Unexplained female infertility (UFI) is a devastating obstetrical condition that affects females who are unable to conceive, without any definitive causes found despite extensive investigations and interventions 12. The approach to UFI is continuously being updated, as the latest evidence describes different potential etiologies with clinical links, including immunological factors 2.

Immunological responses of the uterine mucosa to developing embryos are well regulated, and a successful pregnancy requires proper immune system adaptation for the fetus and placenta 32. Approximately 20% of couples of reproductive age are affected by immune infertility, making it a significant health concern 3.

Immunoglobulins are vital for any immunologic evaluation to reflect the function of humoral immunity 4.A few studies have shown that a successful pregnancy is associated with increased total IgG production in the first trimester, followed by decreased total immunoglobulin concentrations in the second and third trimesters, which results from the immunomodulation of a healthy pregnancy 56.

The complement system consists of a series of proteolytic enzymes and regulatory proteins that play a positive role in various pregnancy stages, such as implantation, fetal development, and labor 7. However, an imbalance in the complement system has been detected in pregnancy complications, and this can induce unfavorable effects on both the pregnant mother and her fetus 87.

Although most international reproductive and obstetric societies agree that successful conception is influenced by a healthy immune system, routine immunological investigations to explore female infertility is not recommended 9.Nevertheless, several societies recommend some immunological testing (mainly autoantibodies) for patients with recurrent pregnancy loss 10. Recently, some societies have suggested some immunological testing for recurrent implantation failure, but with limited evidence or for clinical research purposes 1112. To date, there are no recommendations about testing for total immunoglobulins and complements in most reproductive societies in UFI.

However, in the face of underestimated abnormalities, general immunological laboratory investigations are seldom conducted in infertility centers in the increasing number of cases with UFI. Therefore, this research was conducted to search the prevalence of any possible abnormalities in the levels of total immunoglobulins and complements as biomarkers in patients with UFI in the Kingdom of Saudi Arabia.

This project was a retreospective cross-sectional clinical study. It was carried out on patients with UFI who attended the clinical immunology clinic at the King Abdulaziz University Hospital (KAUH) over a period of four months, from May to August 2022. The KAUH is a tertiary referral center and a large teaching center with 800 beds, located in Jeddah city in the Western zone of the Kingdom of Saudi Arabia.

This study was authorized by the Unit of the Biomedical Ethics Research Committee at KAUH, with a reference number of 331-22. Participants were enlightened about the purposes and procedures of the study, and participation was voluntary and without any offered incentives. A verbal consent was acquired from all participants before any collection of research data.

The inclusion criteria was specified for all participating females with unexplained infertility aged 18 to 50 years old who were consulted by different infertility specialists to identify any potential immunological etiologies. Other possible common causes of infertility (anatomical, genetic, and male partner factors) were excluded. Based on the World Health Organization (WHO), primary infertility occurs when a woman has never achieved a pregnancy, and secondary infertility is when at least one prior pregnancy has been achieved 13.

The criteria for exclusion were any females complainig of infertility with known common etiologies other than disturbed immunological tests, those with deficient immunoglobulins or complements laboratory results, and those who missed follow-ups.

The patients’ demographic, clinical, and laboratory information were recorded from the electronic files of medical records. Data recording was performed with Google spreadsheets for documenting the patients’ demographic data and clinical details, which included type of infertility, pregnancies number, living children, preterm labors, abortions, stillbirths, and assisted reproductive techniques, including in-vitro fertilizations.

Thereafter, the laboratory results of five basic immunological tests on the serum of the included patients that was taken before attempting pregnancy were collected. These were total immunoglobulin M (IgM), immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin E (IgE), complement 3 (C3), and complement 4 (C4). The results of the immunological tests were obtained from the immunology laboratory at the laboratories of KAUH.

