3. Female autoimmune infertility

Women are prone to autoimmune diseases due to hormonally dictated cytokine and chemokine milieu [82] often leading to other autoimmune dysfunctions [83] including reproductive autoimmune failure. Gleicher and co-workers [6] postulated that endometriosis could be an autoimmune disease and studies from our lab show 30% prevalence [84]. Commonly seen serum AAbs are anti-phospholipid, anti-nuclear, anti-thyroid, anti-annexin V, anti-prothrombin, anti-laminin, anti-ZP (Table 3 for entire list), with the high level of NK cells as the risk factors but not as those pathognomonic [85]. However, none of the AAb biomarkers tested were effective [86]. A recent study reported better sensitivity of 6 new biomarkers [87]. With detection of AAbs to steroid producing cells and thyroglobulin in cases with concomitant adrenal or thyroid disease in PCOS, it is now considered an autoimmune disease. However, anti-ovarian antibodies were reported in only one study [7, 88] with no clarity on their role in PCOS pathogenesis [89]. Organ-specific AAbs such as ovary, adrenal and thyroid (endocrine autoimmune) disease are reported to cause infertility due to premature ovarian insufficiency (POI) [90].

Both PCOS and endometriosis are also causative factors of POI. 40–60% women with endometriosis possess anti-ovarian Abs in addition to anti-endometrial Abs [103]. Several AAbs to non-organ specific targets are seen in women with unexplained infertility [104]. Further, 22% of patients with SLE show anti-corpus luteum antibodies and elevated FSH levels typical of POI [57] and 60% POI cases are of autoimmune origin [105, 106]. POI is typically detected late with both non-organ and organ-specific antibodies in conjunction with an autoimmune disease thus evading a specific and accurate biomarker for diagnosis and prognosis [107, 108]. Whether AAbs are causative of or a by-product of underlying disease is unclear.

Nevertheless, elaborate animal models of the disease as well as case studies have provided relevant data. Day three neonatal thymectomy mouse model showed that multi-organ autoimmune disease prevails. Immunization with a single antigen causes oophoritis alone while those to multiple antigens completely compromises ovarian function. Additionally, concomitant presence of the autoantigens was mandatory [109].

Efforts to identify target autoantigens based on discovery of an ovary specific autoantigen by ELISA, immunofluorescence or immunohistochemistry approach were unfruitful. This interference was due to non-specific reactivity of natural albumin antibodies [110]. Attempts to identify target autoantigens using sera and proteomics approach were fruitful enough to identify several somatic proteins: alpha actin, alpha actinin-4, heat shock proteins 70 and 90β in 30% of POI and 26% of IVF-ET failure cases [100, 111, 112]. Of these, 47% cases showed presence of AAbs to HSP90β. Reactivity of these antibodies was seen against several follicular components (Table 4). Note, besides oocyte the corpus luteum seems to be a major cellular target while HSP90β the molecular target contributing to early POI (bold and italics in Table 4) [111]. AAbs to MATER led to assuming it to be an ovary specific target [113] however, these

AAbs were also seen in idiopathic hypoparathyroidism cases only in context of autoimmune

Autoantibodies Compartment Reference

mucus

FSH, β-subunit Serum [9, 12, 91–93]

Thyroid peroxidase [94] Alpha enolase [95]

Syntaxin 5 [86]

Selenium binding protein 1 [96] Heat-shock protein 90-β Serum [97] LH receptor [98, 99] α-Actin Serum [100, 101]

Stomatin-like protein 2 [84, 87]

Double stranded DNA – [89] Angiotensin II type 1 receptor agonistic autoantibodies Serum [102]

[50]

43

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Zona pellucida (ZP3, ZP2) Peritoneal, follicular fluids, cervical-vaginal

Anti-phospholipid Cervical, serum

Anti-cardiolipin Serum Anti-HAL Peritoneum

17α-hydroxylase, desmolase (P450-side chain cleavage)

3β-hydroxysteroid dehydrogenase

Antinuclear autoantibodies (ANA) SMOOTH muscle autoantibodies (SMA)

Anti annexin 5

21-hydroxylase

Anti-endometrial Abs

Aldehyde dehydrogenase

Cancer antigen 125 (CA125) Cancer antigen 19.9 (CA19.9)

Tropomodulin 3 (TMOD3) Tropomyosin 3 (TPM3)

α-Actinin-4 HSPA5 (HSP70)

Serine/threonine-protein kinase (PDIK1L)

Though a 75–90% accuracy was observed in ELISA assays using immunodominant epitopes from the identified targets, the AAbs were also present in normal population, highlighting the

polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome [114].

