A Ten year Clinicopathological Study of Female Genital Tuberculosis and Impact on Fertility

Methods: A total number of 68 cases of gynaecological tuberculosis affecting different parts of female genital tract from 56 patients were selected. The age range of the patients was 17-36 years with mean age of 25.6 years. The diagnostic procedures used included endometrial curettage and biopsy, histopathological examination, culture and Mycobacterium Tuberculosis Polymerase Chain Reaction (MTB PCR), laparoscopy, Hysterosalpingography (HSG) and Ultrasonography (USG). Most of the specimens received were biopsies of endometrial curettage for evaluation of infertility. In 7 cases, specimens of total hysterectomy with bilateral salpingo-oophorectomy were submitted with lesions involving multiple sites.


IntRODUctIOn
Genital tuberculosis (TB) predominantly affects individuals below 40 years of age and peak age frequency ranges between 21 to 30 year of age. 1 The disease is responsible for 5% of all female pelvic infections and occurs in 10% cases of pulmonary tuberculosis.Gupta et al, studied 40 infertile women with genital tuberculosis and found pulmonary tuberculosis in 9 of them and positive Mantoux test results in two patients. 2Tuberculosis of female genital tract although uncommon in western world, is still prevalent in developing countries like India, Pakistan. 3Most cases were detected in infertile women in the reproductive age group and sterility was the predominant motive for consultation. 4,5nital tuberculosis of women occurs secondary to primary disease in the lungs, lymph nodes, urinary tract, bones, joints, and bowel and spreads to genital organs.The spread is usually by haematogenous or lymphatic route.Sexual transmission of the disease is also documented but direct spread from other intraperitoneal foci is very rare.

