#### **2.10 Measurement of urinary albumin and creatinine levels**

Albumin and creatinine levels were measured in first morning urine samples. Albumin levels were assessed with an ICT-EIA as described above but using a specific anti-albumin monoclonal antibody as described in previous report [Umehara, 2009] and creatinine levels were assessed by reaction with picric acid using a commercial kit (Creatinine-test Wako, Wako Pure Chemical Industries, Ltd., Osaka, Japan).

#### **2.11 Laboratory tests**

Plasma lipoproteins, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG) and HbA1c levels were analyzed consecutively using standard laboratory techniques by Bio Medical Laboratories, Inc. (Tokyo, Japan).

#### **2.12 OGTT test**

Eleven healthy young subjects (3 males and 8 females) ingested 75 g glucose. Blood samples were then obtained at 0, 60 and 120 min and urine samples at 0 and 120 min following ingestion. The levels of blood glucose and insulin, as well as levels of urinary insulin, were measured.

#### **2.13 Statistical analysis**

All data are presented as means ± SD. The detection limit of ICT-EIA or ELISA for insulin was expressed as the minimal amount of insulin which gives a significant bound enzyme activity (fluorescence or absorbance signal) after subtraction of the background signal (no insulin). A significant difference from the background was confirmed using Student's t-test (P < 0.001, n = 5).

Statistical analysis was performed using SPSS version 20.0.0. Correlations were performed using Spearman's correlation coefficient. Differences between two and three groups were compared using the Mann-Whitney U test and the Kruskal-Wallis test, respectively.

#### **3. Results**

88 Trends in Immunolabelled and Related Techniques

with DNP-biotinyl-BSA or a DNP-biotinyl-Ab-1 IgG conjugate, and 30 fmol Ab-2 Fab'--Dgalactosidase conjugate (Complex formation). Thereafter, one polystyrene bead coated with affinity-purified IgG (anti-DNP-BSA) was added to the mixture and incubated for 30 min (Entrapment). After removal of the incubation mixture, the polystyrene bead was washed twice with buffer A and then incubated in 150 l buffer A containing 2 mM *eN*-2,4-DNP-Llysine for 30 min (Elution). After removal of the polystyrene bead, one streptavidin-coated polystyrene bead was added to the eluate and incubated for 30 min (Transfer). The bead was then washed, and the bound -D-galactosidase activity was assayed fluorometrically with 4 methylumbelliferyl--D-galactoside (0.2 mM) as a substrate for 20 h at 30 °C [Hashida, 1995; Ishikawa, 1983b]. All incubations with polystyrene beads were performed with shaking at 210

To compare the assay of urinary insulin with assay of other urinary hormones derived from serum, ICT-EIAs for resistin were performed using a similar method as that described above for the ICT-EIA for insulin, including similar volumes of standard and samples, concentration of antibody conjugates, buffer, temperature and incubation times, except that, in the ICT-EIA for resistin, both DNP-biotinyl-BSA anti-resistin Fab' (184305) and anti-

Albumin and creatinine levels were measured in first morning urine samples. Albumin levels were assessed with an ICT-EIA as described above but using a specific anti-albumin monoclonal antibody as described in previous report [Umehara, 2009] and creatinine levels were assessed by reaction with picric acid using a commercial kit (Creatinine-test Wako,

Plasma lipoproteins, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG) and HbA1c levels were analyzed consecutively using

Eleven healthy young subjects (3 males and 8 females) ingested 75 g glucose. Blood samples were then obtained at 0, 60 and 120 min and urine samples at 0 and 120 min following ingestion. The levels of blood glucose and insulin, as well as levels of urinary insulin, were

All data are presented as means ± SD. The detection limit of ICT-EIA or ELISA for insulin was expressed as the minimal amount of insulin which gives a significant bound enzyme activity (fluorescence or absorbance signal) after subtraction of the background signal (no insulin). A significant difference from the background was confirmed using Student's t-test

standard laboratory techniques by Bio Medical Laboratories, Inc. (Tokyo, Japan).

strokes/min at room temperature [Hashida, 1995; Ishikawa, 1983b].

resistin Fab'(184320)-β-D-galactosidase antibody conjugates were used.

**2.10 Measurement of urinary albumin and creatinine levels**

Wako Pure Chemical Industries, Ltd., Osaka, Japan).

**2.11 Laboratory tests**

**2.13 Statistical analysis**

(P < 0.001, n = 5).

**2.12 OGTT test**

measured.

#### **3.1 IEMA for insulin**

To compare the newly developed ICT-EIA with other methods of insulin assay we performed an IEMA for insulin as outlined in Fig. 1. Insulin was simultaneously incubated with a mixture of a monoclonal (Mc) Ab-1 Fab' conjugated with DNP-biotinyl-BSA (Mc-DNP) and a monoclonal (Mc) Ab-2 Fab'--D-galactosidase conjugate (Mc-Gal), resulting in the formation of an immune complex consisting of all three components. This immune complex was then trapped onto a streptavidin-coated solid phase. The solid phase was washed to eliminate unbound conjugates and the bound -D-galactosidase activity was measured fluorometrically.

Using this assay the detection limit for insulin was 0.01 µU, which was 3-fold lower than that of a conventional ELISA (Fig. 2).

The open symbols indicate the specifically bound peroxidase activity in the conventional ELISA. The closed symbols indicate the specifically bound -D-galactosidase activity in the IEMA performed using monoclonal capture and enzyme-conjugated antibodies

Fig. 2. Calibration curves for insulin assay using IEMA and ELISA.

#### **3.2 ICT-EIA for insulin**

The ICT-EIA for insulin was performed as outlined in Figure 1. Insulin was simultaneously incubated with a mixture of Mc-DNP and Mc-Gal, to form an immune complex as described

Development of an Ultra-Sensitive Enzyme Immunoassay for Insulin

preserved (Fig. 5).

enzyme-labeled antibody (Mc-Gal) was used.

for the remainder of the study.

and Its Application to the Evaluation of Diabetic Risk by Analysis of Morning Urine 91

formation of the immune complex in ICT-EIA (Mc-Gal & Pc-DNP). The resulting fluorescence signal decreased linearly in accordance with insulin dilution. The detection limit for insulin was 0.0003 µU (12 fg; 1.8 amol) (Fig. 4), which was 30-fold lower than that of IEMA. When a polyclonal antibody was used for enzyme labeling and a monoclonal antibody for capture (Pc-Gal & Pc-DNP), or when both antibodies were the same polyclonal antibody, the insulin detection limit increased, but the linearity with insulin dilution was

The open symbols indicate the specifically bound -D-galactosidase activity in the ICT-EIA performed as described in Fig. 3. The closed symbols indicate the specifically bound -D-galactosidase activity in the ICT-EIA in which a combination of a polyclonal capture antibody (Pc-DNP) and a monoclonal

Figure 5 shows typical calibration curves of the ICT-EIAs for insulin using various combinations of polyclonal/monoclonal capture and enzyme-conjugated antibodies. The detection limit and sensitivity for insulin using a polyclonal capture antibody and a monoclonal enzyme labeled antibody (Pc-DNP & Mc-Gal) was 0.0003 µU (0.3 nU) and 0.15 µU/ml of urine, respectively, using 2-µl samples for complete recovery of the added insulin. This detection limit was 100-fold lower than that of a sensitive ELISA and of IEMA using Mc-DNP & Mc-Gal (Fig.2 and 3). The assay range (CV < 10) for insulin in urine was 0.5- 150 µU/ml, using urine samples with a volume of 2 µl. Based on these data the ICT-EIA using a polyclonal capture antibody and a monoclonal enzyme labeled antibody was used

Fig. 4. Calibration curves for insulin assay using two different ICT-EIAs.

**3.3 Improved ICT-EIA for insulin and limit of urinary insulin detection**

for the IEMA method. This immune complex was first trapped onto a solid phase coated with anti-DNP IgG. Subsequently, the solid phase was washed to eliminate unbound conjugates and the immune complex was then specifically eluted from this solid phase using DNP-Lys and transferred onto a second solid phase coated with streptavidin. Finally, the bound -D-galactosidase activity was measured fluorometrically.

When the same two mouse monoclonal antibodies that were used for IEMA were used for ICT-EIA, the fluorescence signal from the bound -D-galactosidase activity in the ICT-EIA was strongly decreased compared to that obtained in the IEMA at low insulin concentrations and was not linear with the lowest insulin concentrations. This effect resulted in a detection limit for insulin of 0.03 µU, which was 3-fold higher than that of the IEMA (Fig. 3). Similar results were obtained even if the high molecular weight -D-galactosidase (540 kDa) enzyme with which the antibody was conjugated was exchanged for a lower molecular weight enzyme such as peroxidase (40 kDa) or alkaline phosphatase (120 kDa) (data not shown).

The closed symbols indicate the specifically bound -D-galactosidase activity in the IEMA as described in Fig. 1 using monoclonal capture (Mc-DNP) and enzyme-conjugated (Mc-Gal) antibodies respectively. The open symbols indicate the specifically bound -D-galactosidase activity in the ICT-EIA using the same two monoclonal antibodies that were used for IEMA.

Fig. 3. Calibration curves for insulin assay using IEMA and ICT-EIA.

