What is reproductive immunology?

Reproductive immunology is an area of reproductive medicine that focuses on the interactions between the immune system and reproductive system. Reproductive immunologists study the role of immune cells and immune molecules at different stages of reproduction: menstrual cycle, fertilization, embryo, implantation, early and late pregnancy, and labor and delivery.ⁱ

Why is immune function relevant to reproductive function?

The main function of the immune system is to protect the health of the host by distinguishing between "self" and "non-self" (or foreign) molecules within the human body. "Non-self" entities include bacteria, viruses, and other organisms.ⁱⁱ

After conception, 50 percent of genes within an embryo and its cells are from the egg and 50 percent are from the sperm. During pregnancy, some immune activity will be suppressed so that the embryo will not be recognized as "foreign" by the pregnant person's body (given that the embryo contains genetic material different from the pregnant person). This process is called maternal-fetal immune tolerance.ⁱⁱⁱ

This paradoxical immune response to prevent rejection of the semi-foreign embryo (and, prior to fertilization, the sperm) is regulated by a variety of factors, including changing levels of estrogen and progesterone throughout the menstrual cycle. These two important sex hormones influence the presence of immune cells (e.g., white blood cells), and the secretion of immune molecules (cytokines and chemokines) to create a balanced immune environment in the uterus.^iv,v

The process of embryo implantation itself involves an active immune response, including local inflammation.^vi During the window of implantation, which typically falls on days 19 to 23 of the natural menstrual cycle and lasts about 48 hours, several different white blood cells, including T cells and natural killer cells, are present in the endometrium (the interior lining of the uterus).^vii If an embryo is present in the uterus, cells from both the pregnant female, and the embryo will secrete specific immune factors to support the embryos attachment and penetration into the uterine lining.^viii

The "maternal-fetal interface" refers to the point where the uterus and embryonic tissue meet and interact. The immune cells present at this interface promote embryo implantation and prevent rejection of the embryo.^ix

How does the immune system affect fertility?

Immune responses in the male and female reproductive tract are highly regulated by sex hormones. In the uterus, estrogen and progesterone regulate communication between the immune system and uterine tissues to allow embryo implantation and survival of the fetus until birth.^x

Immunological infertility (or immune infertility) refers to the inability to conceive or maintain a healthy pregnancy due to contributions from immune-related factors. It has become an increasingly recognized, but poorly understood, issue in infertility. One example involves the female immune system attacking sperm, preventing fertilization. The female reproductive tract can react to semen, leading to an immune response and rejection of the foreign sperm or semen components before fertilization can occur. This activated immune response typically involves immune proteins (anti-sperm antibodies, or ASA) that target sperm or components within the seminal fluid.^xi

Immune-mediated infertility has also been brought to the forefront of treating females with recurrent implantation failure (RIF) or recurrent pregnancy loss (RPL). Alterations in immune cells have been found in both these populations, although the full implications of changes in immune cells are still being investigated. Abnormal function of a female’s immune system can also relate to chronic inflammatory diseases such as endometriosis, which occurs in 30 to 50 percent of infertile females, and chronic endometritis.^xii Some researchers have estimated up to 20 percent of RPL cases have been linked to irregularities in the maternal immune system.^xiii

Despite some individuals experiencing immunological-associated infertility, the immune system is not an enemy of people trying to conceive. In fact, an active immune response is important for successful implantation. In addition, when there are immunological imbalances, they are not always due to an overactive or inflammation-promoting immune system.^xiv

When would a patient see a reproductive immunologist?

Females with unknown causes of infertility, including patients who have undergone in vitro fertilization (IVF), and have a history of RIF or RPL, as well as patients with non-autoimmune disorders who are trying to conceive, may seek assistance from a reproductive immunologist (RI).^xv RIs may help these individuals through additional testing and by suggesting certain immunotherapies before subsequent fertility treatment.

What does a reproductive immunologist do?

An RI is a medical doctor who specializes in the field of reproductive immunology. These providers focus on immunological clinical care to help improve reproductive outcomes. Reproductive immunologists often provide immune testing and may prescribe immunotherapy. Some of these therapies include immunosuppressive drugs like TNFα inhibitors (e.g., Humira®), immunomodulatory therapies such as intravenous immunoglobulins (IVIg), and corticosteroids (e.g., prednisolone) to be used during assisted reproductive technology (ART) treatment. However, there is also debate as to whether immunotherapy add-ons to fertility treatment have any impact on outcomes, as discussed below.

