The correlation between antithyroid antibodies, fecundity and pregnancy outcome is quite debatable and conflicting. Previously, meta-analysis of 4 prospective studies that included 1098 subfertile women undergoing IVF noted a significant two fold higher risk of miscarriage of subfertile euthyroid women with TAI compared with a counterpart without TAI[17]. Among those four prospective studies, three of them only measured TPO-Ab, one study recruited participants with unexplained infertility and no history of miscarriage, subfertile women with various etiology and normal thyroid function was reported in another two prospective investigations and the last one specifically excluded women with a history of miscarriage. Under the circumstances of different ART/IVF protocols employed, dissimilar underlying etiologies contributing to infertility and changeable cut-off values concerning euthyroidism and subclinical hypothyroidism, the 2017 American Thyroid Association (ATA) pregnancy guidelines was not able to come to a definite conclusion on the link between thyroid autoimmunity and ART outcomes. What's more, levothyroxine treatment was recommended for subclinical hypothyroidism, defined as a TSH > 2.5 mIU/L, and considered for euthyroid infertile women with thyroid autoimmunity when they attempted to conceive by virtue of assisted reproductive technologies in the 2017 ATA guidelines after weighing the pros and cons of levothyroxine supplement[18].
However, in the past few years following the publication of the 2017 ATA guidelines, two large randomized controlled trials(RCT) assessing the value of levothyroxine on pregnancy outcomes in euthyroid TPO-Ab positive women reported that the use of levothyroxine was not associated with improvements on miscarriage and live birth rate[19, 20]. Though there existed several limitations involving fixed levothyroxine doses, undetermined TSH values during early trimester of pregnancy following medicine supplement, uncertain population compliance and the exclusion of women with recurrent miscarriages or positive for other autoimmune antibodies, the large-sample RCT results were essential and provoking on levothyroxine effectiveness in the specific euthyroid women with thyroid autoimmunity[21]. Furthermore, a recent meta-analysis of six RCTs demonstrated that levothyroxine had no capacity to improve clinical pregnancy outcomes among women positive for thyroid peroxidase antibody. As a matter of fact, of the meta-analysis based on high- to moderate-quality evidence, two trials underwent ART, two studies used fixed levothyroxine doses and one investigation enrolled euthyroid or subclinical women[22]. Thus, further large-scale high-quality investigations on specific population are still urgently needed.
On account of decreased effectiveness of levothyroxine and generalized autoimmune imbalance resulted from thyroid autoimmunity, we retrospectively explored the power of aspirin plus prednisone treatment on euthyroid women with TAI undergoing their first IVF/ICSI procedure. Markedly, there exists a robust, dynamic and responsive immune system for a successful pregnancy. Pregnancy begins in a pro-inflammatory stage allowing implantation and placentation, then shifts to an anti-inflammatory environment which is pivotal for fetal growth, and finally come back to a pro-inflammatory stage available to labor and delivery. A pro-inflammatory process is initiated in the embryo implantation and trophoblast invasion to better promote cell clearance, angiogenesis, cell growth and tolerance, as the pro-inflammatory procedure is characterized by the presence of angiogenic factors, growth and survival factors, and cytokines and chemokines[23]. Following implantation, the female immune system usually induces tolerance towards the embryo, whereas tolerance induction is incomplete in a hyperactive immune system. Subfertile women with autoimmune thyroid disease usually expressed increased level of INFγ from pro-inflammatory Th1 immune cell and reduced secretion of IL-4 and IL-10 from Th2 immune cell compared to controls with no anti-thyroid antibodies, suggesting that the excessive activated pro-inflammatory Th1 cell hampered the subsequent successful pregnancy[24]. Moreover, pinopodes, the spherical protrusions of the epithelial plasma membrane into the lumen, are characterized as classic morphological biomarkers of receptive endometrium favoring implantation. Recently and firstly, a euthyroid Hashimoto's thyroiditis (HT) mice model was established and explored the correlation of HT and endometrial receptivity