We next tested the effect of using a combination of an enzyme labeled monoclonal mouse antibody (Mc-Gal) and a non-affinity purified polyclonal capture antibody (Pc-DNP) for

for the IEMA method. This immune complex was first trapped onto a solid phase coated with anti-DNP IgG. Subsequently, the solid phase was washed to eliminate unbound conjugates and the immune complex was then specifically eluted from this solid phase using DNP-Lys and transferred onto a second solid phase coated with streptavidin. Finally, the

When the same two mouse monoclonal antibodies that were used for IEMA were used for ICT-EIA, the fluorescence signal from the bound -D-galactosidase activity in the ICT-EIA was strongly decreased compared to that obtained in the IEMA at low insulin concentrations and was not linear with the lowest insulin concentrations. This effect resulted in a detection limit for insulin of 0.03 µU, which was 3-fold higher than that of the IEMA (Fig. 3). Similar results were obtained even if the high molecular weight -D-galactosidase (540 kDa) enzyme with which the antibody was conjugated was exchanged for a lower molecular weight enzyme such as peroxidase (40 kDa) or alkaline phosphatase (120 kDa)

The closed symbols indicate the specifically bound -D-galactosidase activity in the IEMA as described in Fig. 1 using monoclonal capture (Mc-DNP) and enzyme-conjugated (Mc-Gal) antibodies respectively. The open symbols indicate the specifically bound -D-galactosidase activity in the ICT-EIA using the

We next tested the effect of using a combination of an enzyme labeled monoclonal mouse antibody (Mc-Gal) and a non-affinity purified polyclonal capture antibody (Pc-DNP) for

same two monoclonal antibodies that were used for IEMA.

Fig. 3. Calibration curves for insulin assay using IEMA and ICT-EIA.

bound -D-galactosidase activity was measured fluorometrically.

(data not shown).

formation of the immune complex in ICT-EIA (Mc-Gal & Pc-DNP). The resulting fluorescence signal decreased linearly in accordance with insulin dilution. The detection limit for insulin was 0.0003 µU (12 fg; 1.8 amol) (Fig. 4), which was 30-fold lower than that of IEMA. When a polyclonal antibody was used for enzyme labeling and a monoclonal antibody for capture (Pc-Gal & Pc-DNP), or when both antibodies were the same polyclonal antibody, the insulin detection limit increased, but the linearity with insulin dilution was preserved (Fig. 5).

The open symbols indicate the specifically bound -D-galactosidase activity in the ICT-EIA performed as described in Fig. 3. The closed symbols indicate the specifically bound -D-galactosidase activity in the ICT-EIA in which a combination of a polyclonal capture antibody (Pc-DNP) and a monoclonal enzyme-labeled antibody (Mc-Gal) was used.

Fig. 4. Calibration curves for insulin assay using two different ICT-EIAs.

#### **3.3 Improved ICT-EIA for insulin and limit of urinary insulin detection**

Figure 5 shows typical calibration curves of the ICT-EIAs for insulin using various combinations of polyclonal/monoclonal capture and enzyme-conjugated antibodies. The detection limit and sensitivity for insulin using a polyclonal capture antibody and a monoclonal enzyme labeled antibody (Pc-DNP & Mc-Gal) was 0.0003 µU (0.3 nU) and 0.15 µU/ml of urine, respectively, using 2-µl samples for complete recovery of the added insulin. This detection limit was 100-fold lower than that of a sensitive ELISA and of IEMA using Mc-DNP & Mc-Gal (Fig.2 and 3). The assay range (CV < 10) for insulin in urine was 0.5- 150 µU/ml, using urine samples with a volume of 2 µl. Based on these data the ICT-EIA using a polyclonal capture antibody and a monoclonal enzyme labeled antibody was used for the remainder of the study.

Development of an Ultra-Sensitive Enzyme Immunoassay for Insulin

with those determined by ELISA (r = 0.924, P < 0.001) (Fig. 6).

**3.7 Comparison with ELISA**

shown.

**in serum and urine**

the relative insulin levels in serum (Fig. 7).

and Its Application to the Evaluation of Diabetic Risk by Analysis of Morning Urine 93

Insulin levels measured in 35 serum samples (3.3-23.1 µU/ml) using ICT-EIA correlated well

The regression equation and the calculated correlation coefficient (n = 35, r = 0.924, P < 0.001) are

Fig. 6. Correlation between serum insulin levels measured by ELISA and by ICT-EIA.

**3.8 Effects of increased blood glucose levels after OGTT on the insulin concentration**

In order to determine whether the urinary insulin level reflects the blood insulin level, insulin levels in both serum and urine from non-diabetic healthy subjects were measured after OGTT using the ICT-EIA. Blood was collected at 0, 0.5, 1.0, 1.5 and 2.0 h, and urine was collected at 0 and 2 h after glucose ingestion. Blood glucose, serum insulin and insulin in the urine were measured (Table 2). Increases in the blood glucose level corresponded to significant increases in the blood insulin level, which were followed by increases in the urinary insulin level two hours later. The total quantity of insulin in urine was calculated based on the urine volume. A strong correlation was found between serum insulin levels at 60 min after glucose ingestion and total insulin values in urine during 2 h after ingestion (r = 0.87, P < 0.001), suggesting that measurement of insulin in urine may be useful for assessing

The closed squares indicate the specifically bound -D-galactosidase activity in the ICT-EIA in which a polyclonal capture antibody (Pc-DNP) and a monoclonal enzyme-labeled antibody (Mc-Gal) were used as described in Fig. 4. The open triangles indicate the specifically bound -D-galactosidase activity in an ICT-EIA in which the polyclonal (Pc-Gal)/monoclonal antibody (Mc-DNP) combination was reversed. The closed triangles indicate the specifically bound -D-galactosidase activity in an ICT-EIA in which the same polyclonal antibody was used for the capture and enzyme-labeled antibody (Pc-DNP & Pc-Gal).

Fig. 5. Calibration curves for insulin assay using three different ICT-EIAs.

#### **3.4 Assay precision**

The reproducibility of ICT-EIA for analysis of insulin in urine was estimated using two or three samples containing different concentrations of insulin (range, 0.5-22.5 µU/ml). The within-assay and between-assay CVs for insulin were 2.5-6.2 (n = 10) and 3.5-7.5 (n = 10), respectively.

#### **3.5 Recovery and dilution tests**

Analytical recovery of exogenously added insulin to 2-µl and 5-µl urine samples (range, 1.2- 25.3 µU/ml) was 92.8-106 and 72.5-103, respectively. Dilution curves for the urine samples paralleled those for standard insulin. Based on these data, 2 µl of urine was used for all experiments described below.

#### **3.6 Specificity of ICT-EIA**

The cross-reactivity of ICT-EIA for insulin and other hormones was evaluated. In the ICT-EIA for insulin, cross-reactions on a molar basis with hIGF-I, hCG and hGH were all <0.1%, indicating that ICT-EIA can specifically measure insulin concentrations.

#### **3.7 Comparison with ELISA**

92 Trends in Immunolabelled and Related Techniques

The closed squares indicate the specifically bound -D-galactosidase activity in the ICT-EIA in which a polyclonal capture antibody (Pc-DNP) and a monoclonal enzyme-labeled antibody (Mc-Gal) were used as described in Fig. 4. The open triangles indicate the specifically bound -D-galactosidase activity in an ICT-EIA in which the polyclonal (Pc-Gal)/monoclonal antibody (Mc-DNP) combination was reversed. The closed triangles indicate the specifically bound -D-galactosidase activity in an ICT-EIA in which the same

The reproducibility of ICT-EIA for analysis of insulin in urine was estimated using two or three samples containing different concentrations of insulin (range, 0.5-22.5 µU/ml). The within-assay and between-assay CVs for insulin were 2.5-6.2 (n = 10) and 3.5-7.5 (n = 10),

Analytical recovery of exogenously added insulin to 2-µl and 5-µl urine samples (range, 1.2- 25.3 µU/ml) was 92.8-106 and 72.5-103, respectively. Dilution curves for the urine samples paralleled those for standard insulin. Based on these data, 2 µl of urine was used for

The cross-reactivity of ICT-EIA for insulin and other hormones was evaluated. In the ICT-EIA for insulin, cross-reactions on a molar basis with hIGF-I, hCG and hGH were all <0.1%,

indicating that ICT-EIA can specifically measure insulin concentrations.

polyclonal antibody was used for the capture and enzyme-labeled antibody (Pc-DNP & Pc-Gal).

Fig. 5. Calibration curves for insulin assay using three different ICT-EIAs.

**3.4 Assay precision**

**3.5 Recovery and dilution tests**

all experiments described below.

**3.6 Specificity of ICT-EIA**

respectively.

Insulin levels measured in 35 serum samples (3.3-23.1 µU/ml) using ICT-EIA correlated well with those determined by ELISA (r = 0.924, P < 0.001) (Fig. 6).

The regression equation and the calculated correlation coefficient (n = 35, r = 0.924, P < 0.001) are shown.

Fig. 6. Correlation between serum insulin levels measured by ELISA and by ICT-EIA.

#### **3.8 Effects of increased blood glucose levels after OGTT on the insulin concentration in serum and urine**

In order to determine whether the urinary insulin level reflects the blood insulin level, insulin levels in both serum and urine from non-diabetic healthy subjects were measured after OGTT using the ICT-EIA. Blood was collected at 0, 0.5, 1.0, 1.5 and 2.0 h, and urine was collected at 0 and 2 h after glucose ingestion. Blood glucose, serum insulin and insulin in the urine were measured (Table 2). Increases in the blood glucose level corresponded to significant increases in the blood insulin level, which were followed by increases in the urinary insulin level two hours later. The total quantity of insulin in urine was calculated based on the urine volume. A strong correlation was found between serum insulin levels at 60 min after glucose ingestion and total insulin values in urine during 2 h after ingestion (r = 0.87, P < 0.001), suggesting that measurement of insulin in urine may be useful for assessing the relative insulin levels in serum (Fig. 7).

Development of an Ultra-Sensitive Enzyme Immunoassay for Insulin

higher than in NDO and NDNO subjects (P < 0.05) (Fig. 9).

