IVF/ICSI outcomes of euthyroid infertile women with thyroid autoimmunity: does treatment with aspirin plus prednisone matter?

Despite significant advances in assisted reproductive technology (ART), including controlled ovarian stimulation, assisted hatching, and pre-implantation genetic testing, implantation remains a long-standing rate-limiting step in in vitro fertilization (IVF) treatments [1]. Successful implantation is dependent on the intricate collaboration between good-quality embryos and a receptive human endometrium, both of which are indispensable requisites [2‐4]. Therefore, when good-quality embryos or even euploid embryos are prepared for the transfer, the endometrium may be responsible for implantation failure [4, 5]. The major factors determining uterine receptivity for implantation and further embryo development are progesterone, estrogens, and the immune system [6]. In addition, the process of reprogramming the maternal immune system from rejection to temporary tolerance towards the fetal (paternally derived) semi-allograft depends on the endocrine-immune interaction [7‐9]. Generally speaking, the immune system of a healthy female induces tolerance towards the embryo, whereas this process fails in a hyperactive immune system, thereby reducing fertility and increasing the risk of miscarriage [7]. Among the various studies investigating immunological mechanisms, thyroid autoimmunity, as a predictor of generalized autoimmune disturbance, has been closely linked to recurrent embryo implantation failure, early pregnancy loss, and adverse pregnancy outcomes [10‐12]. Furthermore, several reports demonstrated that anti-thyroid antibodies did not affect embryo quality but decreased the clinical pregnancy rate, partly because of the impaired maternal immune modulation [13]. When addressing the reproductive challenges faced by the infertile women who are positive for anti-thyroid antibody pursuing pregnancy, prednisone (P) for immunosuppression and aspirin (A) as an antithrombotic agent are frequently and customarily prescribed in clinical practice.

As a therapeutic alternative, corticosteroid hormones in combination with aspirin may potentially benefit blood perfusion to the ovaries and endometrium and decrease local inflammatory reactions to the transfer procedure, thereby inducing a more favorable microenvironment for the transferred embryo [14, 15]. Furthermore, previous investigations have indicated that combined treatment of P + A is effective for women with various autoimmune diseases [16‐19]. However, these trials were published long ago and do not demonstrate the efficacy of this approach in infertile women who are positive only for antithyroid antibodies. Therefore, insufficient evidence exists to determine whether P + A therapy improves the likelihood of a successful pregnancy following ART in thyroid Ab-positive euthyroid women. In addition, some clinicians are more inclined to prescribe combined supplements, even for the first embryo transfer, while others are not.

Hence, the present study aimed to evaluate the effects of aspirin plus prednisone treatment on improving pregnancy outcomes of the first embryo transfer cycle in euthyroid infertile women who only present with positive thyroid autoimmune antibodies.

The correlation between antithyroid antibodies, fecundity, and pregnancy outcomes is quite debatable and conflicting. Previously, a meta-analysis of four prospective studies that included 1098 subfertile women undergoing IVF revealed a significant two-fold increase in the risk of the miscarriage of subfertile euthyroid women with TAI compared with a counterpart without TAI [20]. Among those four studies, three studies measured the TPO-Ab and the remaining study measured both TPOAb and TgAb. Of them, one study only recruited participants with unexplained infertility and without a previous history of miscarriages, whereas the other studies included subfertile women irrespective of the cause of infertility or a previous history of miscarriages. Under the circumstances of different ART/IVF protocols, dissimilar underlying etiologies contributing to infertility, and changeable cut-off values for euthyroidism and subclinical hypothyroidism, the 2017 American Thyroid Association pregnancy guidelines was unable to reach a definite conclusion on the link between TAI and ART outcomes. Moreover, levothyroxine treatment was recommended for subclinical hypothyroidism, defined as a TSH >2.5 mIU/L, and considered for euthyroid infertile women with TAI when they attempted to conceive by ART after weighing the pros and cons of levothyroxine supplement [21].

In the past few years following the publication of the 2017 guidelines, two large RCTs assessing the value of levothyroxine on pregnancy outcomes in euthyroid TPO-Ab positive women reported that the use of this drug did not significantly improve miscarriage rate and live birth rate [22, 23]. Despite several limitations (mainly involving fixed levothyroxine doses, undetermined TSH values during early 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 to specifically evaluate levothyroxine effectiveness in euthyroid women with TAI [24]. Furthermore, a recent meta-analysis including six RCTs demonstrated that levothyroxine could not improve clinical pregnancy outcomes among women who were positive for TPOAb. Indeed, of the meta-analyses that were based on high- to moderate-quality evidence, two trials involved ART, two studies used fixed levothyroxine doses and one investigation enrolled euthyroid or subclinical women [25]. Thus, additional large-scale high-quality research on this particular population is still urgently needed.

