \nfor PCR, resulting in amplification of a 541-bp DNA  fragment (Kox et al . 1994)  \n[29]. This IS 6110  insertion element is almost specific for M. tuberc ulosis complex . \nThese newly developed multiplex PCR  using primers amplifying as described by \nPao et al  1990, Syun-Ichi et al  1993 and Kox et al  1994 [25, 28, 29], with the \nfollowing proposed composition of mixture and condi tion. The multiplex PCR was \ncarried out in a thermal cycler (GeneAmp 2400, Perk in-Elmer Cetus, USA) by \nincubating 2-5 µl of chromosomal dna (approximately 0.2 – 0.4 ng wi th the \nfollowing components (50 µl total vol): 10 mM Tris-HCl ( pH 8.3), 50 mM KCl, \n0.01% gelatin (w/v), 1.5 mM MgCl \n2, 0.2 mM of each of the four deoxynucleoside \ntriphosphates (dATP, dCTP, dGTP, dTTP), 0.2 µM, 0.1 µM and 0.2 µM of primers \nrespectively used in the PCR 1, 2 and 3 and 1 unit of Taq polymerase (Perkin-Elmer \nCetus, USA).  A positive amplified and negative con trol DNA s were run in each \nexperiment.  The reaction mixture was first pre-incubated at 85° C for 5 minutes. The \namplification was performed for 40 cycles of 94° C for 1 minute, 55° C for 1 minute \nand 72° C for 2 minutes. After the last cycle the s amples were incubated for 10 \nminutes at 72° C (final extension). \n \nAnalysis of PCR Products: Ten to fifteen µl of each PCR product was subjected to \nelectrophoresis with 2% and 2.5% agarose gel respec tively and the products were \nvisualized with Ethidium bromide.  The length of th e PCR product was estimated by \npGEM or φX174/ Hae  markers (Promega Corporation, Madison, Wisconsin, USA). \n \nResults  \n \nIt has been observed that among \n517 cases, positive PCR was \nfound in 230 cases (%). After \ntreatment with anti-tubercular \ndrugs, 86 cases conceived. The \nprevious history and clinical \nsituations enhancing infertility \naccording to age distribution of patients undergoin g PCR studies are mentioned \n(below) in Table 1. \n \n \n \nTable 1  \nAge <30 30-40 >40 Total \nCases 221 280 16 517 \nUnexplained infertility 101 143 2 246 \nMild Endometriosis 96 112 10 218 \nRecurrent Abortion 22 24 0 46 \nEctopic Pregnancy 2 1 4 7 \n\nAl Ameen J Med. Sci, Volume 3, No.4, 2010                                                         Gon Chowdhury R. et al  \n© 2010. Al Ameen Charitable Fund Trust, Bangalore  327  \n \n \nThe duration of infertility is also of \nnote, which is mentioned in Table 2: \nPositive cases were subjected to \ntreatment by 3 drugs (Rifampicin, Ethambutal & Ison iazid) for 3 months, followed \nby 2 drugs like Ethambutal & Isoniazid for 3 months . No subsequent PCR study was \nperformed as it is well known that, even dead bacte ria can give +ve TB PCR. The \nstudy was also not done in patient history of previ ous ATD intake.  The AFB smear \nwas positive in two cases. Bactec culture was posit ive in 15 cases but negative in 34 \npatients with PCR positive for tuberculosis. \n \nPregnancy Outcome: Pregnancy outcome is mentioned in following Table 3. Out of  \n230 PCR positive cases, 86 patients \nconceived after treatment with ATD. The \ncompletion of treatment to conception \ninterval was 3 to 12 months. Few patients \nconceived during treatment. Result: 33 \npregnancies were found in patients with \nPCR negative study.  \n \nThe pregnancy rate in post-treatment of +ve TB PCR cases were similar in \nunexplained infertilities and mild endometriosis, and best in case of RSA. \n \nAbortion rate was higher \nin women conceived \nduring or immediately \nafter treatment with ATD. \nTake-home-baby rate was \nmuch high in women \nconceived between six \nmonths to one year, after completion of the treatme nt. These are mentioned in Table \n4: \n \nDiscussion \nThe source of bacteria is mostly the environment. C olonization of MTB may be by \nsexual or asexual mode. Asexual mode may be by haem atogenous spread to \nendometrium, which ultimately remained dormant for long time, and most of bacteria \nwere shed out from others system. The bacteria after colonizing in endometrium may \nproliferate or may remain dormant. They may be eliminated as well. Until and unless \nthe bacteria