Urinary resistin levels are expressed as a boxplot similar to that described in Fig. 8.

Fig. 9. Resistin levels in the urine of NDNO and NDO subjects and of DM patients.

and Its Application to the Evaluation of Diabetic Risk by Analysis of Morning Urine 95

Urinary insulin levels are expressed as a boxplot (box-and-whisker diagram); scale bars indicate sample minimum and maximum, boxes indicate lower quartile, median and upper quartile. DNO (n = 106), NDO (n = 35) and DM (n = 42). Insulin levels are expressed relative to urinary creatinine (Cre). Fig. 8. Insulin levels in the urine of NDNO subjects, NDO subjects and DM patients.

Resistin concentrations were also measured in urine from NDNO and NDO subjects and from DM patients and were determined to be 18.6 ± 20.9, 15.4 ± 17.2 and 30.0 ± 36.2 ng/mg creatinine, respectively (Fig. 9); the resistin concentration in DM subjects was significantly

Blood was collected at 1 h, and urine was collected at 0 and 2 h, after glucose ingestion. The regression equation and the calculated correlation coefficient (n = 23, r = 0.868, P < 0.001) are shown.

Fig. 7. Correlation between serum and urinary insulin levels after OGTT.

\*P < 0.01, compared with that at 0 h.

Urine: 0 h, before OGTT; 2 h, during 2 h after the OGTT.

Table 2. Concentration of glucose and insulin in serum, and of insulin in urine, in the oral glucose tolerance test (OGTT).

#### **3.9 Measurement of insulin and resistin concentrations in urine**

Urine samples were collected in the early morning from fasting NDNO and NDO subjects, and from fasting DM patients. Urinary insulin was measured by ICT-EIA and expressed in terms of µU/mg creatinine. The insulin concentrations determined in urine from NDNO and NDO subjects and from DM patients were 4.3 ± 2.8, 9.0± 5.5 and 13.9 ± 17.6 U/mg creatinine, respectively (Fig. 8); the insulin concentration was significantly higher in DM patients than in NDNO subjects (P < 0.001).

Blood was collected at 1 h, and urine was collected at 0 and 2 h, after glucose ingestion. The regression

0 1 2

equation and the calculated correlation coefficient (n = 23, r = 0.868, P < 0.001) are shown.

Blood glucose (mg/dl) 82 ± 11 135 ± 29\* 118 ± 18\* Serum insulin (µU/ml) 3.1 ± 1.7 33.3 ± 25.9\* 39.1 ± 45.0\* Urine insulin (µU/mg creatinine) 3.0 ± 1.9 - 15.2 ± 8.1\*

Table 2. Concentration of glucose and insulin in serum, and of insulin in urine, in the oral

Urine samples were collected in the early morning from fasting NDNO and NDO subjects, and from fasting DM patients. Urinary insulin was measured by ICT-EIA and expressed in terms of µU/mg creatinine. The insulin concentrations determined in urine from NDNO and NDO subjects and from DM patients were 4.3 ± 2.8, 9.0± 5.5 and 13.9 ± 17.6 U/mg creatinine, respectively (Fig. 8); the insulin concentration was significantly higher in DM

Fig. 7. Correlation between serum and urinary insulin levels after OGTT.

**3.9 Measurement of insulin and resistin concentrations in urine**

Serum and urine levels Time after OGTT (h)

Urine: 0 h, before OGTT; 2 h, during 2 h after the OGTT.

patients than in NDNO subjects (P < 0.001).

\*P < 0.01, compared with that at 0 h.

glucose tolerance test (OGTT).

Urinary insulin levels are expressed as a boxplot (box-and-whisker diagram); scale bars indicate sample minimum and maximum, boxes indicate lower quartile, median and upper quartile. DNO (n = 106), NDO (n = 35) and DM (n = 42). Insulin levels are expressed relative to urinary creatinine (Cre).

Fig. 8. Insulin levels in the urine of NDNO subjects, NDO subjects and DM patients.

Resistin concentrations were also measured in urine from NDNO and NDO subjects and from DM patients and were determined to be 18.6 ± 20.9, 15.4 ± 17.2 and 30.0 ± 36.2 ng/mg creatinine, respectively (Fig. 9); the resistin concentration in DM subjects was significantly higher than in NDO and NDNO subjects (P < 0.05) (Fig. 9).

Urinary resistin levels are expressed as a boxplot similar to that described in Fig. 8. Fig. 9. Resistin levels in the urine of NDNO and NDO subjects and of DM patients.

Development of an Ultra-Sensitive Enzyme Immunoassay for Insulin

was obtained.

if an affinity-purified antibody is used.

and Its Application to the Evaluation of Diabetic Risk by Analysis of Morning Urine 97

were caused by, but the results of these assays were again the same (data not shown). When the same polyclonal antibody, which was not insulin affinity-purified, was used as both the capture and the enzyme labeled antibody for ICT-EIA, a linear standard curve in accordance with insulin dilution was obtained, but the assay was of lower sensitivity than the other ICT-EIA assays (Fig. 5). Subsequently, a combination of one monoclonal and one nonaffinity purified polyclonal antibody to insulin was used and, in this assay, the detection limit of insulin was 1.8 amol and a linear standard curve in accordance with insulin dilution

When an affinity-purified polyclonal rabbit antibody was used in an ICT-EIA for assay of ferritin (450 kDa), 1 zmol (0.001 amol; 1 10-21 moles) of ferritin was detected [Hashida, 1990]. The average number of -D-galactosidase labeled antibody Fab' molecules bound per ferritin molecule was calculated to be 1.0-1.4. In addition, the detection limit of the β-Dgalactosidase activity was 1 zmol. On the other hand, the average number of bound enzyme-labeled antibody molecules per peptide molecule was calculated to be 0.001-0.01. Therefore, the peptide detection limit of this immunoassay was estimated to be between 0.1- 1 attomoles. Thus, it is likely that detection of 0.1 amol (0.02 µU) of a insulin will be possible

As described in the " Introduction ", blood samples have to be collected every 30 min for 2 h to assess the secretion of insulin in the OGTT. If the urinary insulin level reflects the serum insulin level, then the secretion of insulin after glucose ingestion may be easily assessed by measurement of urinary insulin levels. In the ICT-EIA of this study, the urinary insulin levels correlated well with the serum insulin levels at 1 h after glucose ingestion (Fig. 7). Thus, the level of insulin in urine appears to reflect the level in serum suggesting that

A comparison of the insulin levels in urine collected early in the morning from NDNO and NDO subjects and from DM patients showed that the levels were significantly higher in NDO subjects and in DM patients than in NDNO subjects (P < 0.01 and P < 0.001 respectively) (Fig. 8). These results indicate that an increase in insulin levels may be a

We previously developed a highly sensitive ELISA for urinary growth hormone (GH) [Hashida, 1987, Sukegawa, 1988], which showed that assay of GH during nocturia was useful as a supporting diagnostic index of the pituitary dwarf. We have also recently developed an ultra-sensitive ICT-EIA for urinary soluble human insulin receptor ectodomain (sIR) and found that urinary sIR levels correlated well with sIR blood levels [Umehara, 2009]. Furthermore, the levels of urinary sIR in DM patients were significantly higher than those in NDO subjects and DM patients. Therefore, urinary biomarkers can be

In this study, urine samples with micro- and macro-albuminuria were excluded. Urinary insulin and resistin levels were expressed as ratios of urinary concentration to milligrams of urinary creatinine. Urinary insulin levels correlated well with urinary resistin levels (r = 0.426, P < 0.01) (Fig. 10). Further studies with larger numbers of patients will be needed to

determine the cut-off value and criteria for insulin as a diabetic risk biomarker.

measurement of insulin in urine may be useful for assessing insulin secretion.

clinically important biomarker of obesity and diabetes risk.

useful for both physiological and clinical studies.

#### **3.10 Correlation of insulin level to resistin levels in urine**

We analyzed the correlation of urinary insulin level to urinary resistin level in 42 DM patients. The urinary insulin level was significantly and positively correlated to urinary levels of resistin (r = 0.426, P < 0.001) (Fig. 10).

### **4. Discussion**

We developed a new ultra-sensitive technique for the measurement of insulin by employing a non-competitive two-site binding method that utilizes two different types of anti-insulin antibodies and an immune-complex transfer method (ICT-EIA). This new method can measure insulin levels as low as 0.3 nU (12 fg; 1.8 amol), with a sensitivity of 0.15 µU/ml, using 2-µl urine samples. This assay is 100-fold more sensitive than a sensitive ELISA.

Various kinds of peptides can be measured at attomole levels by a non-competitive two-site enzyme immunoassay (ICT-EIA). For this purpose, the peptide molecules to be measured have to have two or more epitopes that are sufficiently separated from each other to allow simultaneous binding of two antibody molecules. A previous study using a single chain peptide provided a hint as to the distance required between epitopes for simultaneous binding of two antibodies. Human α-atrial natriuretic peptide (α-hANP) is a single chain polypeptide consisting of 28 amino acids with a ring structure that is formed by an intramolecular disulfide bound [Hashida, 1988b]. Ten amol of α-hANP were measured by a two-site enzyme immunoassay using a peroxidase-labeled antibody Fab' against the Cterminus of the peptide and a solid phase coated with IgG against the N-terminal half of the ring structure. The distance between the two epitopes recognized by the two antibody molecules in this single chain peptide appeared to correspond to 12-15 amino acids.

Since most polyclonal antibodies used in assays are now being replaced with monoclonal antibodies, a non-competitive two-site binding assay using two kinds of monoclonal antibody has recently been developed. When two kinds of monoclonal antibody were used in the ICT-EIA method, it was possible to measure most proteins at concentrations of less than attomole levels [Hashida, 1988b, 1990 & 1995].

