Abstract
Female genital tuberculosis (GTB) contributes significantly to infertility in low- and middle-income countries. Dissemination of infection from pulmonary and extrapulmonary sites is the major reason for causation of GTB. Additionally, sexual transmission of GTB from male partners has been reported. We selected 81 couples desiring babies from an in vitro fertilization clinic. We used multiplex-PCR for mycobacterial detection in semen of males, in the endometrium of their female counterparts and in the products of conception (POC) from miscarriage. Data interpretation shows that these pregnancies failed owing to sexual transmission of mycobacteria. We noticed by multiplex PCR that mycobacterial infestation in the female can take place in either endometrium or POC from asymptomatic males harbouring mycobacteria in their semen. Therefore, we propose sexual transfer of mycobacteria to be a probable cause of miscarriage. Thus, we suggest multiplex PCR based screening of semen for all males of the couples attempting successful childbirth.
Introduction
GTB is a major concern in low- and middle-income countries; it accounts for 15–20% of all extra-pulmonary TB in India.1–4
Previously, sexual transmission of Mycobacterium tuberculosis from male to female was thought to be uncommon; however recent evidence based on molecular typing and whole genome sequencing from females developing GTB within a few years of their husbands’ diagnosis of GTB indicates sexual transfer of TB.5,6
We demonstrate that both TB and non-tuberculous mycobacteria (NTM) can cause miscarriage when sexually transferred to female partners, who often display no symptoms, and are defined as having latent genital tuberculosis (LGTB). 7 We analyzed PCR data of samples gathered from LGTB females and their respective male counterparts, 8 which indicate sexual transmission of mycobacterium from asymptomatic males.
Methods
165 infertile couples were referred to the Calcutta Fertility Mission from January 2019 to January 2020. Those 81 with ‘unexplained’ infertility were selected for our retrospective study, based on inclusion and exclusion criteria explained in supplementary methods (Fig. S1). All participants had undergone routine tests and widely used multiplex diagnostic PCR for mycobacterial DNA detection. 8 Details on division of patients into groups (A, B, C) based on multiplex PCR data, statistical analyses, and DNA fragmentation index (DFI) technique are included in the Supplemental Material and Fig. S1.
Result
In Group-A, the male partners were asymptomatic, but their semen were GTB + ve. In this group, GTB was not present in the patient's female partner's endometrium, but after abortion, POCs obtained were GTB positive (Tables 1, 2). This group could easily be distinguished from Group B, in which presence of GTB was diagnosed in female partners’ endometrium. In both groups A and B, male partners were asymptomatic carriers of GTB. Both male and female partners of 21 couples in group C were GTB negative and were recruited as controls. Further examination showed that GTB positivity came from three combinations of two sets of amplification products,8–10 one pattern consisting of 165bp and 541bp (Mtb Type-I) (Fig. S2A&B). In contrast, another set of TB strains only yielded 541bp (group Mtb Type-II (Fig. S2A), whereas the NTM organisms with the same set of multiplex primers yielded only a single band of 165bp (Fig. S2A). We found that the Mtb Type-II strain is 100% (21 + 10 out of 21 + 10) transferred from semen to the POC or endometrium (Table 2 and Fig, S3A, B, right bars). However, the transfer of Mtb Type-I strain is only 56% (2 + 3 out of 4 + 5) transferred (Table 2 and Fig, S3A, B, middle bars). Transfer of NTM was also found to be 100% (Table 2 and Fig, S3A, B, left bars).
Distribution of group A category couple with their PCR amplification length.
Significance of symbols-
Distribution of amplification length of GTB under category A, B and C. In case of NTM, we find that 15 males transferred it to 17 females in Group-A and 5 males transferred it to 7 females in Group-B. This apparent anomaly could be explained as, in case of Type-I infested male-partners, there is no way to rule out NTM co-infection by PCR. Precisely, all 15 + 5 of the semen NTM samples were transferred to POC or endometrium. However, we observed the presence of four extra NTM samples in POC or endometrium, which can be explained by speculating that four of the Mtb Type-I samples were mixed with NTM organisms (which were not apparent because of the overlapping presence of 165bp band of NTM). ND: Not Detected. NA: Not Applicable.
Significance of Abbreviations: MPCR1: Multiplex PCR showing 165 bp band (NTM); MPCR2: Multiplex PCR showing 165 and 541 bp bands (Mtb Type I); MPCR3: Multiplex PCR showing 541 bp band (Mtb Type II); M: Male; F: Female. ± SD values are given for age.
Successful PCR amplification can be interpreted despite sperm DNA fragmentation and our PCR assay works reliably irrespective of sperm DNA quality (Fig. S2, DFI analysis in Fig. S4 and Table S1).
Discussion
Recent case-studies have indicated sexual transmission of GTB.5,6 However, these individual cases were too few to argue in favour of female GTB causation by sexual transmission. In those studies, sexual transmission was reported from symptomatic male GTB patients.5,6 In contrast, our study confirms sexual transfer of mycobacteria from many asymptomatic males carrying organisms in semen to produce LGTB in their female partners (n = 60). The transfer to the female partners may result in colonization of either endometrium or POC. Therefore, sexual transfer of mycobacteria (Mtb and NTM) is not uncommon between asymptomatic individuals and seems to be a major cause of miscarriage in our patient cohort.
Following this investigation, we are planning to sequence 16s rDNA loci of mycobacteria present in our samples. These data will give us initial clues on various species of mycobacteria which might be associated with infertility. Following this investigation, by comparison, we should be able to understand the molecular genetic factors augmenting sexual transfer of mycobacteria. A comparison of different types of strains would indicate the genetic loci promoting sexual transfer and subsequent establishment into female genital niches. Thus, this type of follow-up studies may lead to better understanding of the mechanism of the pathogenesis of female GTB.
We recommend that similar studies should be carried out from all strata of Indian society. These studies might give us better epidemiological trends of asymptomatic sexual transfer of GTB.
Even if this trend of LGTB establishment via sexual transmission is not observed universally, the male partners of infertile women should be screened for mycobacterial infestation by semen PCR, before starting IVF protocols.
Supplemental Material
sj-docx-1-tdo-10.1177_00494755221074754 - Supplemental material for Evidence of sexual transfer of mycobacteria from male to female partners reporting to an IVF clinic
Supplemental material, sj-docx-1-tdo-10.1177_00494755221074754 for Evidence of sexual transfer of mycobacteria from male to female partners reporting to an IVF clinic by Abira Datta, Prabuddha Gupta, Basudev Bhattacharya, Arabinda Das, Siddhartha Chatterjee and Asesh Banerjee in Tropical Doctor
Footnotes
Acknowledgements
We sincerely appreciate Dr William R. Jacobs's many invaluable contributions, including the ideas of attacking the problem of sexual transmission of Mtb. We thank him for several useful suggestions and editorial comments on the manuscript as well. We especially acknowledge many invaluable contributions of our esteemed collaborator Late Dr Rajib Gon Chowdhury. We also thank Mr Soumyadeep Ghosh and Dr Shiladitya Ghosh of Amity University Kolkata for his help with manuscript preparation. We are thankful to Ms. Sumana Mukherjee and Dr Anjana Roychowdhury for excellent help with embryological work. Mr Ramyajit Mitra is acknowledged for the final editing of the paper.
Ethical approval statement
The study was approved by Ethics Committee at CFM (Code 2020/5, dated 25th June 2020)
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship and/or publication of this article.
Supplemental material
Supplemental material for this article is available online.
References
Supplementary Material
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