The impact of the presence of antithyroid antibodies
on pregnancy outcome following intracytoplasmatic sperm injection‑ICSI and embryo transfer in women with normal thyreotropine levels
K. Łukaszuk1,2,3 · M. Kunicki3 · P. Kulwikowska1 · J. Liss2 · E. Pastuszek1,2 · M. Jaszczołt4 · B. Me˛czekalski5 · K. Skowron´ski6
Purpose The aim of our study was to investigate the impact of anti-thyroid peroxidase antibodies (Anti-TPO) on pregnancy outcome following the in vitro fertilization and embryo transfer (IVF-ET) in general groups and in sub-groups divided according to AMH level and age.
Methods A total of 114 patients positive for anti-thyroid peroxidase antibodies (Anti-TPO+ group) and 495 infer-tile women negative for anti-thyroid peroxidase antibodies (Anti-TPO− group) undergoing IVF with ICSI from April 2010 to April 2012 were analyzed retrospectively.
Results There were no significant differences in age, BMI, basal FSH, LH, AMH levels and duration of infer-tility between the two main groups. No significant differ-ences in terms of the days of ovarian stimulation, estradiol level in day 8, total gonadotropin dose, number of oocytes retrieved, available embryos and blastocysts, number of
1\ Department of Obstetrics and Gynecological Nursing, Faculty of Health Sciences, Medical University of Gdan´sk, Gdan´sk, Poland
2\ INVICTA Fertility and Reproductive Center, Gdan´sk, Poland
3\ INVICTA Fertility and Reproductive Center, Invicta Złota 6 Str, 00019 Warsaw, Poland
4\ Department of Chemical and Process Engineering Chemical Faculty, Gdansk University of Technology, Gdan´sk, Poland
5\ Department of Gynecological Endocrinology, Poznan
Univeristy of Medcial Sciences, Poznan, Poland
6\ Institute of Computer Science, Faculty of Mathematics, Physics and Informatics, University of Gdansk, Gdan´sk, Poland
embryos transferred nor in rates of fertilization, implanta-tion, clinical pregnancy, live birth and abortion rate between two main groups were found. The only statistically signifi-cant difference among the groups with different anti-TPO antibodies levels was found in basal FSH concentration and BMI. Among the clinical outcomes of IVF with respect to the different anti-TPO levels, the only significant difference was found for the number of oocytes retrieved. Analysis of the baseline parameters in relationship to age catego-ries and AMH levels found significant differences between women positive and negative for thyroid antibodies with respect to basal FSH and LH levels for women >37 years and for basal FSH in AMH <0.6 subgroup.
Conclusions The present study reveals that patients with anti-TPO antibodies showed no significant differences in fertilization, implantation, pregnancy rates, live birth rates and no higher risk for miscarriage following IVF-ET when compared with those negative for anti-thyroid antibodies.
Keywords Anti-thyroid antibodies · TSH · Intracytoplasmatic sperm iniection-ICSI · Miscarriage
Proper functioning of the thyroid is essential for normal growth, sexual development and reproductive functions. Thyroid dysfunction may have a huge effect on female fer-tility. Hypothyroidism and hyperthyroidism are associated with different changes in reproductive functions includ-ing menstrual disorders, anovulatory cycles, infertility and pregnancy wastage [1, 2]. Thyroid evaluation of the infertile couple might be helpful in many circumstances and treatment of thyroid dysfunction may rapidly improve the reproductive functions [3, 4]. Autoimmune thyroid
diseases are common disorders mostly occurring in women 30–50 years old . Worldwide about 2–4 % of women suffer from autoimmune thyroid disorders and this fre-quency increases with age . The immunopathologic pro-cess may be triggered or perpetuated by autoantibodies and immune complexes containing autoantigens and autoreac-tive T lymphocytes . Autoimmunity is associated with many disorders . For example, the higher prevalence of anti-TPO antibodies has been found in women who affected by the premature ovarian failure . However, the presence of anti-thyroid antibodies (ATA) is also frequently encountered in general population (about 10 % of adults), with an increasing incidence (up to 30 %) in older adults [10, 11].
