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Favorable infertility outcomes following anti-tubercular treatment prescribed on the sole basis of a positive polymerase chain reaction test for endometrial tuberculosis

Introduction

Nearly one-third of the world population is infected with Mycobacterium tuberculosis (MTB) of whom only 10% are known to progress to clinical disease (WHO, 2006). Depending upon the localization of the MTB in an organ, a wide spectrum of tubercular disease is encountered in clinical practice of which female genital TB (GTB) is an important manifestation (Schaefer, 1976; Parikh et al., 1997; Aliyu et al., 2004; Dannenberg and Converse, 2011). Damage to the pelvic organs after clinical GTB is well recognized both in the presence of active disease as well as during the process of healing and fibrosis. The diagnosis can be established in such cases with the help of various microbiologic, radiologic and histopathologic tests along with the clinical presentation. The diagnostic tests have high specificity but a low sensitivity even in the presence of active tuberculosis (TB). A battery of tests may be required to arrive at the diagnosis (Tripathy and Tripathy, 1990; Jindal, 2006; Rozati et al., 2006). The fibrosis and scarring which result as a part of healing lead to the loss of function of the Fallopian tubes, and less commonly of ovaries and endometrium. It is therefore desirable to diagnose and treat GTB as early as possible during the subclinical stage to prevent or at least to minimize the damage to the genital organs. Unfortunately, the conventional tests for the diagnosis of TB during the sub-clinical stages have poor sensitivity and specificity. However, recently the detection of MTB DNA by TB-PCR has shown high sensitivity and specificity for the diagnosis of GTB (Baum et al., 2001; Roy et al., 2003; Bhanu et al., 2005; Rana et al., 2011). We have previously shown that the maximum likelihood estimates of sensitivity and specificity for the diagnosis of GTB with a positive TB-PCR in the endometrial samples were 0.59 and 0.92, respectively (Jindal et al., 2010). In a recent study, the authors have shown that 57% of infertile women in whom the presence of TB was suspected on clinical grounds had a positive endo-TB-PCR test, whereas only 9.5% had a positive test where no clinical ground for suspicion were present (Thangappah et al., 2011). Endometrial TB-PCR (endo-TB-PCR) positivity in the absence of symptoms, and without any demonstrable tubal or endometrial damage, raises the possibility of a false-positive TB-PCR test in the absence of any mycobacterial infection. Empirical treatment, especially in the high-prevalence countries is also fraught with the risks of resistance and other side effects of anti-tubercular chemotherapy. On the other hand, a positive endo-TB-PCR test may also imply the presence of sub-clinical, latent or past disease, which could be managed with anti-tubercular treatment (ATT). This is supported by the limited observations made for both genital and other forms of TB in some recent studies (Cheng et al., 2004; Kulshrestha et al., 2011; Thangappah et al., 2011). Short course chemotherapy is effectively used to treat symptomatic GTB (Jindal et al., 1990). However, for infertile women with GTB, assisted reproduction techniques (ARTs) are also required to achieve pregnancy (Soussis et al., 1998; Jindal, 2006; Singh et al., 2008). The present study was undertaken to examine the fertility of infertile women with positive endo-TB-PCR in the absence of demonstrable damage to the endometrium or the Fallopian tubes after the early institution of ATT.

Materials and Methods

The study was undertaken from the year 2006 to 2010 at an IVF center in northern India. Women from all couples seeking treatment for infertility between 2006 and 2008 were screened for inclusion in the study. All couples were investigated and managed according to the standard protocol followed at the center. Tubal and endometrial evaluation was done either by hysterosalpingography (HSG) or laparoscopy and hysteroscopy. Endometrial samples were obtained by endometrial aspiration or curettage, done as a stand-alone test or along with laparoscopy and/or hysteroscopy. One part of the biopsy of endometrial tissue was subjected to histopathologic examination and the second part was sent to the Laboratory for TB-PCR testing. Endometrial samples were obtained by gentle curettage of the endometrium and kept in sterile containers with normal saline to avoid contamination. Histopathologic examination of endometrial biopsies did not reveal any presence of acid fast bacilli, granuloma formation or other findings suggestive of TB.