Based on the decreased effectiveness of levothyroxine and generalized autoimmune imbalance resulting from thyroid autoimmunity, the impact of P + A treatment on euthyroid women with TAI undergoing their first IVF/ICSI procedure was retrospectively explored. A dynamic and responsive immune system is critical for a successful pregnancy—the first trimester begins in a pro-inflammatory stage that allows implantation and placentation, then it shifts to an anti-inflammatory environment, pivotal for fetal growth, and finally returns to a pro-inflammatory stage suitable for labor and delivery [26]. The pro-inflammatory process initiated during embryo implantation and trophoblast invasion better promote cell clearance, angiogenesis, cell growth, and tolerance, as it is characterized by the presence of angiogenic, growth, and survival factors, as well as cytokines and chemokines [26]. Following implantation, the female immune system induces tolerance towards the embryo, whereas tolerance induction is incomplete in a hyperactive immune system. Subfertile women with autoimmune thyroid disease usually express increased levels of IFNγ from pro-inflammatory Th1 immune cells, along with lower IL-4 and IL-10 from Th2 immune cells compared with control patients without antithyroid antibodies. This suggests that excessively activated pro-inflammatory Th1 cells hamper the onset of a successful pregnancy [27]. Moreover, pinopodes, the spherical protrusions of the epithelial plasma membrane into the lumen, are characterized as classic morphological biomarkers of receptive endometrium favoring implantation [27]. Recently, a euthyroid Hashimoto’s thyroiditis mice model was established to explore the correlation between Hashimoto’s thyroiditis and endometrial receptivity defects. The resulting evidence indicated that Hashimoto’s thyroiditis alone inhibited luminal epithelium development, retarded the formation and development of pinopodes, and decreased expression of receptivity markers, thereby inducing a nonreceptive endometrial milieu and leading to implantation failure [28]. Prednisone, a type of glucocorticoid, is readily absorbed from the gastrointestinal tract and used primarily for its anti-inflammatory effects in many disorders [29]. Several trials revealed that low doses of corticosteroids (10 mg/day) improved IVF pregnancy outcomes in women experiencing immunological infertility and recurrent miscarriages, even in patients with a prior history of 19 consecutive miscarriages [16, 17, 30, 31]. Furthermore, by exposing cleavage-stage mouse embryos to 3 and 30 µM concentrations of prednisolone in vitro to assess the embryonic response to direct prednisolone exposure, a recent animal study revealed that exposure to 30 µM prednisolone delayed the embryonic progression, decreased hatching potential, and increased apoptosis in blastocysts. However, 3 µM prednisolone increased proliferation of the inner cell mass, which was incorporated to predict the implantation potential [32]. It is worth mentioning that 3 µM is close to the therapeutic dose and 30 µM is ten-fold higher than the initial level. Experimental evidence in animal models demonstrated that glucocorticoids at higher concentrations could negatively affect oocyte maturation and early embryogenesis. The therapeutic dose of prednisolone reduced post-implantation demise, possibly due to its effects on choriocarcinoma cell lines. Similarly, the latest trial that investigated the role of prednisolone on decidualization and decidual-trophoblast interactions reported that this treatment enhanced trophoblast outgrowth, elevated trophoblast mRNA expression of cell motility gene PLCG1, and altered decidual-trophoblast interactions, yet the clinical consequences of these changes were unknown [33]. Thus, there remains a great need for further research on this topic.

Simultaneously, a low dose of aspirin plays an essential role in improving uterine and ovarian blood flow, enhancing embryo implantation, and sustaining early pregnancy. This stems from its capacity to decrease blood viscosity and increase blood flow, which is secondary to the inhibition of cyclooxygenase-1 and the decreased production of thromboxane-2. In addition, daily low-dose aspirin use is considered safe as it does not affect the menstrual cycle, follicular phase, luteal phase length, or hormone levels across the menstrual cycle [34]. Adjuvant treatment of P + A is recommended to patients with autoantibodies undergoing IVF and its benefits have been demonstrated in several studies [16‐18]. However, these trials were published long ago and do not demonstrate the efficacy of this approach in infertile women who were positive only for antithyroid antibodies.