defects, indicating that HT alone inhibited luminal epithelium development, retarded the formation and development of pinopodes, decreased expression of receptivity markers, thereby inducing a nonreceptive endometrial milieu and leading to implantation failure[25]. Prednisone, a type of glucocorticoid, is readily absorbed from the gastrointestinal tract and used primarily for its anti-inflammatory effects in many system disorders[26]. A substantial amount of trials revealed that low doses of corticosteroids (10 mg/day) had improved the IVF pregnancy outcome in women experiencing immunological infertility and recurrent miscarriages, even with a prior history of 19 consecutive miscarriages[27–30]. Moreover, by virtue of cleavage stage mouse embryos exposing to 3 and 30 µM concentrations of prednisolone(PRDL) in vitro to mimic the possible clinical scenario and to assess the embryonic response to direct PRDL exposure, a recent animal study revealed that exposure to 30 µM PRDL delayed the embryonic progression, decreased hatching potential and increased apoptosis in blastocysts, and yet, 3 µM PRDL exposure increased inner cell mass (ICM) proliferation ability which was incorporated to predict the implantation potential[31]. It was worth mentioning that 3 µM was close to the therapeutic dose and 30 µM to reflect the ten-times higher than the initial level. Experimental evidences in animal models implied that glucocorticoids at higher concentrations could negatively affect oocyte maturation and early embryogenesis. The therapeutic dose of PRDL had the capacity to reduce post-implantation demise, possibly due to its stimulus on choriocarcinoma cell lines. Similarly, the latest trial to investigate the role of prednisolone on decidualization and decidual-trophoblast interactions reported that prednisolone treatment enhanced trophoblast outgrowth, elevated trophoblast mRNA expression of cell motility gene PLCG1, altered decidual-trophoblast interactions and yet the clinical consequences of these changes were unknown[32]. Thus, there remains a great need for further research on this topic. On the other side, with the capacity to decrease blood viscosity and increase blood flow, which is secondary to the inhibition of cyclooxygenase-1 and decreased production of thromboxane-2, low dose of aspirin plays an essential role in improving uterine and ovarian blood flow, enhancing embryo implantation and sustaining early pregnancy. Besides, daily low-dose aspirin use is considerably safe as it did not contribute to any changes to menstrual cycle, follicular phase, or luteal phase length or hormone levels across the menstrual cycle[33]. The adjuvant treatment of aspirin in combination with prednisone is recommended to patients with autoantibodies undergoing IVF as its benefits are demonstrated in several investigations[27, 28, 34]. However, these trials are published from a long time ago and don't reveal the efficacy of medicine on infertile women who present positive only for antithyroid antibodies.
In our study, we applied ranges of < 31 years, 31–37 years, and > 37 years according to the physiological understanding of natural fertility, whose initial decline began at 31 years and the critical age of 37 years recorded as the pivotal age for success rates in treatment programs[35, 36]. Notably, the rough distribution of age was comparable and thus reduced the potential confounding risk of age, as advanced age predisposed to induce increased chance of de novo chromosomal aberrations in oocytes, and, in turn, in the embryo[11, 37, 38]. With regard to ovarian reserve, age, AMH, AFC and bFSH were comparable between women administrated with medicine or not at both fresh and frozen embryo transfer cycles. Additionally, significant parental chromosome abnormality, severe oligoasthenospermia and azoospermatism were definitely excluded in our study as the rate of chromosomal anomaly was 0.24% in normal semen group, 4.7% in moderate-to-severe oligoasthenospermia group and 9.59% in azoospermia group[39]. The consistency between two groups provided confidence for us to come to the final conclusion as 30–50% of implantation failures can be attributed to poor embryo quality and the embryo quality is determined by a number of parameters primarily of the women's age, ovarian reserve, the underlying causes of infertility and sperm quality. In addition, decreased endometrial receptivity is thought to account for similarly two thirds of these failures[5]. Typically, endometrium is the direct or indirect target of antithyroid antibodies, prednisone and aspirin.