However, it has not been possible to measure peptides, insulin, CRH, -ANP, adrenomedullin or PAMP at less than attomole levels with an ultra-sensitive assay (ICT-EIA) using two monoclonal antibodies [Hashida, 2004; Katakami, 2002; Yamaga, 2003]. The major reason for this failure appeared to be due more to a decreased signal rather than because of dilution degree. Two factors have been considered that might contribute to this phenomenon. One factor is insufficient antibody affinity and the other is steric hindrance within an immune complex consisting of two antibodies (each of 50 kDa) and a peptide (6 kDa). Both of these factors would contribute to weak binding of the antibodies to the peptide in the immune complex resulting in dissociation of the peptide from antibody during the transfer from the first to the second solid phase.

We tested a number of combinations of ten different monoclonal anti-insulin antibodies, which varied in affinity and epitope, in the development of the ICT-EIA for insulin, but the results of all of the assays were the same (data not shown). We further examined the study due to the multi-antibody which several kinds of different monoclonal antibodies to insulin

We analyzed the correlation of urinary insulin level to urinary resistin level in 42 DM patients. The urinary insulin level was significantly and positively correlated to urinary

We developed a new ultra-sensitive technique for the measurement of insulin by employing a non-competitive two-site binding method that utilizes two different types of anti-insulin antibodies and an immune-complex transfer method (ICT-EIA). This new method can measure insulin levels as low as 0.3 nU (12 fg; 1.8 amol), with a sensitivity of 0.15 µU/ml, using 2-µl urine samples. This assay is 100-fold more sensitive than a sensitive ELISA.

Various kinds of peptides can be measured at attomole levels by a non-competitive two-site enzyme immunoassay (ICT-EIA). For this purpose, the peptide molecules to be measured have to have two or more epitopes that are sufficiently separated from each other to allow simultaneous binding of two antibody molecules. A previous study using a single chain peptide provided a hint as to the distance required between epitopes for simultaneous binding of two antibodies. Human α-atrial natriuretic peptide (α-hANP) is a single chain polypeptide consisting of 28 amino acids with a ring structure that is formed by an intramolecular disulfide bound [Hashida, 1988b]. Ten amol of α-hANP were measured by a two-site enzyme immunoassay using a peroxidase-labeled antibody Fab' against the Cterminus of the peptide and a solid phase coated with IgG against the N-terminal half of the ring structure. The distance between the two epitopes recognized by the two antibody

molecules in this single chain peptide appeared to correspond to 12-15 amino acids.

Since most polyclonal antibodies used in assays are now being replaced with monoclonal antibodies, a non-competitive two-site binding assay using two kinds of monoclonal antibody has recently been developed. When two kinds of monoclonal antibody were used in the ICT-EIA method, it was possible to measure most proteins at concentrations of less

However, it has not been possible to measure peptides, insulin, CRH, -ANP, adrenomedullin or PAMP at less than attomole levels with an ultra-sensitive assay (ICT-EIA) using two monoclonal antibodies [Hashida, 2004; Katakami, 2002; Yamaga, 2003]. The major reason for this failure appeared to be due more to a decreased signal rather than because of dilution degree. Two factors have been considered that might contribute to this phenomenon. One factor is insufficient antibody affinity and the other is steric hindrance within an immune complex consisting of two antibodies (each of 50 kDa) and a peptide (6 kDa). Both of these factors would contribute to weak binding of the antibodies to the peptide in the immune complex resulting in dissociation of the peptide from antibody

We tested a number of combinations of ten different monoclonal anti-insulin antibodies, which varied in affinity and epitope, in the development of the ICT-EIA for insulin, but the results of all of the assays were the same (data not shown). We further examined the study due to the multi-antibody which several kinds of different monoclonal antibodies to insulin

**3.10 Correlation of insulin level to resistin levels in urine**

levels of resistin (r = 0.426, P < 0.001) (Fig. 10).

than attomole levels [Hashida, 1988b, 1990 & 1995].

during the transfer from the first to the second solid phase.

**4. Discussion**

were caused by, but the results of these assays were again the same (data not shown). When the same polyclonal antibody, which was not insulin affinity-purified, was used as both the capture and the enzyme labeled antibody for ICT-EIA, a linear standard curve in accordance with insulin dilution was obtained, but the assay was of lower sensitivity than the other ICT-EIA assays (Fig. 5). Subsequently, a combination of one monoclonal and one nonaffinity purified polyclonal antibody to insulin was used and, in this assay, the detection limit of insulin was 1.8 amol and a linear standard curve in accordance with insulin dilution was obtained.

When an affinity-purified polyclonal rabbit antibody was used in an ICT-EIA for assay of ferritin (450 kDa), 1 zmol (0.001 amol; 1 10-21 moles) of ferritin was detected [Hashida, 1990]. The average number of -D-galactosidase labeled antibody Fab' molecules bound per ferritin molecule was calculated to be 1.0-1.4. In addition, the detection limit of the β-Dgalactosidase activity was 1 zmol. On the other hand, the average number of bound enzyme-labeled antibody molecules per peptide molecule was calculated to be 0.001-0.01. Therefore, the peptide detection limit of this immunoassay was estimated to be between 0.1- 1 attomoles. Thus, it is likely that detection of 0.1 amol (0.02 µU) of a insulin will be possible if an affinity-purified antibody is used.

As described in the " Introduction ", blood samples have to be collected every 30 min for 2 h to assess the secretion of insulin in the OGTT. If the urinary insulin level reflects the serum insulin level, then the secretion of insulin after glucose ingestion may be easily assessed by measurement of urinary insulin levels. In the ICT-EIA of this study, the urinary insulin levels correlated well with the serum insulin levels at 1 h after glucose ingestion (Fig. 7). Thus, the level of insulin in urine appears to reflect the level in serum suggesting that measurement of insulin in urine may be useful for assessing insulin secretion.

A comparison of the insulin levels in urine collected early in the morning from NDNO and NDO subjects and from DM patients showed that the levels were significantly higher in NDO subjects and in DM patients than in NDNO subjects (P < 0.01 and P < 0.001 respectively) (Fig. 8). These results indicate that an increase in insulin levels may be a clinically important biomarker of obesity and diabetes risk.

We previously developed a highly sensitive ELISA for urinary growth hormone (GH) [Hashida, 1987, Sukegawa, 1988], which showed that assay of GH during nocturia was useful as a supporting diagnostic index of the pituitary dwarf. We have also recently developed an ultra-sensitive ICT-EIA for urinary soluble human insulin receptor ectodomain (sIR) and found that urinary sIR levels correlated well with sIR blood levels [Umehara, 2009]. Furthermore, the levels of urinary sIR in DM patients were significantly higher than those in NDO subjects and DM patients. Therefore, urinary biomarkers can be useful for both physiological and clinical studies.

In this study, urine samples with micro- and macro-albuminuria were excluded. Urinary insulin and resistin levels were expressed as ratios of urinary concentration to milligrams of urinary creatinine. Urinary insulin levels correlated well with urinary resistin levels (r = 0.426, P < 0.01) (Fig. 10). Further studies with larger numbers of patients will be needed to determine the cut-off value and criteria for insulin as a diabetic risk biomarker.

Development of an Ultra-Sensitive Enzyme Immunoassay for Insulin

*Journal of Clinical Laboratory Analysis*, 2, pp161-167

97-119, Kodansha Scientific Ltd., Tokyo, Japan

*Analytical Letters*, 21, pp1141-1154

Amsterdam, Netherlands.

329

and Its Application to the Evaluation of Diabetic Risk by Analysis of Morning Urine 99

Hashida S, Ishikawa E, Kato Y, Imura H, Mohri Z, Murakami Y. (1987). Human growth

Hashida S, Tanaka K, Kohno T, Ishikawa E. (1988b). Novel and ultrasensitive sandwich

Hashida S, Ishikawa E. (1990). Detection of one milliattomole of ferritin by novel and ultrasensitive enzyme immunoassay. *Journal of Biochemistry*, 108, pp960-964 Hashida S, Ishikawa E, Mukoyama M, Nakao K, Imura H. (1991). Highly sensitive two-site

Hashida S, Tanaka K, Yamamoto N, Ishikawa E. (1993). Ultrasensitive enzyme

Hashida S, Kitamura K, Nagatomo Y, Shibata Y, Imamura T, Yamada K, Fujimoto S, Kato J,

and patients with cardiovascular disease. *Clinical Biochemistry*, 37, pp14-21 Ishikawa E, Imagawa M, Hashida S. (1983a). Ultrasensitive enzyme immunoassay using

Ishikawa E, Imagawa M, Hashida S, Yoshitake S, Hamaguchi Y, Ueno T. (1983b). Enzyme-

Katakami H, Hashida S, Oki Y, Murakami O, Ikenoue T, Ono H, Matsukura S. (2002).

Lindström T, Hedman CA, Arnqvist HJ. (2002). Use of a novel double-antibody technique to

immunohistochemical staining. *Journal of Immunoassay*, 4, pp209-227 Ishikawa E, Hashida S, Tanaka K, Kohno T. (1989). Ultrasensitive enzyme immunoassay for

sensitivity. *Developments in Immunology*, 18, pp219-232

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and plasma samples using one extraction method. *Acta endocrinologica*, 106, pp317-

hormone (hGH) in urine and its correlation to serum hGH examined by a highly sensitive sandwich enzyme immunoassay. *Clinica chimica acta*, 162, pp229-235 Hashida S, Ishikawa E, Mukoyama M, Nakao K, Imura H. (1988a). Direct measurement of a-

human atrial natriuretic polypeptide in plasma by sensitive enzyme immunoassay.

enzyme immunoassay (sandwich transfer enzyme immunoassay) for antigens.

enzyme immunoassays for human atrial natriuretic polypeptides. in Atrial and Brain Natriuretic Peptides , *Proceedings of the Kyoto Symposium on ANP (1988),* pp.

immunoassay for peptide. *Methods in neurotransmitter and neuropeptide research*, pp.

