Effect of levothyroxine on the pregnancy outcomes in recurrent pregnancy loss women with subclinical hypothyroidism and thyroperoxidase antibody positivity: a systematic review and meta-analysis

Abstract Objective This study aimed to explore the effects of levothyroxine on pregnancy outcomes and thyroid function in recurrent pregnancy loss (RPL) women with subclinical hypothyroidism (SCH) or thyroperoxidase antibody positivity (TPOAb+). Methods Literature search was performed from inception to 24 June 2022. The heterogeneity for each outcome was evaluated using Cochran’s Q test and quantified with I-squared (I2). Pooled effect sizes were expressed as relative risk (RR) and weighted mean differences (WMD) with 95% confidence intervals (95% CIs). Stability of the results were assessed using the sensitivity analysis. Results Fifteen eligible studies with 1911 participants were included in this meta-analysis. The pooled data showed that levothyroxine decreased premature delivery rate (RR = 0.48, 95%CI: 0.32, 0.72), miscarriage rate (RR = 0.59, 95%CI: 0.44, 0.79), premature rupture of membranes (PROM) rate (RR = 0.44, 95%CI: 0.29, 0.66), and fetal growth restriction rate (RR = 0.33, 95%CI: 0.12, 0.89) in RPL women with TPOAb+. In RPL women with SCH, live birth rate was elevated (RR = 1.20, 95%CI: 1.01, 1.42) and miscarriage rate was reduced (RR = 0.65, 95%CI: 0.44, 0.97) by levothyroxine. In addition, levothyroxine substantially decreased TSH level (WMD = −0.23, 95% CI: −0.31, −0.16) and TPO level (WMD = −23.48, 95%CI: −27.50, −19.47). Conclusions Levothyroxine improved pregnancy outcomes and thyroid function in RPL women with TPOAb+ or SCH, indicating that levothyroxine may be beneficial for RPL women if TPOAb+ or SCH occurs. Future studies are needed to verify our findings.


Introduction
Recurrent pregnancy loss (RPL) is defined as the loss of two or more pregnancies according to the guideline of European Society of Human Reproduction and Embryology (ESHRE) [1].The estimated prevalence of RPL ranges from 1% to 4% in all pregnant women [1,2].Approximately 20% of RPL women have thyroid autoimmunity, which substantially increases the risk of early abortion [2,3].Subclinical hypothyroidism (SCH) and thyroperoxidase antibody positivity (TPOAb þ ) display the thyroid autoimmunity, and women with RPL with SCH or TPOAb þ have an increased risk of poor pregnancy outcomes [4].Therefore, exploring therapies that modulate SCH and TPOAb þ is necessary to improve pregnancy outcomes for RPL women.
Levothyroxine is recommended as a treatment for SCH and TPOAb þ [5].A meta-analysis has shown that levothyroxine therapy can improve pregnancy outcomes in women with SCH and TPOAb þ [6]; however, pregnant women with RPL are not considered in this meta-analysis.Compared to women without pregnancy loss, women with RPL face more psychological burden and have a higher risk of poor pregnancy outcomes, such as stillbirth, pregnancy loss, placental adhesion, uterine infection, and cesarean section [2,7].The efficacy of levothyroxine therapy remains controversial in RPL women with SCH or TPOAb þ .Dal Lago et al. found that the miscarriage rate was decreased and the capacity to conceive was improved in thyroid dysfunction women treated with levothyroxine therapy compared to untreated women [8].However, van Dijk et al. reported no statistical difference in the miscarriage rate and capacity to conceive between TPOAb þ women treated with levothyroxine and untreated women [9].Some studies have reported that there was no effect of levothyroxine on the live birth rate in RPL women with TPOAb þ [9,10], whereas others suggested that levothyroxine therapy increased the odds of live birth [11].
Considering these controversial reports, it is necessary to perform a meta-analysis based on previously published studies reporting the effect of levothyroxine on the pregnancy outcomes in RPL women with SCH or TPOAb þ .Combining and analyzing data on this controversial topic may provide valuable information for the clinical management of RPL women with SCH or TPOAb þ .

Study selection
Studies meeting the following criteria were included: (1) RPL women with SCH or TPOAb þ as the study population; (2) comparing pregnancy outcomes between intervention group and control group (levothyroxine-treated women vs. untreated women/placebo-treated women/women with basic treatment; basic treatment þ levothyroxine-treated women vs. women with basic treatment); (3) designed as cohort studies or randomized controlled trials (RCTs); and (4) published in English or Chinese.Basic treatment included the use of aspirin, dalteparin, and progesterone.
Studies were excluded if: (1) they were not related to the topic; (2) data were incomplete or unable to be extracted; and (3) they were published as case reports, meta-analyses, reviews, or abstracts.