In our study, patients were classified according to their age in three categories: <31 years, 31–37 years, and >37 years, based on our understanding of natural fertility, since its decline begins at 31 years and 37 years old has been recorded as the pivotal age for success rates in treatment programs [35, 36]. Notably, the distribution among age groups was comparable and thus reduced the potential confounding risk of age, as an advanced age increases the chance of de novo chromosomal aberrations in oocytes and, in turn, in the embryo [11, 37, 38]. As for ovarian reserve, age, AMH, AFC, and FSH were all comparable between treated and untreated patients for both fresh and frozen embryo transfer cycles. In addition, couples with significant parental chromosome abnormality, severe oligoasthenospermia, and azoospermatism were excluded from our study as the rate of chromosomal anomaly was 0.24% in the normal semen group, 4.7% in the moderate-to-severe oligoasthenospermia group, and 9.59% in the azoospermia group [39]. A total of 30-50% of implantation failures can be attributed to poor embryo quality and embryo quality was determined by several parameters, primarily the women’s age, ovarian reserve, underlying causes of infertility, and sperm quality. Furthermore, decreased endometrial receptivity was thought to account for two-thirds of these failures [5]. Typically, the endometrium is a direct or indirect target of antithyroid antibodies, prednisone, and aspirin.

Because the harm caused by a single antibody and combined antibodies is not clear, the proportion of positive isolated TPOAb, positive isolated TgAb, and double-positive TPOAb and TgAb were recorded and analyzed in our study, and no significant differences were observed. Moreover, in our study, euthyroidism was defined by a TSH reference value range of 0.35–4.0 mIU/L and the value was comparable between the two groups. The threshold between euthyroid and subclinical hypothyroidism changes over time. Nowadays, the association between elevated maternal TSH concentrations and pregnancy-specific complications appears to be more pronounced when adopting the cut-off point of 4.0 mIU/L, or a population-based reference value than a level of 2.5 mIU/L [40]. 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 [21]. Based on the TSH threshold of 4.0mIU/L and recommendations of the 2017 American Thyroid Association, levothyroxine supplementation was not included in our study.

Interestingly, no association between P + A treatment and reproductive outcomes was observed including clinical pregnancy rate, miscarriage rate, and the live birth rate at the first embryo transfer regardless of embryo type (fresh or frozen). Assessed infertile women exhibited normal thyroid tests and no autoimmune antibodies except anti-thyroid antibodies. These finding have not been replicated in other studies and should be interpreted with caution. In 2009, Revelli et al. performed a retrospective study of 329 euthyroid women who were positive for TPOAb, TgAb, or both. The medication prescribed 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 embryo transfer. This approach was deemed beneficial to pregnancy and implantation rates in contrast with untreated ATA+ patients [18]. In our study, treatment started on the day of embryo transfer and lasted for 2–6 weeks, mainly focusing on improving the micro-immune environment of the implantation site. 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 until the end of the 12th week of gestation increased the pregnancy rate and implantation rate when compared with the control group [19]. The inclusion criteria of this prospective study included inherited and acquired thrombophilia, compound heterozygous polymorphisms, and positive anti-nuclear and anti-thyroglobulin antibodies, which were strong indications for steroid hormone and anticoagulant drugs [19]. Coincidentally, the inclusion criteria coincided with the exclusion criteria in our study, which largely explained the conflicting results. Thus, the effective value of treatment maybe not be evident in women with unaffected thyroid function and only thyroid antibodies when compared with those with multiple types of autoantibodies or a history of recurrent pregnancy loss.

In terms of the mixed correlation between TAI and infertility, a recent review published in 2020 contributed to a better understanding of its relevance. By summarizing and analyzing the latest studies since the 2017 guidelines, this review documented that anti-TPO antibodies were associated with infertility in subsets of women, mainly in those with unexplained infertility or polycystic ovarian syndrome (PCOS), but not in all women [24]. Such a conclusion was primarily dependent on a secondary analysis of data from two multicenter RCTs involving 1468 euthyroid infertile women either with unexplained infertility or PCOS [41]. The weak correlation between TAI and IVF reproductive outcomes of the general infertile population partially explained the negative results of our study. Furthermore, a 2020 meta-analysis of 17 studies pinpointed a statistically significant association between recurrent miscarriage and TAI (odds ratio 1.94; 95% CI, 1.43–2.64). The statistical significance and magnitude of the results remained unchanged following sensitivity analyses [42], similarly to the findings in our previous work [43]. Up to now, for euthyroid infertile women with unexplained infertility, PCOS, or recurrent pregnancy loss, little evidence exists concerning the effect of replacement therapy of P + A. In our study, due to the scarcity of a great number of subjects, stratified research does not achieve valid results at the subgroup level. However, based on the fact that P +A treatment improved adverse IVF reproductive outcomes in women with positive antinuclear antibodies [44], unexplained recurrent pregnancy loss [45, 46], repeated implantation failure [45, 47‐49], and other immune-related antibodies [15‐17, 46, 50, 51], combined treatment is likely to benefit euthyroid infertile women with TAI and unexplained infertility or recurrent pregnancy loss. However, prospective large-sample trials are still required to justify its potency.