Immune testing

Testing involves a range of immune function tests, from limited panels to more extensive ones. Most immune markers are tested using blood samples, although immune testing may also involve uterine biopsies. Many of the tests are performed by both reproductive immunologists (RIs) and reproductive endocrinology and infertility specialists (REIs).

Here are some examples:

• Autoimmune tests
• Immunophenotype blood test
• NK cell testing
• Th1:Th2 assay
• Endometrial immune profiling

Autoimmune tests

Autoimmune tests quantify the blood concentration of certain antibodies that can attack the body’s own cells. Here are some examples:^xvi,xvii

• Antiphospholipid antibodies (anticardiolipin antibodies, lupus anticoagulant, and beta-2 glycoprotein)
• Thyroid peroxidase antibodies (anti-TPO antibodies)
• Anti-nuclear antibodies (ANA)

Not all of these antibody tests will be ordered in all cases; it depends on each patient's particular diagnosis and history. For example, testing for anti-phospholipid antibodies may be indicated for some RPL and RIF patients, while anti-TPO antibodies may be tested in cases of thyroid imbalances.

The most up-to-date clinical guidelines by the European Society of Human Reproduction and Embryology (ESHRE), and the American Society for Reproductive Medicine (ASRM) do not find that there is enough evidence to support the widespread use of immune testing in the general clinical population.^xviii,xix Testing for ANA can be considered for RPL patients for explanatory purposes, but there is still insufficient data regarding ANA screening for patients with RIF.^xx,xxi

Immunophenotype blood test

A blood test called an immunophenotype can also be done to measure the quantity of different types of white blood cells in the uterus or peripheral blood. Irregularly high quantities could potentially cause infertility or pregnancy loss.

For example, some studies indicate that overall levels of B cells (a type of white blood cell), above 12 percent may lead to infertility or RPL.^xxii Another study showed an association between infertility and elevated CD56-positive lymphocytes (immune cells such as NK cells, monocyte, and certain T cells).^xxiii

While these associations have been observed, evidence is mixed on potential effective treatments for any abnormalities. Until more quality and consistent evidence arises, routine immune testing is not currently recommended by the American Society for Reproductive Medicine (ASRM).^xxiv,xxv

NK cell testing

Natural killer (NK) cells are a type of white blood cell that fight viral infection and tumors. NK cells circulate in the bloodstream (where they are called peripheral blood NK cells) but are also found in the uterus during implantation and initial stages of pregnancy (called uterine NK cells, or uNK cells).

It is possible to analyze the concentration and activity of NK cells. This analysis can be done using a blood test to assess peripheral NK cells or by performing a uterine biopsy to assess uNK cells. Because uterine biopsies are much more invasive and expensive, blood testing is often used to represent uNK cells; however, there is debate as to whether the circulating peripheral NK cells are a good indicator of uNK levels and activity.^xxvi

Some evidence indicates that an abnormal presence or activity of NK cells may potentially cause implantation or pregnancy failure.^xxvii Certain studies have shown that elevated levels of NK cells may negatively impact fertility. For example, a study of 137 patients indicated that peripheral NK cell levels above 18 percent may predict poor reproductive outcomes.^xxviii

However, not all evidence supports the link between elevated NK cells and fertility. A meta-analysis of 22 studies published in 2013 reported no significant differences in the percentage of peripheral and uterine NK cells in fertile versus infertile females.^xxix The researchers also reported no significant difference in IVF outcomes between females with and without elevated peripheral NK cells.^xxx A 2022 meta-analysis of 60 studies found higher uNK cell levels for RIF and RPL patients, but they did not observe any difference in pregnancy outcomes.^xxxi

Overall, there is a lack of consensus as to whether a high percentage of NK cells has an impact on reproductive outcomes. The most up-to-date guidelines do not support the widespread use of peripheral or uterine NK cell testing for either the general population or patients with RIF or RPL.^{xxxii,xxxiii,xxxiv,xxxv}

Th1:Th2 assay

The Th1:Th2 assay is a test used to determine the ratio between two types of T helper cells (Th1 and Th2) identifying imbalance in the maternal immune system. T helper cells are a type of white blood cells that are important in activating immune responses.