Outcomes
The outcomes assessed in this meta-analysis were pregnancy outcomes and thyroid function.Pregnancy outcomes included pregnancy rate, live birth rate, premature delivery (at < 37 weeks), miscarriage rate, premature rupture of membranes (PROM), fetal growth restriction, ectopic pregnancy, 1 min Apgar, 5 min Apgar, birth weight (g), and birth height (cm).Thyroid function was assessed through TSH, free thyroxine (FT4), thyroid peroxidase (TPO), and thyroid peroxidase antibody (anti-TPO).

Data extraction
Two reviewers (MJY and YLL) independently extracted the following data from the included studies: the first author, year of publication, country, study design, patients, RPL definition, maternal age, groups, interventions in the groups, sample size, body mass index (BMI), number of previous miscarriages, number of previous live births, and outcomes.Any disagreement was resolved through discussion with a third researcher (YYW).

Quality assessment of included studies
The cohort studies were subjected to the Newcastle-Ottawa Scale (NOS) for quality assessment, which was based on three perspectives: selection of participants, comparability of the groups, and ascertainment of either exposure or outcome of interest [13].The total score of this scale was 9, with 0-3 for poor quality, 4-6 for fair quality, and 7-9 for good quality.
RCT quality was assessed using the modified Jadad scale (allocation concealment added to the original Jadad scale) [14,15].The total score of this scale was 7, and the study quality was defined as poor (1-3 points) and good (4-7 points).

Statistical analysis
All analyses were performed using STATA 15.1 (StataCorp, College Station, TX).The included studies were compared using a standard meta-analysis approach, and the effect size was described as relative risk (RR; for categorical data) and weighted mean difference (WMD; for continuous data).The pooled results were expressed as corresponding 95% confidence intervals (95% CIs).RR and WMD of the outcomes were graphically represented using forest plots.
The heterogeneity of the effect size for each outcome was evaluated using Cochran's Q test and quantified with I-squared (I 2 ).The fixed-effects model was used if I 2 < 50%, and a random-effects model was used if I 2 !50%.Sensitivity analysis was carried out by sequentially eliminating studies to check the potential impact on the pooled effect and to evaluate the stability and reliability of the results.To make our results more reliable, we also excluded two studies with poor quality for re-analysis [16,17].P significance was set at p < .05.

Literature search and characteristics of included studies
A flowchart of the literature search is shown in Figure 1.A total of 345 studies were searched (319 studies from English databases and 26 studies from Chinese databases).Among these, 166 duplicates were excluded.Furthermore, 145 studies were excluded because they did not meet the requirements (n ¼ 93), were published as reviews or meta-analyses (n ¼ 24), case reports (n ¼ 6), and abstracts (n ¼ 18), and were non-English/Chinese articles (n ¼ 4).After reviewing the titles and abstracts, six studies with topics that did not meet the requirements were excluded.Finally, 15 studies (8 cohort studies and 7 RCTs) were included [3,[8][9][10][16][17][18][19][20][21][22][23][24][25][26].Seven studies were assessed as good quality, six studies were assessed as fair quality, and two studies were assessed as poor quality.A total of 1911 participants were included in this meta-analysis, with 981 and 930 participants in the treatment and control groups, respectively.The characteristics and quality assessments of the included studies are presented in Table 1.
Overall effects of levothyroxine on the pregnancy outcomes of RPL women with SCH Table 3 shows the pregnancy outcomes of RPL women with SCH in the levothyroxine treatment and control groups.Three of the 15 studies contributed to the meta-analysis of the live birth rate.We found that levothyroxine therapy significantly increased the rate of live births (RR ¼ 1.20, 95% CI: 1.01, 1.42;I 2 ¼ 0.0%) (Figure 3(A)).The pooled results of the four studies reporting miscarriage rate in RPL women with SCH showed that levothyroxine supplementation was associated with a decreased miscarriage rate (RR ¼ 0.65, 95% CI: 0.44, 0.97; I 2 ¼ 60.4%) (Figure 3(B)).We did not find a statistical difference in the association between levothyroxine treatment and premature delivery rate in RPL women with SCH, with an RR of 0.58 (95% CI: 0.19, 1.78) and I 2 ¼ 0%.
The pregnancy rate, PROM rate, ectopic pregnancy rate, and birth weight were reported in one study [8,22,24].Dal Lago et al. reported that patients with use of levothyroxine had a higher rate of pregnancy compared to those with no use, and RR was 4.68 (95%CI: 3.28, 6.69) [8].In a study by Zhang et al.PROM rate in patients with levothyroxine supplementation was not significantly different from that in patients with no treatment (RR ¼ 0.86, 95%CI: 0.32, 2.30) [24].Yoshihara et al. found that there was no significant difference in ectopic pregnancy rate (RR ¼ 7.07, 95%CI: 0.29, 170.20) and birth weight (WMD ¼ 111.00, 95%CI: À92.14, 314.14) between RPL women with SCH who received levothyroxine treatment and those in the untreated group [22].