In the context of the unclear and indefinite efficacy of both antibodies, we recorded and analyzed the proportion of positive isolated TPOAb, positive isolated TgAb, and double positive TPOAb and TgAb in our study and no significant difference was observed. Moreover, in our study, euthyroidism was defined as the reference value of 0.35-4.0mIU/L for the TSH and the value was comparable between two groups. The borderline of euthyroid and subclinical hypothyroidism is changeable over time. Nowadays, the association between elevated maternal TSH concentrations and pregnancy-specific complications appears to be more pronounced when adopting the the cut-off point of 4.0 mIU/L, or a population-based reference value than the level of 2.5 mIU/L[40]. And newer guidelines suggested that an upper limit of 4.0 mIU/L should be considered diagnostic compared with the previous guideline of 2.5 mIU/L[18]. Based on the borderline of 4.0mIU/L for the TSH and 2017 ATA recommendations, levothyroxine supplement was not included in our study.
Interestingly, we observed no association between prednisone plus aspirin treatment and reproductive outcomes including CPR, MR and LBR at the first embryo transfer regardless of fresh or frozen embryo transfer. Selected and assessed infertile women owned normal thyroid test and no autoimmune antibodies except anti-thyroid antibodies. This finding had not been replicated in other literature and should be interpreted with caution. In 2009, Alberto Revelli et al performed a retrospective study of 329 euthyroid women who were positive for TPOAb, TgAb, or both. The medication was prednisolone (10 mg/d) and aspirin (100 mg/d) from the day of stimulation to 10 weeks of gestational age, and during that period, P was increased to 30 mg/d for 5 days starting from the day of ET. Such a medication approach was acknowledged as of value on pregnancy and implantation rates in contrast with untreated ATA + patients[34].The timing of medication in our study commenced on the day of embryo transfer and lasted for 2-6weeks, mainly focusing on fascinating the implantation micro-immune environment. In a prospective case-control study including 233 consecutive patients, dexamethasone(0.5 mg/d) and acetylsalicylic acid(100 mg/d) starting from the day of embryo transfer and continuing until the end of the 12th week of gestation increased the pregnancy rate and implantation rate when compared with the control group[42].But it was noteworthy that the inclusion of this prospective study was inherited and acquired thrombophilia, compound heterozygous polymorphisms, positive anti-nuclear and anti-thyroglobulin antibodies, the strong indication for steroid hormone and anticoagulant drug[42]. Coincidentally, its inclusion standard was exactly in line with the exclusion criteria in our study, which largely explained the conflicting results. Thus, the effective value of treatment may be not obvious in women with unaffected thyroid function and only thyroid antibodies when compared with those with multiple types of autoantibodies or the history of RPL.
When talking about the mixed correlation between TAI and infertility, a recent review published in 2020 may help us to have a better understanding of its relevance. By summarizing and analyzing the latest studies since the 2017 guidelines, the review documented that anti-TPO Abs were associated with infertility in subsets of women mainly with unexplained infertility or polycystic ovarian syndrome (PCOS), but not all women[21]. Such a conclusion was primarily dependent on a secondary analysis of data from two multicenter RCTs involving 1650 euthyroid infertile women either with unexplained infertility or PCOS[41]. The weak correlation of TAI and IVF reproductive outcomes of general infertile population possibly partially explained the negative results of our study. Furthermore, a 2020 meta-analysis of seventeen studies pinpointed a statistically significant association between RPL and TAI (odds ratio 1.94; 95% CI, 1.43–2.64) and the statistical significance and magnitude of the results remained unchanged following sensitivity analyses[43], similar to the finding of our previous investigation[44]. As for euthyroid infertile women with unexplained infertility, PCOS or RPL, little evidence existed concerning the effect of replacement therapy of A plus P in those specific women. Besides, owning to the paucity of a great number of subjects, stratified research is usually not likely to fulfill at the subgroup level. However, based on the fact that P plus A treatment did play a positive role on improving adverse IVF reproductive outcomes in women with positive antinuclear antibodies[45], with unexplained RPL[46] and with other immune-related antibodies[15]. Combined treatment is likely to exert great influence on euthyroid infertile women with TAI and unexplained infertility or RPL, though it still requires prospective large-sample trials to justify its potency.