Autoimmune abnormalities have been investigated for possible associations with reproductive failures . The miscarriage rate and increased incidence of infertility are associated with higher ATA levels . Various studies pub-lished to date describe the relationship between autoimmune thyroid disease and success of assisted reproductive technol-ogy (ART), especially in vitro fertilization (IVF) and intracy-toplasmatic sperm injection (ICSI). However, the role of ATA is still unclear. Some researchers speculated that women pos-itive for these antibodies had poor outcome of in vitro ferti-lization [14–16]. Other studies did not confirm such correla-tion [17, 18]. It is speculated that poor outcomes in euthyroid women undergoing IVF could be caused by ATA’s effect on zona pellucida. This could result in not only lower fertiliza-tion rates but also decreased quality of embryos. This detri-mental effect could be avoided using ICSI .
Thus, the aim of our study was to investigate the impact of the presence of anti-TPO antibodies on pregnancy out-come following ART cycles with ICSI and fresh embryo transfer. We analyzed the outcomes for groups based on ATA status and subgroups divided according to AMH level and age.
Materials and methods
We retrospectively investigated 665 consecutive infertility patients undergoing their first ICSI cycles at the INVICTA Fertility Clinic from April 1, 2010 until April 30, 2012. The study protocol was approved by the Institutional Review Board of Medical University of Gdan´sk, Poland (KB-9/13), see Additional file 1. All data were de-identified and ana-lyzed anonymously.
All patients included in the study were euthyroid and had TSH level between 0.5 and 2.5 IU/mL as measured during the month preceding the ICSI cycle [20–22]. The median TSH level was 1.47 IU/mL.
Study participants were divided into two main groups:
Anti-TPO + group consisting of 114 women positive for
TPO-Ab (TPO-Ab >34 IU/mL) and Anti-TPO- group with 551 women negative for TPO-Ab (TPO-Ab <34 IU/mL).
None of the women used levothyroxine hormones, both before and during stimulation. We also excluded women with autoimmune diseases, lupus anticoagulant (LAC) or rheumatoid factor (RF).
The descriptive data, including age, body mass index (BMI), basal serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and Anti-Müllerian hor-mone (AMH), were compared between groups (Table 1). Additionally, number of days of gonadotropin (Gn) treat-ment, total gonadotropin dose, number of oocytes retrieved, fertilization rate, number of available embryos, number of embryos available for transfer, clinical pregnancy, implan-tation and spontaneous abortion rates were also recorded and compared between the two groups (Table 2).
Additionally, we arbitrarily divided the Anti-TPO+ group into subgroups according to the level of the antibodies: 35–100, 101–300, 301–900 and >901. We com-pared all baseline patient characteristics and clinical out-come measures among these subgroups (Tables 3, 4).
Baseline patient characteristics and basal hormone lev-els in Anti-TPO+ and Anti-TPO− groups, each divided into subgroups according to age and AMH levels, are shown in Table 5. For age break down, we applied ranges of <31 years, 31–37 years, and >37 years according to La Marca . AMH concentrations were separated into three categories. They were based on the lower (with the value of 0.6 ng/mL referring to the 25th percentile) and upper quar-tiles of our first kit’s validation results of this method. The value 1.4 reflected the manufacturer’s value. We typically present AMH ranges showing the values rather than per-centiles as this is more understandable for patients.
Serum anti-TPO was detected using the analyzer Modu-lar Analytics E170 (Elecsys module Roche Diagnos-tics Poland) by electrochemiluminescent immunoassay (ECLIA). The reference values of antibody concentrations were as follows: anti-TPO 0-34 IU/mL and sensitivity of <5.0 IU/mL.
Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH) were measured by electrochemiluminescent immu-nometric assay using commercial kits (Cobas 6000, e601, Roche Diagnostics Poland). Fasting venous blood samples were collected, between 8:00 am and noon on days 1 and 3 of the menstrual cycle, prior to initiating stimulation. The intra- and inter-assay coefficients of variation (CV) were: 2.5 and 2.3 % for FSH, 2.2 and 2.4 % for LH, and <2.9 % for TSH. Serum AMH concentrations were measured by enzyme-linked immunosorbent assay ELISA (Diagnostic
Table 1 Baseline characteristics of the patients and laboratory outcome measures
Table 2 Clinical outcome measures
|Variables||Anti-TPO+ group (n = 114)||Anti-TPO− group (n = 495)||p valuea|
|Age (years)||35 (33–38)||34 (32–37)||0.12|
|BMI (kg/m2)||22.31 (20.26–23.44)||21.78 (19.72–23.49)||0.25|
|FSH (IU/L)||7.15 (5.3–9.2)||7.6 (6.1–9.34)||0.16|
|LH (IU/L)||5.10 (4.5–7.1)||5.91 (4–7.02)||0.48|
|AMH (ng/mL)||2.9 (1.4–4.9)||2.6 (1.4–5.1)||0.95|
|Duration of infertility||5 (4–8)||5 (4–7)||0.06|
|Cause of infertilityb|
|Anovulation||2.63 %||1.7 %||0.73|
|Tubal factor||11.63 %||9.62 %||0.71|
|Male infertility||23.26 %||26.20 %||0.65|
|Endometriosis||7.02 %||6.51 %||0.98|
|Unexplained||36.86 %||40.64 %||0.52|
|Others||18.60 %||15.53 %||0.83|
Data are presented as median (25th–75th)
BMI body mass index, FSH follicle-stimulating hormone, LH luteinizing hormone, AMH anti-Müllerian hormone
p < 0.01
a Mann–Whitney U test
b Chi-square test
Variables Anti-TPO+ group Anti-TPO− group p valuea
|Stimulation length (days)||9 (7–10)||9 (8–10)||0.06|
|E2 in 8ds||646 (294.5–1681)||731 (210–1695)||0.56|
|Total Gn dose (75 IU-amp.)||20 (17–25)||22 (18–27)||0.44|
|Number of retrieved oocytes||11 (1–13)||12 (3–14)||0.33|
|Fertilization rate||65.1 % (512/786)||60.5 % (2259/3613)||0.18|
|Number of available embryos||4 (2–7)||5 (3–8)||0.31|
|Number of blastocysts available for transfer||2 (1–2)||2 (1–3)||0.37|
|Number of blastocysts transferred||2 (1–2)||2 (1–2)||0.28|
|Clinical pregnancy rate||43.8 % (50/114)||47.5 % (235/495)||0.55|
|Live birth rate||30.4 % (35/114)||34.1 % (169/495)||0.55|
|Implantation rate||25.11 (55/219)||25.28 % (241/953)||0.97|
|Spontaneous abortion rate||6 % (3/50)||12.4 % (29/235)||0.29|
Data are presented as median (25th–75th)
a Mann–Whitney U test
Table 3 Characteristics and basal hormone levels in four subgroups according to the level of anti-TPO antibodies
|Level of anti-TPO antibodies||35–100 (n = 40)||101–300 (n = 23)||301–900 (n = 24)||>901 (n = 28)||p valuea|
|Age (years)||36 (31–38)||34 (33–36)||34 (33–35)||35 (31–42)||0.30|
|BMI (kg/m2)||22.43 (19.94–23.88)||25.31 (22.68–28.27)||20.72 (19.38–21.80)||22.45 (20.96–22.32)||0.04|
|AMH (ng/mL)||3.1||(1.4–3.8)||2.3 (1.6–4.1)||3.4 (1.9–4.9)||3.4||± (0.6–6.2)||0.65|
|FSH (IU/L)||9.2||(5.45–10.9)||7.64 (5.75–7.7)||6.03||(6.03–7)||5.2||(5.1–6.29)||0.003|
|LH (IU/L)||5.1||(4.1–6.84)||5.75 (4.5–9.65)||5.45||(4.05–12.2)||4.5||(4.5–5.1)||0.24|
Data are presented as median (25th–75th)
n number of patients
a The Kruskal–Wallis one-way analysis of variance
|\||J Endocrinol Invest|
|Table 4 Comparison and IVF outcomes between subgroups according to anti-TPO levels|
|Level of anti-TPO antibodies||35–100 (n = 40)||101–300 (n = 23)||301–900 (n = 24)||>901 (n = 27)||p valuea|
|Stimulation length (days)||9||(7–9.25)||9.5 (8–11)||9 (8–9)||8.5 (7–10)||0.32|
|Total Gn dose (75 IU-amp.)||20 (15.75–23)||30 (23–40)||22 (18.5–27)||18 (14–20)||0.37|
|E2 in 8ds||531 (300–1696)||895 (299.25–1691)||999 (318–1765.5)||647 (310–1721)||0.33|
|Number of retrieved oocytes||9||(1.75–12)||8||(1–10.5)||12 (2–14)||12 (1–14)||0.04|
|Number of available embryos||4||(1–5)||3||(1–4)||5 (1–7)||4 (1–5)||0.17|
|Number of blastocysts available for transfer||2||(1–3)||2||(1–3)||3 (2–4)||2 (1–3)||0.18|
|Number of blastocysts transferred||2||(1–2)||2||(2–2)||2 (1–2)||2 (1–2)||0.87|
|Clinical pregnancy rate||50 % (20/40)||47.83 % (11/23)||29.17 % (7/24)||44.44 % (12/27)||0.57|
|Live birth rate||37.5 % (15/40)||34.8 % (8/23)||16.7 % (4/24)||29.6 % (8/27)||0.52|
|Spontaneous abortion rate||15 % (3/20)||0 % (0/11)||0 % (0/7)||0 % (0/12)||0.12|
|Implantation rate||30.3 % (23/76)||25 % (11/44)||17.39 % (8/46)||24.52 % (13/53)||0.59|
n Number of patients
a The Kruskal–Wallis one-way analysis of variance
Systems Laboratories, Webster, TX, USA). The assay limit of detection was 0.06 ng/mL for AMH. The intra- and inter-assay coefficients of variation for AMH were less than 10 %.