A descriptive statistical analysis was performed for the included cases. Frequency number and percentage were extrapolated for categorical factors. The means with standard deviation were computed for the continuous variables. Then, the associations between the different collected variables were measured by the chi-square test. All p-values of < 0.05 were accounted as statistically significant. The software of Statistical Package for Social Sciences (SPSS) version 23 (Armonk, NY: IBM Corporation, USA) was utilized for all data evaluations

This trial was approved by the Research Committee of the Unit of Biomedical Ethics at KAUH with a reference number of 331-22. All participants were educated about the aims and methods of the project. Participation process was voluntary andwithout any offered incentives. From each participant a verbal consent was obtained before any data collection.

A total of 136 cases with UFI, referred from different specialists in infertility across Saudi Arabia, were enrolled from the clinical immunology clinic at KAUH. Of these, 15 cases were excluded, nine because of a loss of revisit, and 6 cases for incomplete laboratory data results. In total, 121 female cases fulfilled the inclusion criteria and provided consent for this study. The ages of participants ranged from 18 to 49 (mean age of 33.9 ± SD 5.6) years old.

The nationalities data of the studied patients were; 103 cases (85.1%) Saudi citizens, and 18 cases (14.9%) non-Saudi residents. Regarding the city of cases’s residence, 70 (57.9%) from Jeddah, 10 (8.2%) from Makkah, 10 (8.2%) from Taif, and 31 (25.6%) from other cities of Saudi Arabia (Table 1).

The background data of infertility showed that secondary infertility was predominant and had been diagnosed in 99 cases (81.8%). There were 74 cases (61.2%) who had no living children, and 47 (38.8%) who had at least one living child. At least one abortion was a prominent feature in 87 cases (71.9%), while 34 cases (28.1%) had no abortions. Regarding IVF procedures, 67 cases (55.4%) had received at least one intracytoplasmic semen injection (ICSI), and 23 cases (19%) had received at least one intrauterine insemination (IUI) (Table 1).

At least one abnormal level of any of the five biomarkers of total immunoglobulins or complements was found in 65 cases, equivalent to 55.1% of the study group. High immunoglobulin levels (hypergammaglobulinemia) were the commonest abnormal immunological marker, including high IgG in 26 patients (22%), followed by high IgA in 14 patients (11.9%), and high IgM in 11 patients (9.3%) (Table 2). The next most common immunological abnormality marker was elevated levels of complements (hypercomplementemia) in 10 cases (9.4%), mainly high C4 in nine of these 10 cases (8.5%) (Table 2). However, abnormally low levels of immunological markers were rare in the studied group, including low C4 in two cases (1.7%), low IgG in one case (0.8%) and low IgM in one case (0.8%).

A statistically significant association was detected between the high C4 group and some parameters of infertility, including primary infertility (p = 0.005), no pregnancy (p = 0.001) and no abortion ( p = 0.047), more so than in the normal group ( Table 3 and Table 4 ) . Moreover, the high IgA group was nearly significantly more associated with a history of at least one abortion than the normal group (p = 0.054) ( Table 4 ).

In a subgroup analysis based on the age of the patients (if less than or equal to 35 years old versus older than 35 years old), there were some statistically significant associations. The high IgG group was significantly more associated with a history of no abortion than the normal group (p = 0.026). Moreover, the high C4 group was significantly associated with primary infertility (p = 0.006), no pregnancy ( p = 0.006), and no abortion (p = 0.031) more than the normal group. In addition, the high IgG group was nearly significantly associated with primary infertility (p = 0.054) and no pregnancy (p = 0.056), more so than the normal group.

UFI, which is mainly associated with repeated abortions or implantation failures, represents an extremely challenging and distressing topic in the field of reproductive medicine. Moreover it places a significant financial and psychological burden on the involoved couples. Recent publications have advocated that an overactive immune system, such as an autoimmune disorder in some women, may expand the struggling of falling pregnant or recurrent abortions risk 14. This advocates a potential greater chance of success through the evaluation of the immune system and applying individualized immune based treatments.

In this study, five different basic immunological laboratory biomarkers were explored in females with UFI. Interestingly, over half of the studied group had at least one abnormal test result for any of the five biomarkers of immunoglobulins or complements. A recent study measured the same five biomarkers, but during the first trimester 15.Up to our knowledge, this study is the first published research that evaluated these five biomarkers of immunoglobulin and complements together before pregnancy.