Additionally, in our study, there existed a phenomenon that no matter what type of embryo transferred, a higher but not significant incidence of abortion occurred in the treated group. As illustrated above, the value of FT3 in the P + A treated group was 2.90 ± 0.39 pg/mL, significantly lower than that of ATA-positive untreated subjects(P = 0.017). Multivariable logistic regression demonstrated the negative role of FT3 played on the incidence of fetus loss at the first fresh embryo transfer. The demonstration was buttressed by a preliminary observational study, which reported that low serum FT3 level compromised the beneficial effect of levothyroxine substitution in women with Hashimoto thyroiditis[47]. It is widely acknowledged that thyroid hormone transporters, receptors and their associated proteins are expressed in the ovary, the early embryo, endometrium, uterus and placenta[7]. Thus, optimal value of T3 is crucial for ovulation and folliculogenesis as T3 in combination with FSH appears to enhance granulosa cell proliferation and inhibit granulosa cell apoptosis via the PI3K/Akt pathway[7, 16]. Simultaneously, the expression of these proteins in the endometrium is changeable throughout the various phases of the menstrual cycle[48]. It's documented that receptive endometrium is accompanied with strengthened expression of thyroid hormone(TH) receptors in normal women[49], whereas weakened expression of proteins concerning thyroid hormone in the uterus is observed at the day of implantation in hypothyroid pregnant rats[50]. By means of the binding of TH receptors on the placenta and endometrium, as well as regulating the invasive potential of extravillous trophoblasts, thyroid hormone is capable to affect implantation and early fetal development[7]. To conclude, the evidence and statement above seemingly suggest the provision of additional levothyroxine supplement in euthyroid infertile women with low but in the normal reference range of FT3. One potential pathogenesis model with regard to the link between thyroid autoimmunity and fertility may offer us a new perspective[21].At early stages of autoimmunity, the main undesirable impact is through a hostile immune environment at the level of the ovary, with TPO as the direct antigen. At this stage, the thyroid hormone response is intact and the levothyroxine treatment is inefficient. As thyroid autoimmunity progresses, an impaired thyroid response to hCG stimulation appears and unable to meet the excess thyroid hormone demand under the condition of pregnancy. In that situation, treatment with thyroid hormone will be beneficial[21]. On the basis of such a new model, to distinguish the different stage is the key to efficient treatment.
Although progress has been made in some areas of the autoimmune disorders, little is known about the comprehensive understanding of the underling mechanism of autoimmune antibodies on reproductive outcome, leading to a barrier to a deeper search of effective treatments. Organs-on-a-chip, claimed as advanced in vitro models of multicellular tissue complexes or functional organ units, may help illuminate the possible intricate connection. Exploiting organ-on-a-chip approaches to modeling decidualization, implantation and placentation, they are capable of in-depth investigating the invasive and remodeling behavior of extravillous trophoblast cells, the procedure crucial of establishing the uteroplacental circulation that provides vascular supply to the growing fetus[51]. Further to that, interaction of antibodies and endometrium, changeable expression of immunological factors as well as glucocorticoid acting point are all standing a chance of being explored.
To conclude, according to the reality of routine thyroid screening, a large number of euthyroid women who test positive for antithyroid antibodies are discovered. Among them, patients underwent the first IVF cycle without the history of recurrent miscarriages or unexplained infertility are not recommended for the combined treatment of prednisone and aspirin. As a whole, there were several advantages in our study. Firstly, by means of establishing strict inclusion and exclusion criteria, we controlled for the possible confounding factor of other autoantibodies and several severe detrimental elements of spontaneous miscarriages to minimize, as much as possible, any patient-related variation in order to concentrate on the effects of aspirin in combination with prednisone on isolated euthyroid infertile women with TAI. Secondly, we only included first-time ART users and analyzed only first cycle ART outcomes to investigate homogeneous good-prognosis population and provide relevant suggestions for targeted subjects. Additionally, our study also had some limitations. Firstly, the current study was inevitably limited by its retrospective nature. Secondly, given the variation of TPOAb and TH concentrations in the context of pregnancy, measurement of longitudinal thyroid parameters during pregnancy was appreciable and yet we did not record the change[52]. While P + A supplement did not improve live birth rate or pregnancy rate in euthyroid TAI women at the first embryo transfer, the potential benefits of P + A supplementation during pregnancy could not be ruled out. Additional randomized clinical trial is required to determine whether P + A yield different results on women who test positive for antithyroid antibodies with recurrent implantation failure or with unexplained infertility. Similarly, it is possible that a higher risk population with increased recurrent pregnancy loss might yield different results.