The Clinical Features and Prognosis Analysis of Pregnant Women with Tuberculosis After in Vitro Fertilization and Embryo Transfer and Natural Fertilization

Objective: To investigate the clinical characteristics and prognosis of women with tuberculosis after in vitro fertilization and embryo transfer (IVF-ET) and natural pregnancy. Design/Methods: Ninety-ve pregnant women with tuberculosis admitted to Shanghai Public Health Clinical Center between February 2013 and July 2020 were retrospectively analyzed. They were divided into 24 cases of IVF-ET with tuberculosis and 71 cases of natural pregnancy group with tuberculosis. Baseline demographic, medical history were collected. We used descriptive statistics to describe demographic, clinical features and prognosis of pregnant women with tuberculosis using Pearson chi-squared, Fisher’s exact tests, or Kruskal-Wallis. Results: The incidence of fever, hematogenous disseminated pulmonary, drug-induced liver injury and fetal adverse outcomes in IVF-ET group were higher than those in natural pregnancy group (p<0.05). The lymphocyte count, hemoglobin level, albumin level and the number of CD4 + T lymphocyte (cid:0) CD8 + T lymphocyte in IVF-ET group were signicantly lower than that in natural pregnancy group (p<0.05). Conclusions: Pregnant women with tuberculosis after in vitro fertilization and embryo transfer have broader lung lesions, more prominent symptoms of systemic poisoning, larger proportion of severe tuberculosis, more prone to anti-tuberculosis drugs induced liver injury and higher proportion of spontaneous abortion and inevitable abortion, which calls for enough attentions.


Background
Tuberculosis remains a global emergency and continues to poses a considerable threat to human health.
In 2019, an estimated 10 million people fell ill with TB, women accounted for 32%; moreover, it was responsible for an estimated 1.2 million deaths globally 1 . Tuberculosis is one of the top causes of death in women of reproductive age and is a common non-obstetric cause of maternal mortality 2, 3 . It has been estimated that, in 2011, 216 500 active tuberculosis cases existed in pregnant women globally and 9 500 active TB cases during pregnancy in China, which is equivalent to 0.7 per 1 000 pregnant women, accounting for 4.4% of global burden among pregnant women 4 . Indeed, Active TB in pregnancy is associated with adverse maternal and fetal outcome 5 .
In contrast to normal pregnancy, more infertility patients prefer to get pregnant by in vitro fertilization and embryo transfer (IVF-ET) .In some patients, the use of progesterone and glucocorticoids is needed to resist IVF-ET rejection, improve the intrauterine environment and guarantee normal embryonic growth, which may in uence cell-mediated immunity and cause TB relapse and dissemination. However, there are only a few reported cased of tuberculosis in IVF pregnancy 6-10 , and these literatures do not describe the difference in clinical characteristics between natural pregnancy and IVF pregnant women with tuberculosis.
In this study, we summarized the clinical characteristics and prognosis of pregnant Chinese women with TB. The purpose of this study is to raise awareness of TB diagnosis during pregnancy and improve the prognosis of tuberculosis in pregnant women.

Materials And Methods
Study design.
Pregnant patients with pulmonary tuberculosis, who were hospitalized in Shanghai Public Health Clinical Center from February 2013 to July 2020, were enrolled. The inclusion criteria were as follows: 1) Cases data were complete. 2) With conform to the diagnostic criteria for tuberculosis in pregnancy. Patient was diagnosed with TB during pregnancy and postpartum. To ensure that TB cases were not omitted during pregnancy, patients diagnosed with TB from pregnancy until 42 days after delivery were included. Women who suffered from TB before pregnancy and conceived during the period of anti-tubercular therapy (ATT ) were excluded from this study.
Information such as age, TB history, history of BCG vaccination, contact history of TB, Education level, clinical symptom, laboratory-test results, radiographic features, therapeutic regimens, and the outcomes of pregnant women and neonates were reviewed in the medical records. Physician contacted the patients via a follow-up phone call. This study was approved by the Ethics Committee of Shanghai Public Health Clinical Center [batch number:2020-S112-03]. And this study was approved by the Ethics Committee of Shanghai Public Health Clinical Center to exempt subjects from informed consent.
Diagnostic criteria and classi cation of TB.
Active TB was diagnosed based on the 2013 WHO guideline [11] . for example: A patient was diagnosed as having de nite TB based on the identi cation of mycobacterium tuberculosis complex in the clinical sample (sputum, body tissue, or body uid), either by culture or molecular method. In the absence of bacteriological con rmation of TB disease, a clinical diagnosis was based on clinical evaluation by a medical doctor, radiography and the clinician prescribed a full course of anti-tubercular therapy (ATT).
Contacts were de ned as individuals who being in the same room as the index patients for more than 6h per week in the 2 weeks preceding the index patient's diagnosis 12 .
In accordance with the WHO de nitions for TB, Pulmonary Tuberculosis (PTB) was de ned as TB involving lung parenchyma, which included military TB 13 ; Extra-pulmonary Tuberculosis (EPTB) referred to presence of mycobacterium tuberculosis in organs, except the lung 13 . Both PTB and EPTB was de ned as PTB not only involving lung parenchyma but also including other organs 13 .
The medical records were analyzed, with descriptive analysis conducted for clinical data. Statistical analysis data were analyzed using the Statistical Package for Social Sciences (SPSS) software (version 23.0). Data were presents as mean ± SD for continuous variables with normal distribution or median (IQR) for continuous variables with a skewed distribution. The ANOVA or Kruskal-Wallis H test was used to compare the difference among groups. Categorical variables were expressed as n(%) and Chi-square test or Fisher's exact test was used to compare the difference. All P-values were calculated with statistical signi cance set to P < 0.05.