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Morishita K, Eto T. (2004). Development of an ultra-sensitive enzyme immunoassay for human proadrenomedullin N-terminal 20 peptide (PAMP) and direct measurement of two molecular forms of PAMP in plasma from healthy subjects

fluorogenic, luminogenic, radioactive and related substrates and factors to limit the

labeling of antibodies and their fragments for enzyme immunoassay and

antigens: technology and applications — a review. *Clinical Chemistry and*

Immunoreactive corticotropin-releasing hormone (CRH) in plasma and hypothalamic incubation media as assessed by a novel and highly sensitive immune complex transfer EIA. *Clincal Pediatrics Endocrinology*, 11(Suppl 17), pp93-

describe the pharmacokinetics of rapid-acting insulin analogs. *Diabetes Care*, 25,

Insulin and resistin levels in the urine of 42 DM patients were measured and are expressed relative to urinary creatinine (Cre). The regression equation and the calculated correlation coefficient (r = 0.426, P < 0.01) are shown.

Fig. 10. Correlation between urinary insulin and resistin levels.

In summary, we have presented a new ultra-sensitive enzyme immunoassay for insulin, the ICT-EIA. This ICT-EIA can specifically measure insulin in urine, without any requirement for sample extraction or concentration. Urinary insulin and resistin levels were increased in patients with diabetes. ICT-EIA may be useful for both physiological and clinical studies of diabetic risk and urinary insulin may be used as a non-invasive maker that is relative to serum insulin. Therefore, measurement of urinary insulin levels will be useful in studies on dietary counseling and exercise guidance for patients with diabetes.

#### **5. Acknowledgments**

This study was supported in part by Grant-in-Aids for the Promotion of Science and Technology in Regional Areas (Life Science) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The authors thank Y. Yamahashi for assistance with experiments.

#### **6. References**

Insulin and resistin levels in the urine of 42 DM patients were measured and are expressed relative to urinary creatinine (Cre). The regression equation and the calculated correlation coefficient (r = 0.426, P <

In summary, we have presented a new ultra-sensitive enzyme immunoassay for insulin, the ICT-EIA. This ICT-EIA can specifically measure insulin in urine, without any requirement for sample extraction or concentration. Urinary insulin and resistin levels were increased in patients with diabetes. ICT-EIA may be useful for both physiological and clinical studies of diabetic risk and urinary insulin may be used as a non-invasive maker that is relative to serum insulin. Therefore, measurement of urinary insulin levels will be useful in studies on

This study was supported in part by Grant-in-Aids for the Promotion of Science and Technology in Regional Areas (Life Science) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The authors thank Y. Yamahashi for assistance with

Aikawa T, Suzuki S, Murayama M, Hashiba K, Kitagawa T, Ishikawa E. (1979). Enzyme

Fyhrquist F, Soveri P, Puutula L, Stenman U-H. (1976). Radioimmunoassay of plasma renin

Glänzer K, Appenheimer M, Krück F, Vetter W, Vetter H. (1984). Measurement of 8-

arginine-vasopressin by radioimmunoassay: development and application to urine

Fig. 10. Correlation between urinary insulin and resistin levels.

dietary counseling and exercise guidance for patients with diabetes.

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activity. *Clinical Chemistry*, 22, pp250-256

0.01) are shown.

**5. Acknowledgments**

experiments.

**6. References**

and plasma samples using one extraction method. *Acta endocrinologica*, 106, pp317- 329

**8**

*Bulgaria*

**Ovarian Biomarkers in Infertility**

Soren Hayrabedyan and Maria D. Ivanova

*Bulgarian Academy of Sciences,*

Ivailo Vangelov, Julieta Dineva, Krassimira Todorova,

*Institute of Biology and Immunology of Reproduction "Acad. K. Bratanov",*

The female infertility is a complex phenomenon that at least in part resides in the manifestation, possible perturbations and interplay of innate and adaptive immunity, with the participation of apoptotic and reactive oxygen species pathways, and some paracrine factors as causative and implementing factors in this process. The spread of the assisted reproduction technologies provided an unseen before opportunity to the intimacy of the ovarian follicle – the oocyte cellular surroundings and the oocyte itself, enabling research and medical community to boost the pursuit of new ovarian function biomarkers in the context of gamete fertilization and embryo development competence. The notion of infertility markers would be presented in a comprehensive viewpoint based on the importance of the folliculogenesis, oocyte-cumulus-granulosa cells interactions and oocyte genesis. This approach is aimed on the provision of non-invasive means for oocyte and

The human ovary can be target of an autoimmune attack in various circumstances, including several organ-specific or systemic autoimmune diseases. One important issue concerning autoimmunity and infertility is the role of autoimmune responses against ovary,

Premature Ovarian Failure (POF) or Primary Ovarian Insufficiency (POI)) is the loss of function of the ovaries before age of 40. It has been estimated that POF affects 1% of the population (Chatterjee et al., 2007). Hormonally, POF is defined by abnormally low levels of estrogen and high levels of follicle stimulating hormone (FSH), which demonstrate that the ovaries are no longer responding to circulating FSH by producing estrogen and developing

One of the reasons to suspect an autoimmune etiology of POF is the frequent association of POF with some organ-specific autoimmune diseases but the absence of associated disease cannot exclude an autoimmune mechanism. Among organ - specific autoimmune diseases, the thyroid and adrenal diseases (Addison's disease) are frequently associated with POF. In

embryo competence evaluation and improved reproductive medicine outcome.

adrenal and thyroid glands in the development of premature ovarian failure.

**2. Autoimmunity and ovarian function impairment**

**2.1 Anti-ovarian antibodies and infertility**

**1. Introduction**

fertile oocyte.

### **Ovarian Biomarkers in Infertility**

Ivailo Vangelov, Julieta Dineva, Krassimira Todorova, Soren Hayrabedyan and Maria D. Ivanova *Institute of Biology and Immunology of Reproduction "Acad. K. Bratanov", Bulgarian Academy of Sciences, Bulgaria*

#### **1. Introduction**

100 Trends in Immunolabelled and Related Techniques

Morton JJ, Padfield PL, Forsling ML. (1975). A radioimmunoassay for plasma arginine-

Mukoyama M, Nakao K, Yamada T, Itoh H, Sugawara A, Saito Y, Arai H, Hosoda K,

Scharpé S, Verkerk R, Sasmito E, Theeuws M. (1987). Enzyme immunoassay of angiotensin I

Sukegawa I, Hizuka N, Takano K, Asakawa K, Horikawa R, Hashida S, Ishikawa E, Mohri

Tikkanen I, Fyhrquist F, Metsärinne K, Leidenius R. (1985). Plasma atrial natriuretic peptide in cardiac disease and during infusion in healthy volunteers. *Lancet*, 2, 66-69 Umehara A, Nishioka M, Obata T, Ebina Y, Shiota H, Hashida S. (2009). A novel ultra-

Uno T, Uehara K, Motomatsu K, Ishikawa E, Kato K. (1982). Enzyme immunoassay for

Yamaga J, Hashida S, Kitamura K, Tokashiki M, Aoki T, Inatsu H, Ishikawa N, Kangawa K,

transfer enzyme immunoassay in rat. *Hypertension research*, 26, pp45-53.

*Journal Endocrinology*, 65, pp411-424

*Communications*, 151, pp1277-1284

and renin. *Clinical Chemistry*, 33, pp1774-1777

*endocrinology and metabolism*, 66, pp1119-1123

mellitus. *Clinical Biochemistry*, 42, pp1468-1475

arginine vasopressin. *Experientia*, 38, pp786-787

vasopressin in man and dog: application to physiological and pathological states.

Shirakami G, Morii N, Shiono S, Imura H. (1988). A monoclonal antibody against N-terminus of a-atrial natriuretic polypeptide (a-ANP): a useful tool for preferential detection of naturally circulating ANP. *Biochemical and Biophysical Research*

Z, Murakami Y, Shimizu K. (1988). Urinary growth hormone (GH) measurements are useful for evaluating endogenous GH secretion. *The Journal of clinical*

sensitive enzyme immunoassay for soluble human insulin receptor ectodomain and its measurement in urine from healthy subjects and patients with diabetes

Morishita K, Eto T. (2003). Direct measurement of glycine-extended adrenomedullin in plasma and tissue using an ultrasensitive immune complex The female infertility is a complex phenomenon that at least in part resides in the manifestation, possible perturbations and interplay of innate and adaptive immunity, with the participation of apoptotic and reactive oxygen species pathways, and some paracrine factors as causative and implementing factors in this process. The spread of the assisted reproduction technologies provided an unseen before opportunity to the intimacy of the ovarian follicle – the oocyte cellular surroundings and the oocyte itself, enabling research and medical community to boost the pursuit of new ovarian function biomarkers in the context of gamete fertilization and embryo development competence. The notion of infertility markers would be presented in a comprehensive viewpoint based on the importance of the folliculogenesis, oocyte-cumulus-granulosa cells interactions and oocyte genesis. This approach is aimed on the provision of non-invasive means for oocyte and embryo competence evaluation and improved reproductive medicine outcome.

#### **2. Autoimmunity and ovarian function impairment**

#### **2.1 Anti-ovarian antibodies and infertility**

The human ovary can be target of an autoimmune attack in various circumstances, including several organ-specific or systemic autoimmune diseases. One important issue concerning autoimmunity and infertility is the role of autoimmune responses against ovary, adrenal and thyroid glands in the development of premature ovarian failure.