Table 3. List of autoantigenic targets against sera of women with reproductive infertility.


vasovasostomy can cause necrospermia and deteriorate sperm count hence IVF-ICSI using

Women are prone to autoimmune diseases due to hormonally dictated cytokine and chemokine milieu [82] often leading to other autoimmune dysfunctions [83] including reproductive autoimmune failure. Gleicher and co-workers [6] postulated that endometriosis could be an autoimmune disease and studies from our lab show 30% prevalence [84]. Commonly seen serum AAbs are anti-phospholipid, anti-nuclear, anti-thyroid, anti-annexin V, anti-prothrombin, anti-laminin, anti-ZP (Table 3 for entire list), with the high level of NK cells as the risk factors but not as those pathognomonic [85]. However, none of the AAb biomarkers tested were effective [86]. A recent study reported better sensitivity of 6 new biomarkers [87]. With detection of AAbs to steroid producing cells and thyroglobulin in cases with concomitant adrenal or thyroid disease in PCOS, it is now considered an autoimmune disease. However, anti-ovarian antibodies were reported in only one study [7, 88] with no clarity on their role in PCOS pathogenesis [89]. Organ-specific AAbs such as ovary, adrenal and thyroid (endocrine autoimmune) disease are reported to cause

Both PCOS and endometriosis are also causative factors of POI. 40–60% women with endometriosis possess anti-ovarian Abs in addition to anti-endometrial Abs [103]. Several AAbs to non-organ specific targets are seen in women with unexplained infertility [104]. Further, 22% of patients with SLE show anti-corpus luteum antibodies and elevated FSH levels typical of POI [57] and 60% POI cases are of autoimmune origin [105, 106]. POI is typically detected late with both non-organ and organ-specific antibodies in conjunction with an autoimmune disease thus evading a specific and accurate biomarker for diagnosis and prognosis [107, 108].

Nevertheless, elaborate animal models of the disease as well as case studies have provided relevant data. Day three neonatal thymectomy mouse model showed that multi-organ autoimmune disease prevails. Immunization with a single antigen causes oophoritis alone while those to multiple antigens completely compromises ovarian function. Additionally, concomi-

Efforts to identify target autoantigens based on discovery of an ovary specific autoantigen by ELISA, immunofluorescence or immunohistochemistry approach were unfruitful. This interference was due to non-specific reactivity of natural albumin antibodies [110]. Attempts to identify target autoantigens using sera and proteomics approach were fruitful enough to identify several somatic proteins: alpha actin, alpha actinin-4, heat shock proteins 70 and 90β in 30% of POI and 26% of IVF-ET failure cases [100, 111, 112]. Of these, 47% cases showed presence of AAbs to HSP90β. Reactivity of these antibodies was seen against several follicular components (Table 4). Note, besides oocyte the corpus luteum seems to be a major cellular target while HSP90β the molecular target contributing to early POI (bold and italics in Table 4) [111]. AAbs to MATER led to assuming it to be an ovary specific target [113] however, these

Whether AAbs are causative of or a by-product of underlying disease is unclear.

testicular sperm is an option [81].

42 Autoantibodies and Cytokines

3. Female autoimmune infertility

infertility due to premature ovarian insufficiency (POI) [90].

tant presence of the autoantigens was mandatory [109].

Table 3. List of autoantigenic targets against sera of women with reproductive infertility.

AAbs were also seen in idiopathic hypoparathyroidism cases only in context of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome [114].

Though a 75–90% accuracy was observed in ELISA assays using immunodominant epitopes from the identified targets, the AAbs were also present in normal population, highlighting the


with concomitant decrease in fertility index along with an increased polymorphonuclear cell infiltration of the ovarian follicles. The infiltration may have contributed to generation of

In normal physiological inflammatory processes like ovulation, follicular atresia, corpus luteum regression and tissue remodeling, the ovarian leukocytes like T cells and macrophages play an important role [117, 118]. Interestingly, NAbs especially, IgM play a role in clearing apoptotic cells, maintaining B cell homeostasis, inflammation, atherosclerosis and autoimmunity. Any drop in IgM levels is associated with ineffective clearance of apoptotic cells culminating into autoimmune disease. Alternatively, strong and persistent recognition of apoptotic cells by such NAbs may overactivate the immune system and cause chronic inflammation [3]. Corticosteroid treatment resolves the ensuing infertility [119]. However, there are no randomized controlled trials (RCT) to date. Our animal studies showed high dose corticosteroid was better able to rescue fertility in mice immunized with immunodominant epitopes of HSPA5 (Table 5). An interesting finding was the epitope spreading observed: AAbs to HSPA5 crossreact with immunodominant epitope (EP6) of HSP90β at high titer [120]. Thus, autoreactivity