Reference intervals in follicular phase were as follows
Serum: FSH: 2.4–9.3 U/L (for follicular phase), LH 2.0-12.0 U/L (for follicular phase), TSH 0.5-4.5 IU/mL.
All women underwent a long protocol of pituitary sup-pression with the GnRH agonist diphereline at a dose of 0.1 mg/day (Pharmacia Upjohn, Kalamazoo, MI, USA). GnRH agonist was administered from day 14–16 of the oral contraception cycle (Ovulastan Polfa Poland). Controlled ovarian hyperstimulation was initiated 14 days later with the administration of urinary gonadotropins (Menopur, Fer-ring, Pharmaceuticals Germany). The initial FSH dose was based on the woman’s age and AMH level .
Follicular growth was monitored using a day 8 ultra-sonographic scan and a serum E2 assay. Ovulation was induced by administration of 5000 IU hCG (Pregnyl, Organon, Oss, the Netherlands) when at least two lead-ing follicles had reached a diameter of 17 mm. The oocyte retrieval procedure was performed 36 h after hCG administration.
Embryo transfer, in all cases, was performed using a soft catheter at the blastocyst stage on day 5 of embryo cul-ture. The number of embryos transferred was determined by the number and quality of the available embryos and by the guidelines of the institution and ASRM (Practice
Committee. Society for Assisted Reproductive Medicine and the American Society for Reproductive Medicine 2004) .
The luteal phase was supported by natural micronized P (Luteina, Adamed, Czosnów, Pl), administered vaginally in three daily doses of 200 mg/day, beginning on the day of oocyte retrieval. The women received daily supplementa-tion with oral 6 mg doses of micronized 17 beta-E2 (Estro-fem, Novo Nordisk, Denmark) during the entire luteal phase. A serum chorionic pregnancy test was performed 14 days after oocyte retrieval.
Clinical pregnancy was defined as the presence of an intrauterine gestational sac as visualized by transvaginal ultrasonography 5–6 weeks after embryo transfer. Implan-tation rate was calculated as the number of sacs with fetal heart beat divided by the total number of embryos transferred. Spontaneous abortion rate was calculated as number of fetal demises per total number of clinical pregnancies.
Statistical analysis was prepared using Statistica 10 (Tulsa OK, USA) software. First, a Shapiro–Wilk test was applied to all groups and variables to evaluate the adjustment to a normal or symmetrical distribution. As our data were non-symmetrically distributed, the median and 25th and 75th percentiles were calculated. Comparisons of quantitative data were performed with the Mann–Whitney U or Kruskal– Wallis test when appropriate. To examine the compatibility of measurable characteristics, Chi-square test was used.
The significance level (α) was defined as 0.01. A value of p < 0.01 was considered statistically significant.