MetHODs
A prospective study was carried out on female genital tuberculosis in tertiary hospital based study done in Medical College and Hospital, Kolkata, India from July, 1998 to June, 2008.Our institutional approval and patients' consent was taken.Females who were suffering from genital tuberculosis were included in this study.Detailed clinical information, HSG/USG findings and relevant investigations were recorded from the case sheets.These included age of the patients, signs and symptoms, routine haematological check up, chest x-ray, Mantoux/Purified Protein Derivative (PPD) skin tests etc.The diagnostic procedures used to detect the lesions included histopathological examination of tissue biopsies, culture and MTB PCR detection of bacteria in tissue and fluid (peritoneal and menstrual).HSG was done in 38 cases and USG in 27 cases.Most of the specimens (43 cases) received were biopsies of endometrial curettage for evaluation of infertility.Patients presented with either tubal involvement (10 cases) or with tubo-ovarian mass (six cases) and subsequently underwent surgical removal.In 7 cases, specimens of total hysterectomy with bilateral salpingoophorectomy were submitted with lesions involving multiple sites.Tissue sections were processed and paraffin blocks were made.Slides were stained by Haematoxylin andEosin (HandE) stain, and Ziehl-Neelsen (ZN) stain routinely.Periodic Acid Schiff (PAS) and other special stains like Gomori's methenamine silver stain were done when necessary to exclude fungal etiology (five cases).
Exclusion criteria were females with proven diagnosis of other causes of infertility.Patients, who had granulomas on biopsy but later on proved to be of nontuberculous origin, were also excluded from our study.Patients who showed negative results for detection of Mycobacterium tuberculosis either in tissue sections (ZN stain) or in culture or in molecular methods (PCR) were excluded from the present study.
For extraction of DNA, decontaminated sediment (200μl) was incubated in a lysis buffer containing 20 mM Tris/HCl (pH 8.3.DNA was purified in phenol and DNA extract (10 μl) was mixed with primers (0.4%μM) and other reagents.After that amplification was carried out for 30 cycles; each consisting of denaturation at 94 ○ C for 2 minutes,annealing at 60 ○ C for 2 minutes and extension at 72 ○ C for 2 minutes.Two pairs of oliginucleotide primers were used for molecular diagnosis.A 123 bp fragment corresponding to a specific mycobacterium tuberculosis complex sequence which was the insertion sequence 6110(IS 6110) and a 383 bp DNA fragment encoding part of the 65 kD mycobacterial surface antigen were amplified by PCR.
Data was compiled and statistical analysis was done using Microsoft Excel.Mean, Median and other relevant statistical data were obtained.
Tuberculous inflammation was seen in endometrium in 38 cases (55.88%).The cervix was the least common site (four cases, 5.88%).Caseation was present in nine of 68 cases (13.23%) and acid-fast bacilli were found in tissue sections in four cases (5.88%) (Table 1).No case of TB vulva and vagina was seen in this study.
The principal histological findings in tuberculous endometritis (38 cases) were presence of the epithelioid cell granulomas.Granulomas were small, isolated lesions scattered throughout the endometrium in most of the cases (31/38).Aggregates of epithelioid histiocytes and with a peripheral collar of lymphocytes were found scattered in the functionalis layer of endometrium (Figure 1).Caseation was found very rarely (one case) in the endometrium and the patient was post-menopausal.Multinucleated giant cells of both Langhans and foreign body type were present in seven cases with disruption of endometrial glands in Grossly, fallopian tubes in tuberculous salpingitis (16 cases) appeared enlarged and slightly oedematous with irregular external surfaces due to adhesions.Fallopian tubes were involved bilaterally in 13 cases and fimbrial ends were closed in six cases.In three cases, the fimbria were everted with a patent orifice, imparting the characteristic "tobacco pouch" appearance.Cut sections revealed serosanguineous fluid (five cases) or blood (one case) or caseous material (three cases) with diffuse or focal mucosal ulceration.The lumen contained clear fluid in four cases (hydrosalpinx) and pus (pyosalpinx) in three cases.The histology of tuberculous salpingitis was not always specific.In the early stage (five cases), the features were of chronic salpingitis with occasional non-caseating granulomas.In this stage, plical adhesions gave the appearance of follicular salpingitis.In advanced stages (11 cases), single or multiple confluents epithelioid granulomas were present in the lamina propria (Figure 2).The tubercles and chronic inflammation involved the muscularis (two cases) and serosa (one case) infrequently (Figure 3).Caseating granulomas were present in five out of 16 cases of tuberculous salpingitis and two cases showed AFB in tissue sections.
Ovarian tuberculosis (10 cases) was usually a sequel of a tubal lesion and presented as a tubo-ovarian mass (Figure 4).Histological examination at the stage of peri-oophoritis (2/10) showed fibrinous exudates on the surface but with extension to the ovarian parenchyma, a multilocular thick-walled abscess developed.This usually involved both the ovary and tube and microscopy revealed necrosis, granulation tissue formation and neutrophilic infiltration.In the long standing cases (8/10), fibrotic adhesions between ovary and surrounding structures, especially fallopian tube occurred; which might play a role in the sterility in these patients.The granulomas were present in the cortical surface (7/10) though diffuse involvement was also seen (3/10).Microscopically, caseation was present in three out of 10 cases of ovarian tuberculosis and one case revealed AFB on ZN stain (Figure 5).Cervical tuberculosis (four cases) was almost always secondary to involvement elsewhere in female genital tract.Macroscopically, the cervix was red, enlarged, friable and ulcerated which led to misdiagnoses of cervical cancer clinically in two cases.The mucosa of the endocervical canal was commonest site (three cases) of involvement.The granulomas consisted of epithelioid cells and lymphocytes as in other sites of genital tuberculosis but caseation was absent.
Epithelioid granulomas suggestive of tuberculosis were found microscopically in 73 cases, of which 68 cases were proven for mycobacterium tuberculosis either by detection of AFB in tissue sections, or by mycobacterial culture on Lowenstein Jensen medium/ BACTEC culture or by MTB PCR.In four cases, diagnoses were confirmed by presence of AFB in tissue sections on ZN stain and in 37 cases by mycobacterial culture (BACTEC /LJ) of tissue or menstrual/ peritoneal fluid.In the remaining 27 cases, the bacteria did not grow in culture and was detected by identification of particular DNA sequence by MTB PCR on agarose gel electrophoresis (Figure 6).The other five cases were excluded from this study due to lack of definite diagnostic evidence.Women with genital tuberculosis were treated with long-term combined antituberculous drug therapy.Surgical treatment was employed for macro-lesional forms which were resistant to medical treatment .Conservative surgery was attempted in nine patients and radical surgery in seven patients.Tubal reconstructive surgery was performed in two patients.After therapy; nine women conceived (seven tubal pregnancies and only two uterine pregnancies), some with multiple pregnancies.Out of the nine pregnancies eight suffered spontaneous abortion.Only one patient had a successful pregnancy who delivered a baby through Caesarean section.