Premature Ovarian Failure (POF) or Primary Ovarian Insufficiency (POI)) is the loss of function of the ovaries before age of 40. It has been estimated that POF affects 1% of the population (Chatterjee et al., 2007). Hormonally, POF is defined by abnormally low levels of estrogen and high levels of follicle stimulating hormone (FSH), which demonstrate that the ovaries are no longer responding to circulating FSH by producing estrogen and developing fertile oocyte.

One of the reasons to suspect an autoimmune etiology of POF is the frequent association of POF with some organ-specific autoimmune diseases but the absence of associated disease cannot exclude an autoimmune mechanism. Among organ - specific autoimmune diseases, the thyroid and adrenal diseases (Addison's disease) are frequently associated with POF. In

Ovarian Biomarkers in Infertility 103

Follicle dysfunction could be associated with inadequate follicular function, for example, due to mutated FSH- receptor gene (Doherty et al., 2002), but also due to autoimmune ovarian disease damaging the maturing follicles, but at the same time leaving the primordial follicles intact. In the majority of the cases, the cause of POF is unknown, but some of the causes can lead to absence of oocytes, whereas others can lead to disordered follicle maturation. However, several studies have suggested that some cases of POF could be a

Strong support for an autoimmune character of isolated POF would be the presence of antibodies to ovarian structures or AOA in the serum of these patients. Evidence favors the presence of an autoimmune disease of the ovary. In some cases AOA has been considered to be a suitable marker for identification of autoimmune POF (Luborsky et al., 1990, 2000, Fénichel et al 1997, Pires et al., 2006) as well as immunological- related infertility in "*in vitro"* fertilization (IVF) patients (Gobert et al., 1992, Ivanova et al. 1994, 1999, Barbarino-Monnier et al., 2003, Pires et al, 2007). Screen for the presence of AOA before the initiation of the IVF cycle could be recommended, since AOA are thought to be associated with poor results in infertility patients included in the programs for assisted reproduction (Pires et al., 2007).

Other ovarian disturbances as Polycystic Ovarian Syndrome (PCOS), endometriosis, and unexplained infertility are associated with anti-ovarian autoimmunity. Approximately 15% to 30% of couples will be diagnosed with unexplained infertility after their diagnostic workup (Practice Committee of the American Society for Reproductive Medicine, 2006). The presence of AOA has been detected in patients with unexplained infertility (Luborsky et., al 1999, 2000, 2002). It has been demonstrated that AOA could be detected several months or years before the onset of the clinical symptoms without affecting the serum FSH or inhibin B levels (Luborsky et al 2000). Collectively these findings suggest that the AOA are

PCOS is of clinical and public health importance as it is very common, affecting up to one in five women of reproductive age. The prevalence of PCOS depends on the choice of diagnostic criteria. One community-based prevalence study using the Rotterdam criteria found that about 18% of women had PCOS, and that 70% of them were previously undiagnosed (Teede et al., 2010). The association of ovarian autoimmunity with PCOS has been previously evaluated (Luborsky et al., 1999). It has been found that the frequency of AOA is similar (25%) among the normally cycling women and PCOS patients. The possible development of humoral autoimmunity and production of AOA consequent to ovarian damage has also been determined in clomiphene citrate-resistant PCOS patients (Alborzi et

Autoimmunity to endometrium and ovary has been studied in patients with endometriosis (Mathur et al., 1982). It has been demonstrated the presence of elevated autoantibody titers to whole ovary, theca cells, granulosa cells and endometrium in the patient sera, and to GLCs in the cervical secretions. Endometriosis and PCOS have been associated with higher values of anti-FSH IgA autoantibodies, and endometriosis also has association with anti-FSH IgG autoantibodies). One of the major epitopes identified was a 78-93 amino acid sequence belonging to part of the human FSH beta-chain (Gobert et al., 2001). This autoantigenic determinant is recognized most frequently by human serum samples from women with AOA. Anti-FSH are thought to be naturally occurring autoantibodies associated with peripheral FSH concentrations, but increased in infertile women with

direct result of autoimmune-mediated damage of the ovaries (Hoek et al., 1997).

independent markers for the future onset of ovarian failure.

al., 2009).

regard to association with adrenal autoimmunity (Addison's disease) three different situations have to be distinguished: POF associated with adrenal autoimmunity, POF associated with nonadrenal autoimmunity, and isolated or idiopathic POF (Forges et al., 2004). Among POF patients with adrenal autoimmunity 4–5% are positive for autoantibodies directed against steroidogenic cytochrome P450 enzymes, such as 21-hydroxylase autoantibodies, 17α-hydroxylase autoantibodies and cholesterol side-chain cleavage enzyme (side-chain cleavage autoantibodies or steroid cell autoantibodies, SCAs)(Chen et al., 1996). Positivity for these autoantibodies identifies POF due to autoimmunity to steroid-producing cells - SCA-POF (Hoek et al., 1997, Betterle et al., 2002, Bakalov et al., 2005). In these patients the ovarian pathology has been associated with selective mononuclear cell infiltration of large antral follicles (Hoek et al., 1997) and with autoimmune destruction of theca cells (Welt et al 2005). The autoimmune damage of the ovarian theca cells and adrenal cortex are thought to be a T cell-mediated process (Hoek et al., 1997) as POF patients with adrenal autoimmunity have predominantly expressed IgG1 isotype SCA (Brozzetti et al., 2010). The predominant IgG1 isotype of the specific autoantibodies can be interpreted as a sign of Th1 immune response (Hawa et al., 2000). The presence of SCA is a risk factor for development of POF. Relatively higher percentage of women with normal menstrual cycle, who have polyendocrinopathy and SCA become susceptible to ovarian failure development within 8– 15 years). The pathophysiological significance of SCA seems to be restricted to patients whose ovarian failure is associated with adrenal autoimmunity. These antibodies are very useful markers of ovarian failure in Addison's disease patients. In 90% of these patients at least one type of SCA can be detected (Falorni et al., 2002) whereas the prevalence of SCA in <10% of cases has been detected in the patients with POF associated with nonadrenal autoimmunity and in the patients with idiopathic POF (Dal Pra et al., 2003).

On the other hand, two-thirds of women with recently diagnosed POF due to SCAassociated autoimmunity have normal serum Anti-Müllerian hormone (AMH) concentrations (La Marca et al., 2010). AMH is expressed by granulose luteinized cells (GLCs) of the ovary during the reproductive years, and controls the formation of primary follicles by inhibiting excessive follicular recruitment by FSH. It therefore, has a role in folliculogenesis and some authors suggest it is a measure of certain aspects of ovarian function (Broer 2011) and useful in assessing conditions such as polycystic ovary syndrome (PCOS) and POF. However, the normal AMH levels in women recently diagnosed for SCA-POF imply that this form of ovarian failure have association with a preserved pool of functioning follicles (La Marca et al., 2010). Indeed, in such patients this is selective autoimmune destruction of theca cells with preservation of GLCs that produce low amounts of estradiol because of lack of substrates. Therefore, the more exact term of this complication, as well as other forms of ovarian failure, would be primary ovarian insufficiency (POI) (Nelson 2009), since the term POI implies more appropriately the continuous nature of the impaired ovarian function.

Among systemic autoimmune diseases, the systemic lupus erithemathosus (SLE) is thought to be most frequently associated with POF (Hoek et al., 1997). Anti- corpus luteum antibodies could represent the first stage of altered ovarian function in SLE patients (Pasoto et al., 1999). An autoantigenic 67 kDa target recognized by autoantibodies from 22% of patients (age<40) with SLE has been described to correlate with elevated serum FSH levels. In general, anti ovarian antibodies (AOA) were found in 84% of the cases of female SLE patients (Moncayo-Naveda et al., 1989).

regard to association with adrenal autoimmunity (Addison's disease) three different situations have to be distinguished: POF associated with adrenal autoimmunity, POF associated with nonadrenal autoimmunity, and isolated or idiopathic POF (Forges et al., 2004). Among POF patients with adrenal autoimmunity 4–5% are positive for autoantibodies directed against steroidogenic cytochrome P450 enzymes, such as 21-hydroxylase autoantibodies, 17α-hydroxylase autoantibodies and cholesterol side-chain cleavage enzyme (side-chain cleavage autoantibodies or steroid cell autoantibodies, SCAs)(Chen et al., 1996). Positivity for these autoantibodies identifies POF due to autoimmunity to steroid-producing cells - SCA-POF (Hoek et al., 1997, Betterle et al., 2002, Bakalov et al., 2005). In these patients the ovarian pathology has been associated with selective mononuclear cell infiltration of large antral follicles (Hoek et al., 1997) and with autoimmune destruction of theca cells (Welt et al 2005). The autoimmune damage of the ovarian theca cells and adrenal cortex are thought to be a T cell-mediated process (Hoek et al., 1997) as POF patients with adrenal autoimmunity have predominantly expressed IgG1 isotype SCA (Brozzetti et al., 2010). The predominant IgG1 isotype of the specific autoantibodies can be interpreted as a sign of Th1 immune response (Hawa et al., 2000). The presence of SCA is a risk factor for development of POF. Relatively higher percentage of women with normal menstrual cycle, who have polyendocrinopathy and SCA become susceptible to ovarian failure development within 8– 15 years). The pathophysiological significance of SCA seems to be restricted to patients whose ovarian failure is associated with adrenal autoimmunity. These antibodies are very useful markers of ovarian failure in Addison's disease patients. In 90% of these patients at least one type of SCA can be detected (Falorni et al., 2002) whereas the prevalence of SCA in <10% of cases has been detected in the patients with POF associated with nonadrenal

autoimmunity and in the patients with idiopathic POF (Dal Pra et al., 2003).

continuous nature of the impaired ovarian function.

patients (Moncayo-Naveda et al., 1989).

