Singleton pregnancy losses before gestational week 22 among patients with autoimmune disorders and methylenetetrahydrofolate reductase polymorphisms

1. Introduction

Maternal health, genetic/epigenetic stability and regular implantation are essential to a balanced maternal-fetal interaction with no obstetrical complications [1, 2]. This necessitates orderly developed cellular structures of the intervillous space that make up the “maternal fetal interface” of the placenta [3, 4, 5, 6, 7]. Maternal risk factors such as autoimmune disorders and Methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms (reduced MTHFR activity) combined with injury of the cellular structures in the maternal fetal interface (placental inflammation) may cause obstetrical complications such as pregnancy loss (PL) before gestational week 22 [8, 9, 10, 11].

Miscarriage is a fetal loss before gestational week (gw) 20 (birthweight ⩽ 500 gr) [12, 13]. Our institution’s PL (miscarriage) rate before gestational week 22 is 10.7% and 73.8% of these occurred at gw ⩽ 10 [12]. Miscarriage rate per pregnancy may vary between 10% to 20%, with differences driven by etiological factors and the timing of their occurrences [13, 14]. Moreover, there is an association between etiological factors and the timing of PLs [15].

Chemical pregnancy (CP) is miscarriage that occurs before the fetus can be visibly detected on an ultrasound (gw 5) and is most likely associated with genetic/epigenetic reasons and/or implantation problems [16]. Blighted ovum (BO) is a pregnancy with a gestational sac but no fetus, probably because of various types of chromosomal abnormalities and/or gene disorders [17]. Biological mechanisms behind CP and BO are still unclear. Moreover, a wide spectrum of maternal risk factors (genetic, metabolic, immunologic, inflammatory, infectious, etc.) are defined for early fetal losses especially those at gw ⩽ 10 [12, 15]. Etiological reasons and maternal risk factors for fetal losses between gw11 and 22 differ relative to those responsible for earlier miscarriages [12, 14, 15]. Systemic diseases, mechanical problems related to genital organs (myoma uteri, Müllerian canal abnormalities, etc.) and epigenetic disorders are more frequently the cause of PL in these patients.

Knowing the clinical characteristics of PLs before gestational week 22 (miscarriage) in the presence of certain risk factors might be an advantage in the management of the patients. In this study, we review types of PL in terms of clinical appearances and temporizations among patients with autoimmune disorders and MTHFR gene polymorphisms.

This retrospective cohort (patients only with autoimmune disorders and/or MTHFR polymorphisms) was generated within the framework of a “miscarriage pre-conception counseling program” that took place from 2015–2019 at Hacettepe University. It consists of 1108 singleton pregnancies in 243 patients with poor obstetric history with at least one PL before 22 nd gestational week. Based on risk factors for PL, patients were categorized into 3 groups as follows: 1) 148 pregnancies in 33 patients with autoimmune disorders only (AD group = autoimmune diseases and autoimmune antibody positivity); 2) 316 pregnancies in 66 patients with MTHFR polymorphisms only (MTHFR group = reduced MTHFR activity); and, 3) 644 pregnancies in 144 patients with both autoimmune disorders and MTHFR polymorphisms (AD + MTHFR group). Autoimmune diseases (systemic lupus erythematosus, Sjogren’s disease, type-1 diabetes mellitus, Hashimoto’s disease, rheumatoid arthritis, Behcet’s disease) and autoimmune antibody positivities (anti-nuclear antibody, extractable nuclear antigen antibody, anti-parietal antibody, anti-smooth muscle antibody) were included to the AD group, while MTHFR polymorphisms, C677T and A1298C, were included to the MTHFR group.

In this study, PL is defined as fetal loss before gw22 (birthweight ⩽ 500 gr). We preferred PL instead of miscarriage (PL before gestational week 20), because we believe that miscarriage terminology does not explain/express all clinical situations clearly (for example CP, CP, BO, TD, PLs between gw20–22). Records are based on the self-definitions and descriptions from patients with variable backgrounds of medical knowledge. Medical care (including genetic evaluations) for previous PLs varied greatly among patients due to differences in facilities among related institutions. Furthermore, insufficient clinical information and the presence of non-uniform and ambiguous information related to the genetic conditions of previous PLs meant we could not focus on etiological factors such as chromosomal abnormalities, gene disorders, and congenital malformations in this retrospective study.