DIscUssIOn
Tuberculosis remains a major global health problem and most of these are pulmonary TB.
Actual frequency of female genital tuberculosis is unknown despite different published data from various countries as it is often discovered incidentally or remain 'undetected' in symptomless patients. 6More than 90% of patients of female genital TB were under 40 years of age which indicated the hormone dependent nature of the disease and corroborated other studies. 7,8The frequency of genital tuberculosis was 0.002% of all patients admitted for gynaecologic diseases. 9n postmenopausal women genital tuberculosis is rare and seen in 1% of patients with postmenopausal bleeding. 10he exact cause of low incidence of the disease in this age group is not known.Most authors believe that an atrophic endometrium is a poor milieu for the growth of mycobacterium tuberculosis.Since 1950, however, the peak incidence has shifted to the perimenopause as infertility has been superseded by abnormal uterine bleeding and pelvic pain as dominant complaint in the developed countries. 9,11Genital tuberculosis is usually an indolent infection and takes years to manifest clinically after initial seeding.
Genital TB may be suspected from the clinical presentation, radiological findings (USG, HSG etc) and

Mondal et al. A Ten year Clinicopathological Study of Female Genital Tuberculosis and Impact on Fertility
other investigations like positive Mantoux test, high sedimentation rate, tuberculous foci on chest x-ray film.On pelvic examination of patients with genital TB, Saracoglu and collegues found that no abnormaliy in 43% of cases, adnexal mass (23.6%), myoma-like lesion (23.6%), adnexal tenderness (4.2%), irregular uterus (1.4%) and cervical polyp (1.4%).
In tuberculous endometritis, caseation is rare in women of reproductive age group. 12In our study; caseation was found in 1 case only, when the patient had amenorrhoea due to menopause.
In that case the granulomas remained within the endometrial tissue long enough for caseation to develop, as there was no periodical loss of endometrium of menstruation.The disease process has to regenerate after menstrual shedding from the basal layer of endometrium with start of each menstrual cycle and the granulomas become well developed and numerous as the cycle progresses.So, biopsy is recommended immediately before the menstrual phase as the granulomas get longest possible time to develop and greater chance of providing accurate diagnosis.In our study, the endometrial glands were usually unaffected but showed poor response to ovarian hormones which might have contributed to the infertility.Nogales-Ortiz et al found abundant lymphoid follicles without granuloma in the endometrium in patients who have had both fallopian tubes involved. 13When TB affects the female genital tract, the fallopian tube is involved in nearly all patients; involvement of the endometrium is usually secondary to tubal disease.But in this study, number of endometrial TB (38 case) is more than fallopian tube TB (16 cases).This might be due to sample biasness, as most of the specimens we received were endometrial biopsies for evaluation of infertility.
The tubal mucosa is most favorable nidus to blood borne tubercle bacilli and endosalpingitis, usually bilateral, is the earliest lesion with transluminal spread to ovary and uterus.The gross pathology of tuberculous endosalpingitis is variable and bilateral involvement is common, owing to typical tendency of the tubercle bacillus to attack each one of bilateral organs in succession. [13]In tubal pregnancy, due to insufficient tissue and blood supply adequate placentation is not possible; most of the pregnancies abort at early stage.Moreover,due to limited distensibility and trophoblastic invasion in the wall of the tube, rupture is very common (>50%), resulting in unsuccessful pregnancies.The most common granulomatous infection of the ovary is tuberculosis and usually secondary to tubal disease. 14pread of organisms from the tube might have affected the surface only and produce either an acute or a chronic peri-oophoritis.Often the ovarian substance was involved subsequently.The ovaries are typically involved as tubo-ovarian mass, which is frequently adherent to the omentum and intestine. 15tensive ulceration occurs in severe cases of cervical tuberculosis and reactive atypical hyperplasia of the cryptal epithelium with florid papilla formation develops; the features can resemble carcinoma macroscopically and microscopically. 16 Z et al used three pairs of oligonucleotide primers to differentiate between mycobacterium tuberculosis and nontuberculous mycobacteria by triplex-PCR from formalin fixed, paraffin embedded tissue. 17 Mondal et al.A Ten year Clinicopathological Study of Female Genital Tuberculosis and Impact on Fertility two cases.Plasma cells were present in three cases of tuberculous endometritis and indicated secondary infection.Granulomas in different stages were found in eight cases of tuberculous endometritis.

figure 4 .figure 5 .
figure 4. Gross photograph showing tuberculous inflammation which presented as Tubo-Ovarian mass.Multiple small nodular lesions were found in fallopian tube and ovary on cut section.

PCR was table 1. tuberculosis of different anatomical sites and percentage (n=68) site number of cases Percentage (%) caseation present Afb present in tissue section (Zn stain)
Photomicrograph showing numerous noncaseating epithelioid cell granulomas and Langhans giant cells in functionalis layer of endometrium [HandE X100].Inset shows high power view of a typical granuloma in endometrium [HandE x 400].