In general, Th1 creates a pro-inflammatory environment, whereas Th2 creates an anti-inflammatory environment.^xxxvi There is some evidence to suggest that higher Th1:Th2 ratios may be associated with subfertility and increased risk of miscarriage.^xxxvii Although, currently, evidence is not sufficient to recommend this test to patients undergoing IVF.^{xxxviii,xxxix,xl}

Endometrial immune profiling  

A relatively recent technology called endometrial immune profiling analyzes the local immune response in the endometrium during the window of implantation using endometrial tissue biopsy. It aims to identify patients with a dysregulated uterine immune environment (both over- and under-reactive) to help design immune-based add-on treatment plans.

In a study by Ledee et al (2020) of 1,738 infertile patients receiving endometrial immune profiling, 28 percent of females had an underactive immune environment, and 45 percent had an overactive environment, according to their criteria for immune dysregulation.^xli Patients with a history of RIF or RPL and an irregular endometrial immune profile had improved clinical pregnancy rates following an immunomodulatory treatment plan. However, the study sample size was small and there are limitations in the study design. The effectiveness of testing certain cytokines and immune factors is debated.^xlii,xliii

Reproductive immunology treatments

For females who are considered to have immune imbalances, as well as for those with unexplained RIF or RPL, immunomodulatory and immunosuppressive therapies are intended to help improve fertility outcomes. However, as discussed below, the effectiveness is not clear or not supported by evidence. These are the therapies that may be considered:^xliv,xlv

• Immunomodulatory intravenous immunoglobulins (IVIg)
• Immunosuppressive drugs, such as prednisone, prednisolone, Humira®, or Prograf®
• Intralipid therapy  

IVIg

Intravenous immunoglobulin (IVIg) therapy is a product containing a mix of antibodies (immunoglobulin G, IgG), derived from thousands of donors. It is administered intravenously (directly into a vein) and used to treat immune deficiencies (e.g., due to cancer treatment), as well as autoimmune and inflammatory disorders.^xlvi IVIg is sometimes used by IVF patients with RPL or RIF, especially if associated with immune causes. IVIg has both anti-inflammatory and pro-inflammatory effects on the immune system.

Some studies have shown altered cellular profiles following IVIg treatment and a potential for improved outcomes in RPL patients with immune abnormalities.^xlvii,xlviii However, no large IVIg randomized clinical trials have shown an improvement in live birth or miscarriage rate in patients with unexplained RPL.^xlix,l

A single-center retrospective study of 143 patients diagnosed with RIF found that there was a higher live birth rate in patients who received IVIg (65.3 percent) compared to those who did not (34 percent). The researchers found that patients diagnosed with severe RIF (individuals who experienced more than five failed good-quality embryo transfers) were among the people who benefited from IVIg treatment.^li Similarly, another study of 202 IVF cycles observed an improvement in live birth rates for individuals receiving IVIg treatment due to immune imbalances, such as NK cell or T helper cells (a type of white blood cell).^lii In contrast, other published studies examining IVIg immunotherapy did not observe an improvement in IVF outcomes.^liii,liv

It is important to note that, although there is some evidence to support the use of IVIg for patients with difficulty conceiving, and some physicians might find this treatment beneficial, current ASRM and ESHRE practice guidelines do not find these studies sufficient to recognize the use of IVIg as a treatment add-on for the general IVF population, or patients with RIF or RPL.^{lv,lvi,lvii,lviii}

The universal protocol for IVIg treatment does not yet exist, but most studies using IVIg administer it in one- to four-week intervals with a dosage of 400 mg/kg. A common protocol for patients with RIF and RPL is at least one IVIg infusion before ovulation or embryo transfer, one infusion after a positive pregnancy test, and then an infusion every 1 to 3 weeks during pregnancy. Clinics can adjust the protocol and dosage for IVIg depending on immune testing results such as NK cell levels, Th1:Th2 ratios, or autoimmune status.^lix

Corticosteroids

Corticosteroids, such as prednisone or prednisolone, suppress the immune system and prevent inflammation. A large systematic review found no benefit in adding corticosteroids to IVF cycles to improve success rates.^lx