enter the internal body surface, it ca nnot be called infection. Mere \npresence of bacteria on external body surface like skin, gut or endometrium, which is \nconnected easily to the external environment, shoul d be called infestation. The \nquestion then arises, whether infestation is pathol ogical. The majority of the invaded \nbacilli are arrested by the natural defense of the human body. Bacilli reaching the \nendometrium are arrested by the macrophages. Severa l factors like the number and \nvirulence of the infecting bacilli, host factors, i ncluding genetic susceptibility, age, \nimmuno-competence, stress, nutrition and co-existing illness influence the outcome  \nTable 2 \nDuration < 5years 5-10 years > 10 years \nNo. of cases 207 281 29 \nTable 3  \nPCR +ve Pregnancy \nTotal 230 86 (37.3%) \nUnexplained Infertility 114 44 (38.5%) \nMild Endometriosis 96 33 (34.4%) \nRSA 18 8 (44.4%) \nEctopic Pregnancy 2 1 \nTable 4  \n Cases Abortion Term  \nPregnancy \nConception by 9 months of  \ninitiation of the treatment                \n10 8 2 \n \nConception after 9 months  \nof initiation of the treatment         \n76 1 75 \n\nAl Ameen J Med. Sci, Volume 3, No.4, 2010                                                         Gon Chowdhury R. et al  \n© 2010. Al Ameen Charitable Fund Trust, Bangalore  328  \n \nof the infection. Tubercle bacilli do not contain o r secrete toxin. The exact basis of \ntheir virulence is not understood, but seems to be related to their ability to survive \nand multiply in macrophages. Various components of the bacillus have been shown  \nto possess different biological activities, which m ay influence the pathogenesis, \nallergy and immunity in the infection. Humans are e vidently able to mount an \neffective defense against the invaded bacteria, as only about a tenth of the infected \ndevelop active tuberculosis. The only specific immu ne mechanism effective is the \ncell mediated type. Humoral immunity appears to be irrelevant. The key cell is the \nactivated CD4+ helper T cell, which can develop along with two different paths – the \nTh1 or Th2 cells, releasing cytokines such as interferon γ (gamma) interleukins 1 and \n2, tumor necrosis factor α (TNF α) and others, exerting different biological effects . \nTh1 dependent cytokines activate macrophages, resulting in protective immunity and \ncontainment of the infection. Th2 cytokines induce delayed type hyper-sensitivity \n(DTH), tissue destruction and progressive disease. The essential pathology in \ntuberculosis is the production in infected tissues of a characteristic lesion, the \ntubercle. This is an avascular granuloma, composed of a central zone containing \ngiant cells, with or without caseation, and a perip heral zone of lymphocytes and \nfibroblasts. Tubercular lesions are primarily of two types – exudative and productive. \nThe exudative type is an acute inflammatory reactio n with accumulation of edema \nfluid, polymorphonuclear leucocytes, and later of l ymphocytes and mononuclear \ncells. This is typically seen when the bacilli are many and virulent and the host \nresponse is more, in the nature of DTH than of prot ective immunity. The productive \ntype of lesion is predominantly cellular, associate d more with protective immunity \nthan DTH. Endometrium is the normal site where embr yo implants, to give rise to a \nhealthy intrauterine pregnancy. Endometrial part of  implantation involves secretion \nof several molecules or markers generated by hormon al, biochemical, genetic and \nimmunomodulatory changes within the endometrium its elf. Immuno-modulatory \nchanges are brought about by cytokines and growth f actors. They have a wide range \nof family members. Cytokines consist of different m embers of interlukin family, \nwhile the growth factors consist of VEGF (Vascular Endothelial Growth Factor), \nTNF (Tumor Necrosis Factor), LIF (Leukemia Inhibiti ng Factor) etc. Some of these \ncytokines and growth factors help in implantation, while others antagonize the \nprocedure. If immuno-modulatory response is favorab le, helpful cytokines and \ngrowth factors will appear, and successful