On the other hand, two-thirds of women with recently diagnosed POF due to SCAassociated autoimmunity have normal serum Anti-Müllerian hormone (AMH) concentrations (La Marca et al., 2010). AMH is expressed by granulose luteinized cells (GLCs) of the ovary during the reproductive years, and controls the formation of primary follicles by inhibiting excessive follicular recruitment by FSH. It therefore, has a role in folliculogenesis and some authors suggest it is a measure of certain aspects of ovarian function (Broer 2011) and useful in assessing conditions such as polycystic ovary syndrome (PCOS) and POF. However, the normal AMH levels in women recently diagnosed for SCA-POF imply that this form of ovarian failure have association with a preserved pool of functioning follicles (La Marca et al., 2010). Indeed, in such patients this is selective autoimmune destruction of theca cells with preservation of GLCs that produce low amounts of estradiol because of lack of substrates. Therefore, the more exact term of this complication, as well as other forms of ovarian failure, would be primary ovarian insufficiency (POI) (Nelson 2009), since the term POI implies more appropriately the

Among systemic autoimmune diseases, the systemic lupus erithemathosus (SLE) is thought to be most frequently associated with POF (Hoek et al., 1997). Anti- corpus luteum antibodies could represent the first stage of altered ovarian function in SLE patients (Pasoto et al., 1999). An autoantigenic 67 kDa target recognized by autoantibodies from 22% of patients (age<40) with SLE has been described to correlate with elevated serum FSH levels. In general, anti ovarian antibodies (AOA) were found in 84% of the cases of female SLE Follicle dysfunction could be associated with inadequate follicular function, for example, due to mutated FSH- receptor gene (Doherty et al., 2002), but also due to autoimmune ovarian disease damaging the maturing follicles, but at the same time leaving the primordial follicles intact. In the majority of the cases, the cause of POF is unknown, but some of the causes can lead to absence of oocytes, whereas others can lead to disordered follicle maturation. However, several studies have suggested that some cases of POF could be a direct result of autoimmune-mediated damage of the ovaries (Hoek et al., 1997).

Strong support for an autoimmune character of isolated POF would be the presence of antibodies to ovarian structures or AOA in the serum of these patients. Evidence favors the presence of an autoimmune disease of the ovary. In some cases AOA has been considered to be a suitable marker for identification of autoimmune POF (Luborsky et al., 1990, 2000, Fénichel et al 1997, Pires et al., 2006) as well as immunological- related infertility in "*in vitro"* fertilization (IVF) patients (Gobert et al., 1992, Ivanova et al. 1994, 1999, Barbarino-Monnier et al., 2003, Pires et al, 2007). Screen for the presence of AOA before the initiation of the IVF cycle could be recommended, since AOA are thought to be associated with poor results in infertility patients included in the programs for assisted reproduction (Pires et al., 2007).

Other ovarian disturbances as Polycystic Ovarian Syndrome (PCOS), endometriosis, and unexplained infertility are associated with anti-ovarian autoimmunity. Approximately 15% to 30% of couples will be diagnosed with unexplained infertility after their diagnostic workup (Practice Committee of the American Society for Reproductive Medicine, 2006). The presence of AOA has been detected in patients with unexplained infertility (Luborsky et., al 1999, 2000, 2002). It has been demonstrated that AOA could be detected several months or years before the onset of the clinical symptoms without affecting the serum FSH or inhibin B levels (Luborsky et al 2000). Collectively these findings suggest that the AOA are independent markers for the future onset of ovarian failure.

PCOS is of clinical and public health importance as it is very common, affecting up to one in five women of reproductive age. The prevalence of PCOS depends on the choice of diagnostic criteria. One community-based prevalence study using the Rotterdam criteria found that about 18% of women had PCOS, and that 70% of them were previously undiagnosed (Teede et al., 2010). The association of ovarian autoimmunity with PCOS has been previously evaluated (Luborsky et al., 1999). It has been found that the frequency of AOA is similar (25%) among the normally cycling women and PCOS patients. The possible development of humoral autoimmunity and production of AOA consequent to ovarian damage has also been determined in clomiphene citrate-resistant PCOS patients (Alborzi et al., 2009).

Autoimmunity to endometrium and ovary has been studied in patients with endometriosis (Mathur et al., 1982). It has been demonstrated the presence of elevated autoantibody titers to whole ovary, theca cells, granulosa cells and endometrium in the patient sera, and to GLCs in the cervical secretions. Endometriosis and PCOS have been associated with higher values of anti-FSH IgA autoantibodies, and endometriosis also has association with anti-FSH IgG autoantibodies). One of the major epitopes identified was a 78-93 amino acid sequence belonging to part of the human FSH beta-chain (Gobert et al., 2001). This autoantigenic determinant is recognized most frequently by human serum samples from women with AOA. Anti-FSH are thought to be naturally occurring autoantibodies associated with peripheral FSH concentrations, but increased in infertile women with

Ovarian Biomarkers in Infertility 105

those with ovarian cancer have autoantibodies to ovarian antigens. Luborsky et al., 2011 showed for the first time that antibodies to mesothelin, a well-characterized ovarian cancer

The antibody-mediated autoreactivity directed towards the granulosa cells has been reported in the sera of POF patients (Damewood et al., 1986, Kelkar et al., 2005). AOA in POF patients showed immunoreactivity against the granulosa cells from both the pre-antral and the antral follicles (Damewood et al., 1986). In small percentage of the anti-ZP positive POF sera, a strong autoreactivity directed to granulosa cells has also been detected (Horejsi et al., 2000, Kelkar et al 2005). A 51 kDa protein has been identified as an additional target of SCA (Winqvist et al., 1998). It has been localized in the placenta and in the GLCs by the sera

We have developed an ELISA assay to assess the prevalence of anti-granulosa cell antibodies and we have estimated it to be 28.7% in the sera and 44.90 % in the follicular fluids (FFls) of infertile patients undergoing controlled ovarian hyper-stimulations (COHS) for IVF-ET (embryo transfer) compared to 9.1% and 6.67% in the fertile women sera and FFls respectively (Vangelov et al., 2005,2006,2009).For the isolation of granulosa cells, pooled follicular fluids were collected individually for each patient, centrifuged at 200 x g and purified using HISTOPAQUE® -1077 (Sigma Diagnostics, Inc.) gradient centrifugation approach (for details, see Vangelov et al., 2005). The results obtained by this ELISA assay disclosed that antibodies against granulosa cell-antigens occurred more frequently in infertile women undergoing IVF-ET than in fertile normally menstruating women (controls) at the same age, inferring to the conclusion that the detection of anti-granulosa cell antibodies serve as an indicator for their involvement in immunological infertility. Auto-antibodies against GLCs antigens may be considered as a part of the heterogeneous group of AOA (Ghazeeri & Kutteh 2001). The prevalence of anti-granulosa cell autoantibodies in the subgroup with low fertilization rate suggests a negative impact of the anti-granulosa cell autoantibodies on the fertilization outcome. It was suggested that the binding of these antibodies to follicular cells might be a defective signal in the fertilization process (Horejsi et al., 2000). Anti-granulosa

In addition to the mentioned above, the identification of putative granulosa cell antigens recognizable by the antibodies in the infertile women sera, but not by the fertile controls sera is of huge importance. Four undefined GLC autoantigens - 110 kD, 70-80 kD, 47 kD and 37 kD have been identified as autoantigenic targets for the anti-GLCs autoantibodies using immunoblotting assay developed by us (Vangelov et al., 2005). The results from immunoblotting revealed that: 3 patient's sera and none control sera reacted with protein band with MM 37 kD; 14 patient's sera and 3 control sera - at 47 kD; 10 patient's sera and 1 control serum - at 70-80 kD and 8 patient's sera and 3 control sera - at 110 kD. Four patient's sera reacted with all above protein bands. The band with approximate MM of 47 kD was the most frequently occurring antigen in infertile women and the band of 37 kD reacted only

**2.2 Cellular immunity, pro-inflammatory cytokines and apoptosis in infertility**

The diagnosis of an autoimmune mechanism involvement in infertile patients relies not only on the presence of autoantibodies directed to ovary, but also on the dynamics of immune cell populations and their soluble products. Natural killer cells (NK cells) constitute a major

antigen, occur in some of the women with epidemiologic risk for ovarian cancer.

of patients with adrenal insufficiency and associated ovarian failure.

cell autoantibodies could play a role as a marker of fertilization failure.

with sera from infertile patients studied.

dysregulation of immune reactions and repeatedly performed in vitro fertilization (IVF) (Haller et al., 2005).

Significant progress was made towards the identification of candidate biomarkers involved in human ovarian autoimmunity. The presence of anti-oocyte antibodies has been demonstrated in the sera of POF patients (Damewood et al., 1986) with or without associated autoimmune diseases (Luborsky et al., 1990) and in the sera of infertile patients (Pires et al., 2006, 2007), as well as in mice after neonatal thymectomy (Tong & Nelson 1999). In the mouse model of autoimmune ovarian failure, the autoantigenic oocyte protein has been identified and called Maternal Antigen That Embryos Require (MATER), (Tong & Nelson, 1999). Using specific and sensitive immunoassays (Pires et al., 2007) demonstrated that AOA in infertile women identified an immunodominant antigen- heat shock protein 90 beta, which was localized predominantly in the oocyte cytoplasm. Therefore, anti-HSP90 antibodies could be used as one of the diagnostic markers for ovarian failure and thereby infertility (Pires et al., 2007).