PCR testing

Endo-PCR test was arranged with Reliance Life Sciences Pvt. Ltd. Mumbai, a national laboratory providing services all over India, accredited by the American College of Pathologists, with a proficiency test score for TB of 100% in 2007 and 2008. Stringent criteria were used at the laboratory to avoid contamination of results. The test was run in duplicate. Nested PCR against the most conserved region insertion sequence 6110 gene was done employing a Fastprepw sample preparation system (BioMedicals, Cambridge, UK) for mycolic acid cell wall lysis to extract DNA. The PCR assay gave a clear band of 123 base pairs (bp), indicating positivity of a sample. The assay used a cellular gene to rule out false negativity of the samples, with the cellular gene confirming no general DNA degradation in the sample and absence of PCR inhibitors in the sample. One positive and one negative clinical samples were used in every assay to validate the assay and confirm the results. A blank reagent containing no DNA was used to check contamination during PCR by the absence of any PCR fragment in the gel. The assay was also validated by direct sequencing of the PCR product indicating .95% homology with MTB, using NBLAST (www .ncbi.nlm.nih.gov/ blast). The quoted sensitivity of the test is almost 100% and specificity 96 – 99%, with a lower detection limit of 100 TB bacilli/ml (Nolte et al., 1993; Folgueira et al., 1996; Takahashi and Nakayama, 2006)

Patients

Of 3108 infertile couples who reported between 2006 and 2008, the study included 443 (14.2%) women of, 40 years of age who had no symptoms other than infertility, and without any evidence of endometrial or tubal damage on HSG or laparoscopy and hysteroscopy. The reasons for exclusion of the remaining 2665 (85.8%) cases were TB-PCR not done (1244); presence of a tubal factor (552); tubal evaluation not done (248); severe male factor (132); severe and moderate endometriosis (101); endometrial factor (76); previous history of ATT (168); age .40 or previous oophorectomy (91) and those who did not report for followup evaluation (53). Among 443 women who were included, 169 (38.15%) with PCR positive test constituted the study group (Group I) and 274 (61.85%) PCR negative, the control group (Group II). Follow-up assessment was continued for at least 2 years after recruitment in the study up to end of 2010.

Treatment

The study Group I, received standard short course daily ATT consisting of the intensive phase of 2 months of four drugs (isoniazid, H 300 mg; rifampicin, R 450 – 600 mg; ethambutol, E 800 – 1200 mg and pyrazinamide, Z 1200 – 1500 mg) followed by the maintenance phase of 4 months comprising the same doses of isoniazid and rifampicin (2HRZE, 4HR). The first-line treatment for non-tubal infertility consisted of ovulation induction along with IUIin PCR negative Group II and also in PCR positive Group I, after completion of ATT. IVF was undertaken for refractory infertility for 32 of Group I and 45 of Group II women; the remaining 150 women did not opt for IVF and wished to wait longer for various personal reasons. In brief, the IVF procedure consisted of employing stimulation protocols using GnRH analogs and urinary or recombinant gonadotrophins. Final maturation trigger was given when at least three lead follicles were .16 mm. Ovum pick up was done 36 h after hCG. All metaphase-2 oocytes were injected with sperm by intracytoplasmic sperm injection. Embryo transfer was done on second/third day and a maximum of three embryos were transferred. Pregnancy was defined as the presence of viable gestational sac on ultrasound examination at 3 – 4 weeks after embryo transfer.

Statistical methods

Details of all cases were recorded on a structured format and analyzed with the help of registered version of SPSS version 13. Group comparisons were made using x2 test (for categorical variables) or Student t-test (for scalar variables). Statistical significance was assessed at P , 0.05. Probability of spontaneous pregnancy (without any IUI or IVF) during the follow-up period was calculated by Kaplan –Meier method, and formal comparisons between different groups were performed using the log-rank test.



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