implanta tion will take place. \nImmunologically, this is known as T helper 2 (Th2) response. On the other hand, if \nharmful immuno-modulators develop, the response is said to be T helper 1 (Th1) \nresponse, resulting in failure of implantation and pregnancy will not occur. In \nsuccessful implantation, the Th1 sub-set of cytokines, namely TNF α, IL2 of the CD4 \ncell line are down-regulated, where as the cytokine s of the Th2 sub-set are up-\nregulated. Apart from T (Thymus) lymphocyte mediate d helpful or harmful \ncytokines and growth factors, similar type of harmf ul or helpful antibodies may also \ndevelop through immuno-modulation of B (Bone Marrow) lymphocytes. Helpful and \nharmful antibodies are known as asymmetric and symm etric antibodies respectively. \nPresence of MTB in the endometrium though paucibaci llary, which is otherwise \ncalled MTB infestation, probably excites an inflamm atory process, may be mild,  \n \n\nAl Ameen J Med. Sci, Volume 3, No.4, 2010                                                         Gon Chowdhury R. et al  \n© 2010. Al Ameen Charitable Fund Trust, Bangalore  329  \n \nwhich induces the production of adverse cytokines a nd antibodies, resulting in Th1 \nresponse. It is well known that for successful impl antation, Th2 bias is specifically \nneeded.  Inflammatory environment in the endometrium may inhibit down-regulation  \nof Th1 bias, so also up-regulation of Th2 bias. Thi s leads to pre-ponderence of \nharmful cytokines and antibodies of Th1 series in t he endometrium, making it non-\nreceptive to embryo, intending implantation. These cause, either so, are called \nunexplained infertility, recurrent implantation fai lure or recurrent abortion. \nMoreover, simple effort of Th1 response to mount up  to Th2 bias may activate the \ndormant bacilli, which activates inflammatory proce ss, further leading to more Th1 \nresponse, and thereby negative impact on implantati on. The improvement of \nendometrial environment after treatment with ATD is  evident by pregnancy rate in \npositive TB PCR cases (86 out of 230 cases - 37.4%) . The period taken into account \nfor pregnancy to occur in these cases was one and a half year, from the completion of \nthe treatment, which is otherwise two years, from the initiation of the ATD treatment. \nIt has been observed, that the patients who conceiv ed during or immediately after \nmedical treatment by anti-tubercular drugs mostly a bort. Take-home-baby rate was \nhigher in patients who conceived later. This indica tes that, probably it takes longer \nfor the inflammatory process to subside, even after  the bacilli are eliminated. In \nmany cases, probably some form of serious damage ma y happen, resulting in \npermanent non-receptive endometrium. Sub-endothelia l blood flow may also be \naffected by the presence of MTB in endometrium, as it may down-regulate VEGF \nconcentration. This may be other cause of implantat ion failure. Mild endometriosis \nmost often is not considered to be the cause of inf ertility. These patients are grouped \nunder unexplained infertility. In this study, mild to moderate endometriosis with \ngood tubo-ovarian relations suffering from infertil ity showed substantial TB PCR \npositive cases. This raises a suspicion, whether en dometriosis and tubercular \nbacillary invasion, either in the form of infestati on or infection are different spectra \nof the same disorder of immuno-competence. Samples from endometrial aspirates, \nendometrial biopsies and the fluid from the Pouch o f Douglas, from 25 women with \ninfertility, suspected to be suffering from genital  TB on laparoscopic findings were \nstudied for the presence of the mpt64 gene of M. tu berculosis by Bhanu et al. [30]. \nPresence of M. tuberculosis DNA was detected in 56% of cases by PCR as compared \nto 1.6% smear-positive and 3.2% culture-positive ca ses. PCR was positive in all \nwomen with laparoscopic findings suggestive of TB, in 60% of those with probable \ndiagnosis, in 33% of those with incidental findings and even in one case with normal \nlaparoscopic findings [30]. Other authors have also  observed PCR to be