In this study, the most predominant immunological abnormality was the increased levels of immunoglo bulins (hypergammaglobulinemia), mainly IgG, followed by IgA and IgM, in nearly half of the studied group. Hypergammaglobulinemia is seen in some infections, inflammatory diseases, autoimmune conditions, and plasma cell disorders 416. The impacts of hypergammaglobulinemia on infertility, IVF success, and pregnancy are not yet clearly defined, but if these immunoglobulins coexist with autoantibodies, they may impair fertility 1417.

This study revealed a potential association between high IgG and a history of (primary infertility, no pregnancy and no abortion in younger age groups) and a near association between high IgA and a history of abortion. Preconception hypergammaglobulinemia was suggested as a risk factor for low pregnancy rates with IVF 18. Contrary to another study, there was no relationship found between preconception immunoglobulins and recurrent abortions 19.

As expected, in this study, low levels of immunoglobulins (hypogammaglobulinemia) were found to be rare, with only one case of low IgG and one case of low IgM. Hypogammaglobulinemia is an uncommon clinical finding associated with some rare immunodeficiency disorders 416. Reduced levels of IgG in the first trimester have been linked to recurrent abortions 20.

The second most predominant immunological abnormality found was high levels of complements, mainly high C4, in 9.4% of the studied group. Hypercomplementemia is seen in many inflammatory disorders as acute phase reactants 416. Interestingly, the group studied in this investigation showed a relationship between preconception high C4 and a history of primary infertility, no pregnancy, and no abortion. There are few studies that have linked preconception hypercomplementemia and recurrent abortions and suggest that it may predict subsequent abortion 1921.

In this studied group, hypocomplementemia was rare; low C4 was only detected in two cases (1.7%), while no participant had low C3, which is less than what has been reported in the literature. There are many studies that document preconception hypocomplementemia, more with C4 than C3, with recurrent abortions at somewhat higher rates (6–10%) and more if there are associated autoantibodies22232425. Hypocomplementemia is seen in immune complex diseases, which indicate consumption and disease activity or, rarely, a genetic deficiency416.

This research project had a few limitations, such as the use of convenient sampling from a specific clinical immunology clinic, a few deficient patient data points, and a small sample size of cases. Hence, interpreting these immunological investigations in cases with UFI requires further large-scale, highly standard-controlled research projects in the future.

The detection of an abnormality in any of the general immunological investigations may help in the establishment of a guideline as to when and in which backgrounds of infertility to order and consider these immunological biomarkers. This might shift the perspective of experts in the field of infertility to establishing a proper clinical link between the immune system and the potential causes of UFI.

In conclusion, this study focused on the prevalence of five general immune biomarkers in a convenient sample of patients with UFI. Abnormal levels of at least one immunoglobulin or complement were a common finding in more than half of these patients. Among these, high immunoglobulins (IgG, IgA, IgM) and high C4 were the predominant immunological abnormalities. A potential relationship between high IgG, IgA, and C4 and lower pregnancy rates was noted. Identifying abnormal general immune responses of the mother to her fetus may advance the clinical investigational approach of UFI. Further large and randomized controlled trials for a promising clinical application of these general immunological evaluations in UFI are necessary.

The authors would like to express their appreciation to all infertility experts who referred patients to the allergy and immunology clinic at KAUH. Moreover, we want to express our gratitude for the support from Ekthar Medical Clinics for their cooperation in recruiting some patients and providing some of the missing and required additional laboratory investigations that considered the essence of this study.

Immune system aberrations can interfere with normal embryo implantation and may lead to infertility. The authors illustrate that cases of unexplained female infertility (UFI) may have associated abnormalities in the total levels of immunoglobulins and/or complements. Additional management steps are necessary to address these abnormalities and their potential comorbidities.

Healthy maternal immune system homeostasis is crucial for success conception and the delivery of normal fetuses.

Several obstetrical guidelines are somewhat uncertain about the evidence for screening general immunological tests in cases of UFI.

Total immunoglobulins and complements abnormalities may be detected in some cases of UFI.