Table 5 The BMI and basal
hormone levels in Anti-
TPO+ and Anti-TPO− group in
age and AMH level subgroups
|Variables||Anti-TPO+ group||Anti-TPO− group||p valuea|
|Age sub groups|
|BMI (kg/mL)||24 (19.26–21.63)||20.78 (18.78–25.00)||0.70|
|FSH (IU/L)||8.1||(7.4–9.2)||6.92 (5.25–8.1)||0.12|
|AMH (ng/mL)||3.35 (2.4–6.2)||3.5||(2.27–6.05)||0.81|
|BMI (kg/m2)||21.88 (20.20–26.22)||21.43 (19.72–23.23)||0.84|
|LH (IU/L)||5.7||(4.32–9.1)||6.01 (4.2–7.1)||0.82|
|AMH (ng/mL)||3.05 (1.85–4.9)||3.4||(1.8–5.6)||0.02|
|BMI (kg/m2)||22.32 (21.63–23.88)||22.79 (20.52–24.29)||0.17|
|LH (IU/L)||4.5||(4.2–5.1)||5.17 (3.2–6.6)||0.03|
|AMH level sub groups|
|Age (years)||36 (33–38)||39 (34–46)||0.71|
|BMI (kg/m2)||22.84 (21.45–23.01)||23.79 (22.11–23.8)||0.72|
|FSH (IU/L)||5.70 (5.37–6.0)||15.4(7.3–22.3)||0.01|
|LH (IU/L)||8 (6.98–9.43)||5.2||(4.6–10)||0.87|
|Age (year)||38 (33.5–42)||37 (34–39)||0.25|
|BMI (kg/m2)||22.41 (22.32–23.44)||21.28 (19.26–22.86)||0.13|
|Age (years)||34 (32–42)||32 (29–34)||0.06|
|BMI (kg/m2)||21.30 (20.32–23.87)||21.27 (19.83–23.88)||0.97|
|FSH (IU/L)||6.5||(5.1–10.9)||7.03 (5.5–18.5)||0.28|
|LH (IU/L)||5.35 (4.3–7.1)||5.9||(3.87–7.2)||0.13|
Data are presented as median (25th–75th)
Data are presented as median (min–max)
a Mann–Whitney U test
There were no significant differences in age, BMI, basal FSH, LH, AMH levels and duration of infertility between the two main groups (Table 1). Additionally, no signifi-cant differences were found between the two main groups in terms of the days of ovarian stimulation, estradiol level on day 8, total gonadotropin dose, number of oocytes retrieved, available embryos, number of embryos trans-ferred nor in rates of fertilization, implantation, clinical pregnancy and spontaneous abortion rate (Table 2).
Analysis of the subgroups established based on the ATA levels showed that the only baseline characteristics with
statistically significant differences were the basal FSH con-centration and BMI (Table 3). As for clinical outcomes of ICSI for the same subgroups, the only significant difference was found with respect to the number of oocytes retrieved (Table 4).
The BMI and basal hormone levels were analyzed for the subgroups based on age and AMH levels (Table 5). We found significant differences between women positive and negative for thyroid antibodies with respect to basal FSH and LH levels for women >37 years and for basal FSH in AMH < 0.6 subgroup. Figure 1 presents the clinical out-comes in Anti-TPO+ and Anti-TPO- group in relation to the age and AMH level subgroups.
Fig. 1 The clinical outcomes in Anti-TPO+ and Anti-TPO− group in relation to the age and AMH level subgroups
Figure 2 shows the distribution of antibodies in age sub-groups and in AMH level subgroups.
Statistical analysis showed no association between the anti-TPO antibodies levels and the woman’s age or AMH level (p = 0,0830, p = 0,5336, respectively).
In the present study, we did not observe any negative effect of the presence of anti-TPO antibodies in women under-going ICSI with embryo transfer cycles. Our study group included only euthyroid women with TSH level between 0.5 and 2.5 IU/mL. The cutoff points were chosen accord-ing to data showing that the vast majority of healthy indi-viduals have the TSH levels below 2.51mIU/mL . Our threshold is also in accordance with the study presented by Busnelli et al. and the recent study presented by Weghofer et al. [27, 28]. The latter paper described establishment of low and high normal TSH levels. Additionally, it is worth noting that there is an ongoing debate regarding the need to use pregnancy cutoffs for TSH to diagnose subclinical hypothyroidism (SCH) [9, 29].
The women did not take levotiroxine during the study. According to recent meta-analysis by Velkeniers et al., such
supplementation could improve clinical pregnancy out-come in women with subclinical hypothyroidism and/or thyroid autoimmunity undergoing ART .
In all cases, we used our standard long ovulation induc-tion protocol together with ICSI for fertilization . The significance level of alpha defined as 0.01 was chosen due to the large number of analyzed variables.