Results
Baseline characteristics and onset time of pregnant women with tuberculosis on admission The total number of pregnant women with tuberculosis in Shanghai Public Health Clinical Center between February 01, 2013 and July 31, 2020 was 134. After applying the exclusion criteria, the nal cohort consisted of 95 pregnant women with tuberculosis. Among the 95 cases, there are 24 cases with IVF-ET and 71 cases with natural fertilization ( gure 1). The baseline characteristics of pregnant women with tuberculosis after in vitro fertilization and embryo transfer (IVF-ET) and natural pregnancy are compared ( Table 1).The average age of was 29.17±4.18 in IVF-ET group, while 27.08±5.27 in natural fertilization group (p=0.082). Indeed, the proportion of double embryos in IVF-ET group is signi cantly higher than that in natural group (37.5% vs. 1.4%, p<0.05), but the other elements such as TB history, contact history of TB, BCG vaccination status and education level did not reach statistical signi cance (p>0.05). The proportion of HBsAg positive in two groups was same (4.2% vs. 4.2%) and the HCV antibody of the two groups was negative. The onset period of patients with IVF-ET was at a median of 115th day of pregnancy (ranged 92th-141th day), while the onset period of patients with natural fertilization was at a median of 147th day of pregnancy (ranged 68th-219th day), there is no statistical signi cance (p>0.05) ( gure 2).

Symptoms of TB in pregnant women
The symptoms of TB in pregnant women with tuberculosis after in IVF-ET and natural pregnancy are shown in Table 2. The incidents of fever among pregnant women with IVF-ET was signi cantly higher than that among pregnant women with natural fertilization (79.2% vs. 35.2%, p<0.05). The ratio of respiratory symptom (cough, cough with phlegm, and shortness of breath), the incident of failure to gain weight, headache and night sweating was higher in IVF-ET group than those in natural fertilization, but the difference did not reach statistical signi cance (p>0.05). however, the ratio of cough with blood was lower in IVF-ET group than that in natural fertilization (0% vs. 12.7%, p>0.05). 266.00) in IVF-ET group were signi cantly lower than that in natural pregnancy group (p<0.05) ( Table 3). Chest X-ray or CT images from the IVF-ET group showed a higher proportion of diffuse military in ltration than those from the natural pregnancy group (87.5% vs. 12.7%, p<0.05). In addition, a higher proportion of pregnant women in the IVF-ET group had coexisting pulmonary tuberculosis with extrapulmonary tuberculosis (45.8% vs. 12.7%, p<0.05). Although the lymphocyte count of pregnant women in the IVF-ET group is low, the positive rate of T-spot/TST is higher than that in natural pregnancy group, and the difference did not reach statistical signi cance (91.7% vs. 83.1%, p>0.05). The proportion of pathogenic diagnosis and the proportion of treatment options are similar in two groups (Table 3, supplementary table   1).