Overall effects of levothyroxine on the thyroid function of RPL women with TPOAb þ
Table 4 shows the thyroid function of RPL women with TPOAb þ in the levothyroxine treatment group and the control group.Of the 15 studies, three reported TSH levels.The pooled results showed that levothyroxine treatment substantially decreased TSH levels (WMD ¼ À0.23, 95% CI: À0.31, À0.16;I 2 ¼ 32.5%).Three studies reported FT4 levels, and pooled data showed no statistical significance between the two groups (WMD ¼ 0.03, 95% CI: À0.01, 0.06; I 2 ¼ 0%).The pooled results of three studies reporting the TPO level showed that levothyroxine supplementation reduced the level of TPO (WMD: À23.48, 95%CI: À27.50, À19.47).Mosaddegh et al. found that anti-TPO levels were lower in a levothyroxine treatment group than in the control group [19].After excluding two studies with poor quality, we obtained similar results (Supplementary Table S2).

Sensitivity analysis
Sensitivity analysis was performed to evaluate the influence on the effect estimate by sequentially omitting each study.For all outcomes, the pooled RRs or WMD of the remaining studies did not change after eliminating each study one by one (data not shown).

Discussion
Fifteen studies were included in this meta-analysis.
The pooled results showed that levothyroxine treatment was associated with a decreased rate of premature delivery and PROM, and not associated with the live birth rate in RPL women with TPOAb þ .Moreover, live birth rate was elevated and miscarriage rate was reduced by levothyroxine in RPL women with SCH.In addition, we found that levothyroxine supplementation decreased the levels of TSH and TPO in RPL women with TPOAb þ .RPL is an important reproductive problem, and 8-12% of abortions are caused by endocrine factors, including thyroid dysfunction [27].Autoimmunity is the main cause of thyroid dysfunction in pregnant women, and TPOAb is a sensitivity index [28].TPOAb þ not only leads to the decline of thyroid physiological function, abnormal embryonic development, and early abortion, but also activates autoimmune factors to promote endometrial receptivity changes caused by the deposition of autoimmune complexes, thereby increasing the difficulty of embryo implantation [29].In addition, the incidence of SCH is increased in RPL patients, and the risk of subsequent adverse pregnancy outcomes of RPL patients with SCH is also elevated [30].The American Society for Reproductive Medicine (ASRM) did not make a clear recommendation for the treatment of SCH or TPOAb þ in RPL women [31].ESHRE stated that treatment of SCH with levothyroxine may reduce the miscarriage rate, but this treatment decision should be weighed against the potential risks, and that there was not enough evidence to support treatment in RPL women with isolated thyroid autoimmunity outside of clinical trials [1].A review by Dong et al. indicated that the benefits of levothyroxine for RPL women with SCH were not supported by current studies, while their findings were not quantitatively analyzed [4].Considering the lack of clear recommendation and evidence-based analysis, we performed a meta-analysis based on available studies.After quantitative analysis, association between levothyroxine and live birth rate was not observed in RPL women with TPOAb þ , which was supported by the previous studies [9,10].However, the pooled results showed that levothyroxine supplementation increased the live birth rate in RPL women with SCH.A study showed no association in the live birth rate between levothyroxine treatment group and the control group [22], whereas Leng et al. have reported that levothyroxine increased the live birth rate in RPL women with SCH [32].Our findings indicated that levothyroxine supplementation may be beneficial for RPL women with SCH.In the future, more studies are needed to further verify our findings.
A previous meta-analysis by Rao et al. included non-RPL women with TPOAb þ , and found the beneficial effect of levothyroxine on the decrease of premature delivery rate [6].Another meta-analysis by Di Girolamo et al. reported that the risk of premature delivery was lower in non-RPL women with TPOAb þ receiving levothyroxine supplementation [33].In our meta-analysis, we found the levothyroxine decrease the premature delivery rate in RPL women with TPOAb þ .This may be explained by that levothyroxine can improve the fiber tension of fetal membranes in early pregnancy, improve local body resistance, and prevent viral infections that can cause premature birth [16].Di Girolamo et