Thyroid autoimmunity is commonly found with other systemic autoimmune diseases [121, 122] and is associated with anti-phospholipid syndrome (APS) due to anti-phospholipid antibodies [123] which in turn mediate recurrent miscarriages common to APS [124]. Thus women with thyroid autoimmunity and APS have greater risk of recurrent miscarriages mandating screening for anti-phospholipid antibodies. AAbs to ANA (12%), ANCA (20%), AECA (24%), ACLA (8%), anti-dsDNA (0%), β2 microglobulin (14%), and anti-HLA antibodies (10%) have been reported among Indian RSA patients [125]. This indicates that women with thyroiditis,

At least 20–30% of POI cases have an additional autoimmune disorder [126] including several endocrinopathies, thyroid diseases, Addison's disease, rheumatoid arthritis and polyglandular

> Delay in vaginal plug

Not determined

– 44%

30% 36.4%

Preimplantation

30% 24% 32% 44%

Fertility reduction

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45

Effect of corticosteroid treatment

loss

estrus cycle

Not determined

Not significant

Not determined

Table 5. Effect of autoantibodies on fertility and extent of rescue with corticosteroid therapy.

antibodies against the EP6 peptide [115, 116].

to HSP90β could have diagnostic and prognostic value.

AutoAb target Cellular target Effect on

corpus luteum

theca and corpus luteum

developing embryo

oocytes of later-stage small follicles

Alpha actinin-4 Ooplasm, theca and

HSPA5 Ooplasm, granulosa,

HSP90-beta (EP6) Granulosa cells,

MATER/NALP5 (parathyroid autoantigen)

Alpha actin Ooplasm, granulosa and theca,

endometriosis, SLE, APS also run the risk of repeated miscarriages.

Table 4. List of antigens and cellular targets detected using sera of women with premature ovarian insufficiency (POI) and in vitro fertilization-embryo transfer (IVF-ET); compiled from [97].

fact that these were NAbs. These were also validated to induce aPOI in a mouse model. The immunodominant epitopes tested were able to induce POI and alter ovarian cytoarchitecture. Folliculogenesis was severely affected at each developmental stage with gross lack of mature Graafian follicles and a persistent corpus luteum [101].

AAbs to a single immunodominant epitope (EP6) HSP90β led to 9% dissociated oocytecumulus complexes, granulosa cells undergoing apoptosis, 48% empty follicles, and 12% degenerated follicles. These animals demonstrated significant pre- and post-implantation loss with concomitant decrease in fertility index along with an increased polymorphonuclear cell infiltration of the ovarian follicles. The infiltration may have contributed to generation of antibodies against the EP6 peptide [115, 116].

In normal physiological inflammatory processes like ovulation, follicular atresia, corpus luteum regression and tissue remodeling, the ovarian leukocytes like T cells and macrophages play an important role [117, 118]. Interestingly, NAbs especially, IgM play a role in clearing apoptotic cells, maintaining B cell homeostasis, inflammation, atherosclerosis and autoimmunity. Any drop in IgM levels is associated with ineffective clearance of apoptotic cells culminating into autoimmune disease. Alternatively, strong and persistent recognition of apoptotic cells by such NAbs may overactivate the immune system and cause chronic inflammation [3]. Corticosteroid treatment resolves the ensuing infertility [119]. However, there are no randomized controlled trials (RCT) to date. Our animal studies showed high dose corticosteroid was better able to rescue fertility in mice immunized with immunodominant epitopes of HSPA5 (Table 5). An interesting finding was the epitope spreading observed: AAbs to HSPA5 crossreact with immunodominant epitope (EP6) of HSP90β at high titer [120]. Thus, autoreactivity to HSP90β could have diagnostic and prognostic value.

Thyroid autoimmunity is commonly found with other systemic autoimmune diseases [121, 122] and is associated with anti-phospholipid syndrome (APS) due to anti-phospholipid antibodies [123] which in turn mediate recurrent miscarriages common to APS [124]. Thus women with thyroid autoimmunity and APS have greater risk of recurrent miscarriages mandating screening for anti-phospholipid antibodies. AAbs to ANA (12%), ANCA (20%), AECA (24%), ACLA (8%), anti-dsDNA (0%), β2 microglobulin (14%), and anti-HLA antibodies (10%) have been reported among Indian RSA patients [125]. This indicates that women with thyroiditis, endometriosis, SLE, APS also run the risk of repeated miscarriages.