In women undergoing IVF-ET, the relatively high inci-dence of autoantibodies in serum may be attributed to the poor IVF outcome [7, 16, 31, 32]. Our study compared the results of ICSI in Anti-TPO positive and Anti-TPO negative women. Data presented in the literature have consistently reported a reduction in implantation rate and an increase in early miscarriage rate in women with thyroid autoim-munity in comparison to negative controls [19, 33–35]. Stagnaro-Green et al.  were the first to describe the association between anti-thyroid antibodies and pregnancy loss. Shortly thereafter, Glinoer et al.  reported a spe-cific association between anti-thyroid autoantibodies and miscarriages. Also Zhong et al. reported that fertilization, implantation and pregnancy rates are lower in anti-TPO positive group than in anti-TPO negative group . This was consistent with data reported by Kim et al.  where anti-TPO-positive infertile women had a lower pregnancy rate than negative controls (26.3 vs 39.3 %).
Fig. 2 The distribution of anti-bodies in age sub groups and in AMH level sub groups
Our results showed a lack of any statistically sig-nificant differences in fertilization rate, number of avail-able embryos, implantation and clinical pregnancy rates between the two main groups and among subgroups divided according to age or AMH level. The discrepancy between our results and Zhong’s and Glinoer’s results can be more easily explained by referring to immunologic mechanism of action of these antibodies. Even though this mechanism has not yet been fully described, it is speculated that anti-TPO antibodies may bind to either the surface of the egg and/or embryo and interfere with fertilization and subse-quent embryo development [19, 33]. Our seemingly con-tradictory results seem to confirm this hypothesis. While binding of anti-TPO antibodies to the surface of the egg can impede sperm’s access during natural fertilization (and classical IVF), it should not affect the quality of fertiliza-tion using ICSI which was used for all patients in our study. Our results are consistent with the results from Muller et al.  and Tan et al.  studies.
The increased risk of miscarriages or recurrent miscar-riages in ATA-positive euthyroid women had been dem-onstrated in literature. The previous study of Kutteh et al. showed that 22.5 % of women miscarried were positive for ATA . The same conclusions were made by Zhong Y
et al. who showed that the percentage of spontaneous abor-tions in a group of women positive for anti-TPO was higher than in the control group (respectively, 26.9 and 11.8 % p = 0.002) . A recent meta-analysis by Toulis et al.  also found the presence of ATA to be associated with an increased risk of miscarriage in subfertile women who conceived through IVF. However, in another meta-analysis, miscarriage rate was not found to be higher in IVF preg-nancies achieved in ATA-positive women compared with ATA-negative women .
In the present study, there were no statistically signifi-cant differences in miscarriage rates between two main groups and among the subgroups.
In this study, no correlation was found between the pres-ence of anti-TPO and spontaneous abortion. Our findings are consistent with those obtained by Karacan et al.  where implantation rates, miscarriage rates and ongoing pregnancy rates did not differ significantly between the ATA-positive group and the ATA-negative group. Addition-ally, the study presented by Mintziori et al. found no evi-dence that increased TSH concentrations or the presence of TAI determined before IVF affect the live birth rate in euthyroid women. That study included women with TSH level of 0.5–4.5 IU/mL .
In our opinion, the presence of antibodies against the thyroid gland should be treated as a secondary biomarker of autoimmune disease, but not as a real cause of miscar-riage .
The strength of our study is a large sample size. We also presented data with regard to the Anti-TPO a-TPO and AMH which, although not novel, increase the value of our data. These results are in accordance with the data presented by Polyzos et al. who showed no association between thyroid autoimmunity—TAI and AMH levels . Moreover, we presented the relationships between differ-ent antibody levels and clinical outcome measures. The limitation is that we did not measure thyroglobulin anti-bodies (Tg-Ab). According to some data, the presence of these antibodies could be as high as 5 % in women with infertility . Another issue that could be raised as a limi-tation is that we did not include women with TSH levels above 2.5 IU/mL but as we stated on the beginning we only included women with low to normal TSH levels. It is worth to point out that some studies show conflicting results with regard to clinical outcome both in women below 2.5 as above that threshold [28, 44]. Further studies should be car-ried out in that field.
Acknowledgments The authors wish to thank the laboratory staff of Invicta Fertility Centre for the technical support in blood collecting and hormonal analyses. We wish to thank Mrs. A. Knight for native-speaker language editing.
Compliance with ethical standard
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval All procedures performed in the studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards
Informed consent Informed consent was obtained from all individ-ual participants included into the study.
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