al. also explored the effect of levothyroxine on the other pregnancy outcomes, such as miscarriage, and found no difference in the risk of miscarriage between the two groups [33].Our meta-analysis found that levothyroxine decreased the miscarriage rate in RPL women with TPOAb þ or SCH.This difference may be caused by different study population (non-RPL women in Di Girolamo et al. study, and RPL women in our study).Our findings have been supported by the studies from Zhang et al. and Leng et al. [24,32] We also found that levothyroxine decreased the PROM rate.This finding may be explained by that levothyroxine can promote the regeneration and repair of apoptotic tissue cells and improve the proliferative activity of chorionic or amniotic sac cells [16].
TPO is an important enzyme that catalyzes thyroid hormone [28].Due to its cytotoxic effect, TPOAb þ damages normal thyroid cells and inhibits the secretion and synthesis of thyroid hormones, resulting in an abnormal increase in serum TPO levels [28].A study has shown that RPL women with TPOAb þ status can undergo an abnormal increase in TSH secretion in early pregnancy, and FT4 levels in the maternal body continue to decline with the progress of gestational weeks; thus, thyroid function appears as an autoimmune disorder, leading to hypothyroidism and adverse pregnancy outcomes [34].In this systematic review and meta-analysis, a study reported lower anti-TPO levels in the levothyroxine-treated group than in the untreated group [19].Similarly, Okuroglu et al. found that the dose of levothyroxine supplementation was positively associated with titers of TPOAb þ [35].The pooled results of the meta-analysis showed that both TSH and TPO levels decreased following levothyroxine supplementation.This finding was similar to that of Duan et al. and Li et al. [16,25] The decrease in TPO may be related to levothyroxine improving the function of thyroid follicular epithelial cells and reducing the release of autoantibodies caused by immune disorders, and the decrease in TSH indicates the stability of thyroid hormone levels in the body [16].Our findings indicated that levothyroxine may be effective in improving thyroid function in women with RPL and TPOAb þ .
Our meta-analysis aimed to analyze controversial reports on the effect of levothyroxine on pregnancy outcomes in RPL women with SCH and TPOAb þ .The pooled data showed that levothyroxine treatment improved pregnancy outcomes and thyroid function in the target population, which may provide a clinical reference for managing RPL women with SCH and TPOAb þ .Our meta-analysis had some limitations.First, the search strategy included only database search and did not include a search of reference lists of retrieved articles or a hand search of recent journal publications or expert contact.This may exclude several relevant studies.Second, the definitions of SCH or TPOAb þ were different in the included studies in our meta-analysis.This may lead to selection bias in the participants.Due to the limitations of the included studies, we could not unify the definitions.Third, the measurement time of the levels of indicators assessing thyroid function (TSH, FT4, TPO, and anti-TPO) was different in the included studies, which may affect the robustness of the analysis results.Fourth, the dose of levothyroxine is different in the included studies, which is difficult to determine whether overtreatment.Future studies should be performed to further explore the effect of the dose of levothyroxine on the pregnancy outcomes in RPL women with RPL or TPOAb þ .

Conclusion
In conclusion, our meta-analysis suggests that levothyroxine therapy improves pregnancy outcomes and thyroid function in RPL women with TPOAb þ or SCH, indicating that levothyroxine may be beneficial for RPL women if TPOAb þ or SCH occurs.Our findings need to be verified by more studies in the future.

Figure 1 .
Figure 1.The flowchart for selecting studies.

Figure 3 .
Figure 3. Forest plots of studies examining the association between levothyroxine and live birth rate (A) and miscarriage rate (B) in RPL women with SCH.

Table 1 .
The characteristics of the included studies.

Table 1 .
Continued.TPO: thyroid peroxidase; anti-TPO: thyroid peroxidase antibody; PROM: premature rupture of membranes; T: treatment group; C: control group.Note.The basic treatment included the use of aspirin, dalteparin, didroxyprogesterone, folic acid, vitamin E tablets, or progesterone.

Table 2 .
Overall effects of levothyroxine on the pregnancy outcomes of RPL patients with TPOAb þ .