We also did not use/evaluate the variables such as vaginal bleeding, pain, vomiting, etc. due to non-uniformity of the available data. Termination of pregnancies (TOPs) due to the genetic disorders (gene disorders and chromosomal abnormalities) were accepted as PL, while TOPs due to unwanted pregnancies were not included to PL group. In this study, PLs were classified into the subgroups: a) Chemical Pregnancy (CP), b) Blighted Ovum (BO), c) gw ⩽ 10, d) gw11–14, e) gw15–22, f) Ectopic Pregnancy (EP), g) Trophoblastic Diseases (TD). Characteristics of this cohort let us to regrade the PL cases (other than CP, BO, EP, and TD) in term of gestational weeks which is important in our clinical practice. We also evaluated Pre-Prenatal Screening Period (PPSP) fetal losses (gw 11) which are defined as the summation of CPs, BO cases, gw ⩽ 10 fetal losses, EPs and TDs.

Demographic and clinical data were obtained from the electronic database of the Division of Perinatology, Hacettepe University. Obstetric histories of the patients were compared using Beksac Obstetrics Index (BOI): [number of living child + ( π /10)]/gravida [18]. PL numbers and BOI values of patients with ⩽ 4 and ⩾ 5 gravidities were compared based on the assumption that the threshold of complications such as PL increases markedly at gravidity ⩾ 5 (Institutional protocol).

Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS, version 23). Data were presented as number, percentage and mean ± standard deviation. Groups were compared using the Pearson Chi-square test or Fischer’s exact test. For numerical data (age, BOI and pregnancy loss numbers), normality of the data was checked with Shapiro-Wilks test. Normally-distributed data were compared with an ANOVA test, and non-normally distributed data were analyzed with non-parametric Mann Whitney-U and Kruskal Wallis-H tests. P -values 0.05 were considered as statistically significant. The study was approved by the Hacettepe University Ethics Committee with number GO 19/1064.

3. Results

Table 1 shows the demographic and clinical characteristics of the patients. AD, MTHFR and AD + MTHFR groups were similar in terms of age, gravida, and parity ( p values were 0.890, 0.586, and 0.05, respectively). There was no statistically significant difference between these groups in terms of number of PLs ( p = 0.781) (Table 1). PL rate before gw22 among singleton pregnancies with autoimmune disorders and/or MTHFR polymorphisms was 35.8% (Table 2).

PL rates were 39.2% (58/148), 33.2% (105/316), and 36.3% (234/644) in AD, MTHFR and AD + MTHFR groups, respectively ( p = 0.421) (Table 2). Correspondingly, the rate of PPSP fetal losses were 84.8%, 75.9%, and 77.8% in AD, MTHFR, and AD + MTHFR groups respectively ( p = 0.264) (Table 2). Number of PL of CP, BO, gw ⩽ 10, gw11–14, gw15–22, EP, and TD subgroups were shown in Table 3.

Mean ranks, maximum and minimum values, and median values were given in Table 4. No statistically significant difference was found between PL subgroups when mean rank of PL numbers were compared in terms of the presence of autoimmune disorders only, MTHFR polymorphisms only and autoimmune disorders plus MTHFR polymorphisms by non-parametric test ( $p>$ 0.05).

We investigated the distribution of patients in terms of BOI values and gravidity numbers for each study group (Table 1). Mean BOI values were 0.39 ± 0.024, 0.46 ± 0.21, and 0.41 ± 0.21 for AD, MTHFR and AD + MTHFR groups, respectively ( p = 0.203). Patients with gravidity ⩽ 4 versus those with gravidity ⩾ 5 had statistically significant differences in PL numbers and BOI values ( 0.001 for both) (Fig. 1). The clinical findings seem to be more complicated in patients with gravidity ⩾ 5.

OPEN IN VIEWER

Autoimmune disorders and MTHFR polymorphisms are known to be the risk factors for the injury of vascular structures of the placenta and the cellular components of intervillous space (placental inflammation) [8, 9]. Thus, they are associated with obstetric complications related to placental inflammation, including PL [2, 8, 9, 10]. MTHFR reduces inactive Methylenetetrahydrofolate to Methyltetrahydrofolate, the coenzyme of methionine synthase (together with vitamin B12) that converts homocysteine to methionine. This pathway is vital for the methylation of nucleotide/DNA, which is an important component of regular DNA synthesis [10, 19]. MTHFR polymorphisms have been associated with congenital abnormalities, chromosomal abnormalities, and PLs [9, 11, 19]. One-carbon metabolism and genome interaction are also affected by folate-associated pathologies. Threonine, part of one carbon metabolism, plays a role in the DNA methylation pathway, and a decrease in this metabolite may also be the cause of related pathophysiological events such as PL [9, 10]. Further, MTHFR related hyperhomocysteinemia is a risk factor for the endothelial injury of the vascular structures of the placenta and the cellular structures of the intervillous space where fetal perfusion takes place [2, 9].