Corticosteroids may be prescribed to RIF or RPL patients with increased NK cell activity. The study published in the Journal of Reproductive Immunology (2018) reported that only 55 percent of RIF patients responded positively to prednisone use (as indicated by reduced uNK cell activity).^lxi The researchers concluded that only a portion of RIF patients may benefit from corticosteroid treatment. Additionally, no study to date has evaluated how prednisone therapy affects pregnancy rates in RIF patients, although a randomized trial is ongoing.^lxii

For patients with unexplained RPL, prednisone could potentially be beneficial when given in combination with other treatment. A double-blind placebo randomized controlled trial including 160 patients with RPL reported that patients who received prednisone, low-dose aspirin, and heparin had a higher ongoing pregnancy rate (70 percent) compared to those who received heparin and aspirin alone (9 percent).^lxiii Importantly, this was a small study. There are no larger trials to date evaluating the use of prednisone for unexplained RPL.

That said, corticosteroids may be useful in females with RPL and known cellular immune abnormalities. A study published in the Journal of Reproductive Immunology (2021), evaluated whether immunomodulatory treatment (10 mg daily of oral prednisone, alone or combined with 400 mg/kg of IVIg) and anticoagulant treatment (40 mg daily of Lovenox®, and 81 mg daily of aspirin), improved IVF outcomes for 197 females with RPL or RIF who had known cellular immune abnormalities and thrombophilia (a blood clotting disorder commonly associated with autoimmune diseases).^lxiv They observed that for all patients who received immunomodulatory treatment, there was a statistically significant improvement in live birth rate per fresh or frozen embryo transfer cycle.

Prednisone may also be useful for male factor infertility. Studies have shown improved sperm parameters in pregnancy rates for males with anti-sperm antibodies after treatment with prednisone.^lxv Corticosteroids can also improve sperm counts in males with low sperm concentration (oligospermia) and signs of local information on ultrasound.^lxvi

Most research studies on corticosteroid immunotherapy for fertility treatment are retrospective, have small sample sizes, often with non-ideal control groups, and are subject to biases. Current ASRM guidelines do not support the routine use of corticosteroids during IVF stimulation and embryo transfer.^lxvii

Intralipid therapy

Intralipid therapy involves the IV infusion of sterile fat motion (20 percent fat emulsion diluted in 250 mg of saline), which is suggested by several studies to suppress NK cell count and activity in patients with RIF and RPL.

A randomized controlled trial by Singh et al (2019) evaluated 105 females with a history of failed implantation in previous IVF cycles.^lxviii They found higher clinical pregnancy rates in individuals who received two intralipid doses compared to those who did not (34 percent vs. 14 percent).

Another randomized controlled trial in females with secondary infertility, found 144 females receiving intralipids on the day of egg retrieval had a higher live birth rate compared to 154 females not receiving intralipids (37.5 vs. 22. 4 percent).^lxix

In contrast, a 2019 clinical trial and a 2020 meta-analysis did not observe any significant difference in pregnancy outcomes for those using intralipid compared to those that did not use intralipids.^lxx A prospective study, examining intralipids for embryo transfer in females aged 40 to 42 years was cancelled due to no live births observed in the intralipid group (versus 30 percent live birth rate in individuals not receiving intralipids).^lxxi

Overall, there is a lack of research on intralipid treatment, and the limited studies have conflicting results.^lxxii Therefore, both ASRM and ESHRE find that there is not enough evidence to support the routine use of intralipid therapy for patients undergoing infertility treatment.^lxxiii,lxxiv

How to find a reproductive immunologist

Finding an RI can be difficult in certain countries. Some fertility clinics conduct immune testing themselves or are associated with specific reproductive immunology programs with RIs. If the latter is the case, a patient’s fertility doctor may refer her to the RI if indicated or upon her request. Other females undergoing assisted reproduction may choose an RI based on personal recommendations or online searches. Individuals and couples interested in reproductive immunology should check with their IVF clinics for more information.

Conclusion

The interplay between the reproductive and immune system is complex and our understanding is evolving with ongoing research. For all couples and individuals hoping to achieve a healthy pregnancy, it can be useful to learn the basics. Doing so will enable patients undergoing IVF to understand which treatments are evidence-based, and potentially consider consultation with a reproductive immunologist if they suspect an immune system dysfunction related to infertility. It will also help them know what questions to ask and what testing and treatments are available.