At least 20–30% of POI cases have an additional autoimmune disorder [126] including several endocrinopathies, thyroid diseases, Addison's disease, rheumatoid arthritis and polyglandular


Table 5. Effect of autoantibodies on fertility and extent of rescue with corticosteroid therapy.

fact that these were NAbs. These were also validated to induce aPOI in a mouse model. The immunodominant epitopes tested were able to induce POI and alter ovarian cytoarchitecture. Folliculogenesis was severely affected at each developmental stage with gross lack of mature

Table 4. List of antigens and cellular targets detected using sera of women with premature ovarian insufficiency (POI)

Condition Age at detection Cellular target Molecular target

 Oocyte, corpus luteum 30 38 45, 90 24 90, 97 Oocyte, theca, corpus luteum 97 Oocyte, theca 90 Theca 120 Oocyte 90

 Oocyte 55 36 97 Oocyte of primordial follicle 70, 75 Oocyte 70 Granulosa, corpus luteum 30, 45

28 120 39 30, 90 Oocyte 120 50, 75, 90 Oocyte, granulosa 80, 97 Oocyte 120 Theca 45, 97 Oocyte 90, 120 30, 50, 90

33 Oocyte 90, 97 30 Zona pellucida 45

90

POI 22 Oocyte, theca, corpus luteum 90

44 Autoantibodies and Cytokines

33 Ooplasm and nucleus of oocyte, theca

IVF-ET 29 Oocyte, corpus luteum 97

AAbs to a single immunodominant epitope (EP6) HSP90β led to 9% dissociated oocytecumulus complexes, granulosa cells undergoing apoptosis, 48% empty follicles, and 12% degenerated follicles. These animals demonstrated significant pre- and post-implantation loss

Graafian follicles and a persistent corpus luteum [101].

and in vitro fertilization-embryo transfer (IVF-ET); compiled from [97].

syndrome with greater prevalence of thyroid autoimmunity (14–27% at initial diagnosis) and thyroid peroxidase AAbs [127, 128]. At least 10% women with Addison's disease manifest AAbs to 21- or 17-hydroxylase and autoimmune oophoritis [129]. Thyroid peroxidase antibodies (TPO Abs) are also prevalent in PCOS cases. Thus, these along with HSP90β could be included in an antibody detection panel.

to be NAbs [148–151]. Obtaining clarity on role of AAbs will guide further treatment modalities for patients with AI [93, 101, 152]. Global high dose immunosuppressive therapy seems to be the only effective option for autoimmune reproductive failure despite its shortcomings

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47

Targeted interventional therapy by inducing antigen-specific tolerance is another option [155, 156]. Till such a time as a definitive therapy is available, pan autoimmune disease diagnostic panels can be designed using autoantigenic targets (recombinant proteins or peptides) such as β2-glycoprotein I and HSP90β (EP6) [151, 157–159] followed by management with corticosteroid therapy. A loss of reactivity to key autoantigens (predetermined to affect ovarian function) would serve as

The very lack of any organ-specific biomarker till date along with the preponderance of NAbs indicates that warped self-tolerance would lead to AI. AAbs in females alone appear to be significant in AI. Fertility studies need to be undertaken to gauge effect of such AAbs identified thus far and immunodominant epitopes gleaned could prove useful to design a pan autoimmune disease diagnostic peptide array to manage AI. Global immunosuppressant

The work was co-funded by Indian Council of Medical Research and Dept. of Biotechnology,

\*, Purvi Mande2 and Asmita Choudhury<sup>2</sup>

2 Department of Medicine, UMass Medical School, Worcester, MA, United States

1 National Institute for Research in Reproductive Health, Indian Council of Medical Research,

\*Address all correspondence to: kaushikikadam@gmail.com

biomarkers to better manage immunosuppressant therapy.

therapy and IVF-ICSI are the only current hope for such couples.

[153, 154].

5. Conclusion

Acknowledgements

Conflict of interest

Author details

Kaushiki M. Kadam<sup>1</sup>

Mumbai, India

Govt. of India.

None.

In women with endometriosis, use of biomarkers including CA-125 for diagnosis of endometriosis was prohibited [130, 131]. However as per recent guidelines, use of biomarkers has been recommended for both diagnosis and disease monitoring [132] and is still a researchable area. Anti-endometrial antibodies exist but their sensitivity and accuracy varies from 0 to 100% [131, 133, 134].