Additionally, in our study, it was observed that regardless of the embryo type being transferred, the incidence of abortion was higher but not significant in the treated group than in control patients. 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 thyroid antibodies-positive untreated subjects (P = 0.017). Multivariable logistic regression demonstrated the negative role of FT3 in fetal loss incidence at the first fresh embryo transfer. This was consistent with a preliminary observational study that reported that low serum FT3 levels compromised the beneficial effect of levothyroxine substitution in women with Hashimoto’s thyroiditis [52]. It is widely acknowledged that thyroid hormone transporters, receptors, and their associated proteins are expressed in the ovary, early embryo, endometrium, uterus, and placenta [7]. Simultaneously, the expression of these proteins in the endometrium is dynamic throughout the various phases of the menstrual cycle [53]. It has been documented that receptive endometrium is accompanied by an increased expression of thyroid hormone receptors in normal women [54], whereas decreased expression of thyroid-related proteins in the uterus was observed on the day of implantation in hypothyroid pregnant rats [55]. By binding to thyroid hormone receptors on the placenta and endometrium, as well as regulating the invasive potential of extravillous trophoblasts, thyroid hormone can affect implantation and early fetal development [7]. Furthermore, an optimal T3 value is crucial for ovulation and folliculogenesis, as T3 in combination with FSH enhances proliferation of granulosa cells and inhibits apoptosis of granulosa cell via the PI3K/Akt pathway [7, 56]. To conclude, the aforementioned evidence seemingly suggests that additional levothyroxine should be supplemented in euthyroid infertile women with low but yet normal values of FT3. A novel pathogenesis model of the link between thyroid autoimmunity and fertility may offer us a new perspective [24]. During the early stages of autoimmunity, the main detrimental effects comprise the hostile immune environment impacting the ovary, with TPO as the direct antigen. At this stage, the thyroid hormone response is still intact and levothyroxine treatment is inefficient. As thyroid autoimmunity progresses, thyroid response to hCG stimulation is impaired and thus unable to meet the high thyroid hormone demand during pregnancy. In that situation, treatment with thyroid hormone would prove beneficial [24]. Based on this potential model, distinguishing the different stages is key to achieving an efficient treatment regimen.

Although progress has been made in some areas of autoimmune disorders, little is known about the underlying mechanisms of autoimmune antibodies on reproductive outcome, which represents a challenge for effective treatment research. Organs-on-a-chip, advanced in vitro models of multicellular tissue complexes or functional organ units, may help illuminate this intricate connection. Exploiting organ-on-a-chip approaches to model decidualization, implantation, and placentation would enable an in-depth study of the invasive and remodeling behavior of extravillous trophoblast cells, and of uteroplacental circulation that provides vascular supply to the growing fetus [57]. Furthermore, the interaction between antibodies and endometrium and variable expression of immunological factors, as well as glucocorticoid targets, all should be explored.

Our study has several advantages. Firstly, by establishing strict inclusion and exclusion criteria, the possible confounding factors of other autoantibodies and severe detrimental elements of spontaneous miscarriages were controlled for, which allowed us to minimize any patient-related variation and concentrate solely on P + A effects on isolated euthyroid infertile women with TAI. Secondly, only first-time ART users were included and analyzed to investigate a homogeneous, good-prognosis population and provide relevant suggestions for targeted subjects. However, our study also had some limitations. Firstly, it was inevitably limited by its retrospective nature. Secondly, given the variation in TPOAb and TH concentrations in the context of pregnancy, the measurement of longitudinal thyroid parameters during pregnancy was reasonable and necessary, but the changes were not recorded [58]. While the P + A supplement did not improve the live birth rate or pregnancy rate in euthyroid TAI women at the first embryo transfer, the potential benefits of P + A supplementation during pregnancy cannot be ruled out. Additional RCTs are required to determine whether P + A would yield different results on women who test positive for antithyroid antibodies with recurrent implantation failure or with unexplained infertility. Similarly, a higher-risk population with increased recurrent pregnancy loss might be affected differently.

To conclude, according to the reality of routine thyroid screening, many euthyroid women who test positive for antithyroid antibodies were discovered. Among them, patients who underwent the first IVF cycle without a history of recurrent miscarriages or unexplained infertility were not recommended for the combined treatment of prednisone and aspirin.