Antibodies, cytokines, inflammatory interleukins, and degraded cellular structures present in autoimmune disorders all lead to placental inflammation and poor pregnancy outcome along with vascular/thrombotic problems [2, 20, 21, 23]. Furthermore, autoimmune disorders are thought to be associated with miscarriage [24]. In this study, we did not want to focus on searching for recurrent miscarriages, but preferred to concentrate on PL (miscarriage) rates per pregnancy due to the characteristics of the data. Autoimmune and metabolic disorders are risk factors not only for PL but also for other obstetrical complications, such as fetal growth restriction, preeclampsia, and premature contractions. This wide spectrum of potential complications is most likely generated depending on the severity of the immunologic or/and metabolic instability.

PL rate per pregnancy was reported to be 10% to 20% in different study populations [12, 13, 14]. We believe in the importance of preconception counseling in women who have experienced PL. Etiology based management is necessary to achieve better results [13, 14, 15, 16, 17]. It is estimated that 50%–60% of spontaneous abortions are fetuses with chromosomal abnormalities [25]. Most of the miscarriages are random and non-recurrent, caused by a segregation error in zygote formation or other epigenetic, mechanical, and inflammatory reasons [26]. In contrast, most chromosomal abnormalities appearing in recurrent abortions are due to abnormal chromosomal segregation during parental gametogenesis and parents carrying balanced translocations [27, 28, 29]. Unbalanced translocations account for approximately 1% of the spontaneous miscarriages [28, 30]. Trisomy of chromosomes 13, 18, 21, 22, and X chromosome monosomy are the other reasons of miscarriages [29, 30]. Thus, there are several etiological reasons considered for PLs, including genetic (chromosomal, structural, etc.), epigenetic, inflammatory, infectious, and mechanical factors [13, 14, 15, 16, 17].

Our institution’s PL rate before gw22 was 10.7%, and 73.8% of the cases were gw ⩽ 10 PLs [12]. In this study, our concern was to review the type of PLs in terms of their clinical appearances and temporizations among patients with autoimmune disorders and MTHFR polymorphisms. PL rate of patients with autoimmune disorders and/or MTHFR polymorphisms was 35.8% and 79.3% of cases were PPSP fetal losses. PL rates were 39.2% (58/148), 33.2% (105/316), and 36.3% (234/644) in AD, MTHFR and AD + MTHFR groups, respectively ( p = 0.421).

Grand gravidity and multiparity are risk factors for obstetrical complications and are associated with an increased prevalence of maternal and neonatal complications [31]. In this study, we evaluated the distribution of patients in terms of BOI values and gravidity numbers for each study group. BOI is used to evaluate the obstetrical histories of the study groups [8, 9, 18]. No statistically significant differences between study groups in number of PLs or in BOI values were observed. There are various screening methods for the prediction of various obstetrical complications but none of them were as simple as BOI which is using the number of living child and gravida [32, 33]. BOI can be successfully used in various types of maternal disorders and risk factors for statistical comparisons and the advantage of BOI is its usability at the beginning of the pregnancy and even during the course of preconception counseling [9, 23, 34]. Assuming that the threshold of complications such as PL increase markedly at gravidity ⩾ 5, we compared PL numbers and BOI values of patients with ⩽ 4 and ⩾ 5 gravidities and found statistically significant differences between patients with gravidity ⩽ 4 and gravidity ⩾ 5 in terms of PL numbers and BOI values ( 0.001 for both).

The main limitation of this study result from the characteristics of the retrospective data. In this study, recordings were based on the self-definitions and descriptions of the patients, who varied in medical knowledge. Medical care (including genetic evaluations) provided for each PL also varied greatly. Furthermore, insufficient clinical information and the presence of non-uniform and unclear knowledge related to the genetic conditions of the lost fetuses meant we could not consider etiological factors such as chromosomal abnormalities, gene disorders, and congenital abnormalities. Finally, variables such as vaginal bleeding, pain, vomiting, etc. were also discarded due to non-uniformity in the collected data.

In conclusion, PL rate of patients with autoimmune disorders and/or MTHFR polymorphisms was 35.8% and 79.3% of cases were PPSP fetal losses. Knowing PPSP fetal loss rate is an advantage in preconception counseling and in the management of early gestational problems (bleeding, pain, etc.). In addition, PLs were more likely to occur in patients with gravidity ⩾ 5.