Discussion
To our knowledge, no other studies have compared the clinical features and TB treatment outcome among pregnant women after IVF-ET and naturally pregant women. In this study, we observed similar baseline characteristics (such as: age, TB history, history of BCG vaccination, contact history of TB, education level, HBsAg positive and HCV antibody positive) of pregnant women with tuberculosis after in vitro fertilization and embryo transfer (IVF-ET) and natural pregnancy. The onset period of patients with IVF-ET was earlier than the time of patients with natural fertilization (115th vs. 147th, p>0.05).
Generally speaking, TB symptoms during pregnancy were not much different from those of nonpregnancy TB. Cough, cough with phlegm and fever are still the main clinical manifestations. But we found the incidents of fever among pregnant women with IVF-ET was signi cantly higher than that among pregnant women with natural fertilization, which suggests that pregnant women with tuberculosis after IVF-ET are more likely to have systemic symptoms (tuberculosis toxemia).
During pregnancy, the mother uses a complex network of hormones, immune cells and cytokines to immunoregulate the various physiological processes of pregnancy. The speci c and non-speci c immune tolerance between the mother and the fetus are the main factors for maintaining the success of the pregnancy. Hormones such as progesterone, estrogens and human chorionic gonadotropin have very important effect on early pregnancy which play essential roles in the immune crosstalk at the maternalfetal interface 15,16 . Studies have shown speci c immunity is suppressed in pregnant women, including the decrease in the number of T cells, the decline in T-cell functions and the change Th1/Th2 cytokine towards Th2 bias, which enhance the maternal-fetal immune tolerance but impair responses against some pathogens [17][18][19] . IVF is widely used to treat infertility. The clinical pregnancy rate and embryo growth rate are closely related to the receptivity of the endometrium, and are affected by ovarian steroid hormones, particularly estradiol and progesterone 20 .In the process of controlled ovarian hyperstimulation (COH) and luteal phase, progesterone needs to be injected daily 21 . Glucocorticoids, acting as immunomodulators to in uence cell-mediated immunity and reduce in ammation, have been used to improve folliculogenesis and the intrauterine environment 22 . Therefore, once a woman becomes pregnant, especially after IVF-ET, the changes in the immune function of the body are not conducive to the control and elimination of mycobacterium tuberculosis. Our study also shows the lymphocyte count, CD4 T cell count, CD8 T cell count of pregnant women in the IVF-ET group were signi cantly lower than those in natural pregnancy group and the proportion of military tuberculosis of pregnant women in the IVF-ET group at admission were higher than those in natural pregnancy group.
In the published literature, there are few studies on the side effects of tuberculosis treatment in pregnant women, especially liver toxicity. Our study showed severe hepatotoxicity was signi cantly more frequent in pregnant women after IVF-ET compared to those in natural pregnancy. It is speculated that it may be related to pregant women after IVF-ET who are mostly twin pregnancies leading to liver overload, and pregnant women are mostly hematological disseminated tuberculosis patients, with severe symptoms of infection and poisoning, and DILI is more likely to occur. Temporary drug withdrawal or change antituberculosis treatment regimen due to DILI was more frequent in pregant women after IVF-ET than those in nature fertilization. The usual recommendation is restart with rifampin with or without ethambutol, then add isoniazid after 3-7 days, and to continue treatment, if liver function tests are normal. In case of prolonged or severe hepatotoxicity, add other second-line medication (Levo oxacin, Linezolid, Prothionamide, etc.) and discontinue pyzinamide 23 .
A 2017 meta-analysis 5 showed tuberculosis in pregnancy has a greater impact on fetus than on pregnant women, compared with pregant women without TB, pregnant women with active TB tended to have a higher risk of death(OR 4.1, 95%CI 0.65-25.2), was associated with increased odds of preterm birth (OR 1.7, 95%CI 1.2-2.4) and perinatal death (OR 4.2, 95%CI 1.5-11.8). Pregnancy with hematogenous disseminated tuberculosis can cause chorioamnionitis due to severe tuberculosis toxemia and mycobacterium tuberculosis spreading along the blood to infect the placenta 24,25 , leading to miscarriage (spontaneous abortion, inevitable abortion) and fetal death. We also found that the proportion of arti cial abortion among naturally pregnant tuberculosis patients is higher, mainly because most of them have unplanned pregnancies, and the others were worried about the side effects of drugs, although there is evidence that the use of these rst-line antituberculous drugs in pregnancy are considered safe for the mother and the foetus 26 .
Our study had several limitations. First, the study is a retrospective study, and it is impossible to determine whether maternal tuberculosis was infected during pregnancy or caused by recurrence of old lesions in the body, and this may involve different interventions, especially for women who are about to undergo IVF-ET. If active tuberculosis is caused by re-infection, the measures taken are to protect pregnancy women during pregnancy. If it is caused by the reignition of old lesion, detailed tuberculosis screening work is required before IVF-ET. If infertility is caused by congenital tuberculosis, antituberculosis treatment should be initiated immediately and if congenital tuberculosis and active tuberculosis in other parts are ruled out, women infected mycobacterium tuberculosis might need to receive preventive treatment. These measures may reduce the incidence of tuberculosis during pregnancy and abortion rate of pregnant women. We will do further research on these issues next.

Declarations
Ethics approval and consent to participate This study was approved by the Ethics Committee of Shanghai Public Health Clinical Center [batch number:2020-S112-03] .The retrospective nature of the study resulted in a waiver regarding the signing of the informed consent form. And this study was approved by the Ethics Committee of Shanghai Public Health Clinical Center to exempt subjects from informed consent.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request Competing interests data. Wei Huang drafted the initial version of the manuscript, which was subsequently revised and approved for submission by all authors. Figure 1 Flow diagram of patients with tuberculosis in pregnancy in the study IVF-ET: in vitro fertilization and embryo transfer ATT: anti-tubercular therapy