more \nsensitive than histopathology and culture [31, 32]. Rozati et al. [33] observed PCR to \nbe positive in 43.1% of suspected cases of female g enital tuberculosis, in contrast to \n5.2%, 7.8% and 11.5% detection rates with AFB stain ing, culture and \nhistopathology, respectively. Presence of at least 10,000 organisms/ ml in the sample \nis required for microscopic detection of AFB. Cultu re is more sensitive, requiring as \nlittle as 100 organisms/ ml, while PCR may be positive with only 1-10 organisms/ ml \n[30]. However, PCR has its own disadvantages. It can give false-negative results, due \nto contamination with heparin or to a high salt con centration of the specimen, which \nmay interfere with PCR results. As it cannot distin guish between live and dead  \n \n\nAl Ameen J Med. Sci, Volume 3, No.4, 2010                                                         Gon Chowdhury R. et al  \n© 2010. Al Ameen Charitable Fund Trust, Bangalore  330  \n \nbacilli, there is a small risk of false-positive re sult [30]. In our series, we get a \npositive PCR of 44.5% in this study, and contrast t o 30.06% in culture and 4.8% in \nAFB smear, though the number of culture and smear p erformed were small. This \nindicates that the detection of the presence of MTB  bacilli is much more with PCR \nstudy, with standard technique. \nConclusion \nThe influence of tubercle bacteria in fertility is well known, because tuberculosis of \nthe genital organs has long been accepted as an imp ortant cause of sterility in this \ncountry. With increasing health consciousness, impr oved immunization status and \neasy access to diagnose and treatment of tuberculos is have diminished the incidence \nof frank tuberculosis. Tubercular bacterial invasio n to the uterus, in the form of \ninfestation, not infection, has been strongly recog nized in the present study. It has \nalso been found that mere presence of bacilli in th e endometrial layer affects fertility \nstatus of women, as infertility or recurrent reprod uctive failure. Proper treatment \nreverses the situation to great extent. The immunol ogical changes happening during \nbacterial invasion may be associated with other imm uno-compromised disorders like \nendometriosis. This requires a further structured s tudy, to come to a definite \nconclusion about the disease. \n \nReferences \n1. Punnonen R., Kiilholma P., Meurman L. Female genita l tuberculosis and consequent \nInfertility. Int J fertile  1983; 28:235-238  \n2. Nogales-Ortiz F.,Ildefonso T.,Nogales FF: The patho logy of genital tuberculosis. Obstet \nGynecol 1979; 53: 422-428  \n3. 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Development of a  new sensitive and efficient \nmultiplex polymerase chain reaction (PCR) for ident ification and differentiation of \ndifferent mycobacterial species; Tropical Medicine and International Health  2003; 8 (2): \n150-157  \n28. Pao CC, Yen TS, Jinn-Bang Y et al. Detection and id entification of Mycobacterium  \ntuberculosis by DNA amplification; J Clin Microbiol 1988; 28: 1877–1880  \n29. Kox LLF, Rhienthong D, Medo Miranda N et al. A more  reliable PCR for detection of \nMycobacterium tuberculosis in clinical samples; J Clin Microbiol 1994; 32: 672–678  \n30. Bhanu NV, Singh UB, Chakraborty M et al. Improved d iagnostic value of PCR in \ndiagnosis of female genital tuberculosis leading to  infertility. J Med Microbiol ; 2005; \n54:927-931  \n31. Jindal UN. An algorithmic approach to female genita l tuberculosis causing infertility; Int \nJ Tuberc Lung Dis  2006; 10: 1045-1050  \n32. Abebe M, Lakew M, Kidane D, Lakew Z, Kiros K, Harbo e M. Female genital \ntuberculosis in Ethiopia. Int J Gynecol Obstet  2004; 84: 241-246  \n33. Rozati R, Sreenivasagari R, Rajeshwari CN. Evaluati on of women with infertility and \ngenital tuberculosis; J Obstet Gynecol India; 2006,  56: 423-426 \n \n*All correspondences to: Dr. Siddhartha Chatterjee,  Calcutta Fertility Mission, Pushpanjali Apartment, \n102C Ballygunge Place, Kolkata-700019 Phone No: 913 3 2281 2813; e-mail: sidchat54@gmail.com , \nsid2512@vsnl.net","source_license":"CC0","license_restricted":false}