Anti-Zona pellucida autoantibodies bind to the oocyte Zona pellucida (ZP), and therefore, could prevent sperm-zona interaction (Kyurkchiev et al., 1988, Barber & Fayrer Hosken, 2000). Zona pellucida proteins play critical role in oocyte fertilization. Anti-ZP autoantibodies have been linked with human female infertility (Ivanova et al.1994, 1999, Mardesic et al., 2000, Kelkar et al., 2005, Koyama, 2010). Intra cytoplasmic sperm injection (ICSI) is recommended option for IVF therapy of women with anti-ZP autoantibodies (Mardesic et al., 2000). Anti- ZP autoantibodies may induce autoimmune-mediated ovarian damage with follicular depletion and fertility suppression due to impaired ZP formation (Lloyd et al., 2010) and disruption of the gap junctions between the oocyte and granulosa cells, damaging the bidirectional communication necessary for normal folliculogenesis (Calongos et al., 2009).

In addition, novel immunotherapy strategy has been proposed recently for treatment of autoimmune ovarian disease (Li et al., 2008). The continuous presence of physiologicallyexpressed autoantigen is critical for both tolerance maintenance and autoimmune disease pathogenesis, thus the outcome is determined by the integrity of regulatory T cells, whereas deficiency in the regulatory T-cell function is associated with elevated responsiveness to autoantigen and to environmental stimuli that could potentially promote the autoimmune disease and memory (Tung et al., 2005). Sundblad et al., 2006 showed the presence of circulating autoantibodies directed towards an antigen of approximately 50 kDa, identified subsequently as alpha-enolase. It has been demonstrated that 21 of 110 women with autoimmune POF express this autoantibody, whereas 60 control subjects proved negative. Aldehyde (retinal) dehydrogenase 1A1 (ALDH1A1) and selenium-binding protein 1 (SBP1) are identified as unique autoantigenic targets associated with unexplained infertility (Edassery et al., 2010).

More recently, three immunodominant ovarian autoantigens, α-actinin 4 (αACTN4), heat shock 70 protein 5 (HSPA5) and β-actin (ACTB), have been identified using AOA -positive sera from women with idiopathic POF and women undergoing IVF (Mande et al., 2011). These autoantigens are localized in different structures of the ovary such as the ooplasm of the oocyte, theca, granulosa, corpus luteum and zona pellucida. All the above antigens were found to be expressed in the ooplasm throughout follicular development, and all of them were expressed specifically in the oocyte except αACTN4. Both women with infertility and

dysregulation of immune reactions and repeatedly performed in vitro fertilization (IVF)

Significant progress was made towards the identification of candidate biomarkers involved in human ovarian autoimmunity. The presence of anti-oocyte antibodies has been demonstrated in the sera of POF patients (Damewood et al., 1986) with or without associated autoimmune diseases (Luborsky et al., 1990) and in the sera of infertile patients (Pires et al., 2006, 2007), as well as in mice after neonatal thymectomy (Tong & Nelson 1999). In the mouse model of autoimmune ovarian failure, the autoantigenic oocyte protein has been identified and called Maternal Antigen That Embryos Require (MATER), (Tong & Nelson, 1999). Using specific and sensitive immunoassays (Pires et al., 2007) demonstrated that AOA in infertile women identified an immunodominant antigen- heat shock protein 90 beta, which was localized predominantly in the oocyte cytoplasm. Therefore, anti-HSP90 antibodies could be used as one of the diagnostic markers for ovarian failure and thereby

Anti-Zona pellucida autoantibodies bind to the oocyte Zona pellucida (ZP), and therefore, could prevent sperm-zona interaction (Kyurkchiev et al., 1988, Barber & Fayrer Hosken, 2000). Zona pellucida proteins play critical role in oocyte fertilization. Anti-ZP autoantibodies have been linked with human female infertility (Ivanova et al.1994, 1999, Mardesic et al., 2000, Kelkar et al., 2005, Koyama, 2010). Intra cytoplasmic sperm injection (ICSI) is recommended option for IVF therapy of women with anti-ZP autoantibodies (Mardesic et al., 2000). Anti- ZP autoantibodies may induce autoimmune-mediated ovarian damage with follicular depletion and fertility suppression due to impaired ZP formation (Lloyd et al., 2010) and disruption of the gap junctions between the oocyte and granulosa cells, damaging the bidirectional communication necessary for normal folliculogenesis

In addition, novel immunotherapy strategy has been proposed recently for treatment of autoimmune ovarian disease (Li et al., 2008). The continuous presence of physiologicallyexpressed autoantigen is critical for both tolerance maintenance and autoimmune disease pathogenesis, thus the outcome is determined by the integrity of regulatory T cells, whereas deficiency in the regulatory T-cell function is associated with elevated responsiveness to autoantigen and to environmental stimuli that could potentially promote the autoimmune disease and memory (Tung et al., 2005). Sundblad et al., 2006 showed the presence of circulating autoantibodies directed towards an antigen of approximately 50 kDa, identified subsequently as alpha-enolase. It has been demonstrated that 21 of 110 women with autoimmune POF express this autoantibody, whereas 60 control subjects proved negative. Aldehyde (retinal) dehydrogenase 1A1 (ALDH1A1) and selenium-binding protein 1 (SBP1) are identified as unique autoantigenic targets associated with unexplained infertility

More recently, three immunodominant ovarian autoantigens, α-actinin 4 (αACTN4), heat shock 70 protein 5 (HSPA5) and β-actin (ACTB), have been identified using AOA -positive sera from women with idiopathic POF and women undergoing IVF (Mande et al., 2011). These autoantigens are localized in different structures of the ovary such as the ooplasm of the oocyte, theca, granulosa, corpus luteum and zona pellucida. All the above antigens were found to be expressed in the ooplasm throughout follicular development, and all of them were expressed specifically in the oocyte except αACTN4. Both women with infertility and

(Haller et al., 2005).

infertility (Pires et al., 2007).

(Calongos et al., 2009).

(Edassery et al., 2010).

those with ovarian cancer have autoantibodies to ovarian antigens. Luborsky et al., 2011 showed for the first time that antibodies to mesothelin, a well-characterized ovarian cancer antigen, occur in some of the women with epidemiologic risk for ovarian cancer.

The antibody-mediated autoreactivity directed towards the granulosa cells has been reported in the sera of POF patients (Damewood et al., 1986, Kelkar et al., 2005). AOA in POF patients showed immunoreactivity against the granulosa cells from both the pre-antral and the antral follicles (Damewood et al., 1986). In small percentage of the anti-ZP positive POF sera, a strong autoreactivity directed to granulosa cells has also been detected (Horejsi et al., 2000, Kelkar et al 2005). A 51 kDa protein has been identified as an additional target of SCA (Winqvist et al., 1998). It has been localized in the placenta and in the GLCs by the sera of patients with adrenal insufficiency and associated ovarian failure.

We have developed an ELISA assay to assess the prevalence of anti-granulosa cell antibodies and we have estimated it to be 28.7% in the sera and 44.90 % in the follicular fluids (FFls) of infertile patients undergoing controlled ovarian hyper-stimulations (COHS) for IVF-ET (embryo transfer) compared to 9.1% and 6.67% in the fertile women sera and FFls respectively (Vangelov et al., 2005,2006,2009).For the isolation of granulosa cells, pooled follicular fluids were collected individually for each patient, centrifuged at 200 x g and purified using HISTOPAQUE® -1077 (Sigma Diagnostics, Inc.) gradient centrifugation approach (for details, see Vangelov et al., 2005). The results obtained by this ELISA assay disclosed that antibodies against granulosa cell-antigens occurred more frequently in infertile women undergoing IVF-ET than in fertile normally menstruating women (controls) at the same age, inferring to the conclusion that the detection of anti-granulosa cell antibodies serve as an indicator for their involvement in immunological infertility. Auto-antibodies against GLCs antigens may be considered as a part of the heterogeneous group of AOA (Ghazeeri & Kutteh 2001). The prevalence of anti-granulosa cell autoantibodies in the subgroup with low fertilization rate suggests a negative impact of the anti-granulosa cell autoantibodies on the fertilization outcome. It was suggested that the binding of these antibodies to follicular cells might be a defective signal in the fertilization process (Horejsi et al., 2000). Anti-granulosa cell autoantibodies could play a role as a marker of fertilization failure.

In addition to the mentioned above, the identification of putative granulosa cell antigens recognizable by the antibodies in the infertile women sera, but not by the fertile controls sera is of huge importance. Four undefined GLC autoantigens - 110 kD, 70-80 kD, 47 kD and 37 kD have been identified as autoantigenic targets for the anti-GLCs autoantibodies using immunoblotting assay developed by us (Vangelov et al., 2005). The results from immunoblotting revealed that: 3 patient's sera and none control sera reacted with protein band with MM 37 kD; 14 patient's sera and 3 control sera - at 47 kD; 10 patient's sera and 1 control serum - at 70-80 kD and 8 patient's sera and 3 control sera - at 110 kD. Four patient's sera reacted with all above protein bands. The band with approximate MM of 47 kD was the most frequently occurring antigen in infertile women and the band of 37 kD reacted only with sera from infertile patients studied.

#### **2.2 Cellular immunity, pro-inflammatory cytokines and apoptosis in infertility**

The diagnosis of an autoimmune mechanism involvement in infertile patients relies not only on the presence of autoantibodies directed to ovary, but also on the dynamics of immune cell populations and their soluble products. Natural killer cells (NK cells) constitute a major

Ovarian Biomarkers in Infertility 107

patients with ovarian failure did not lower the elevated number of peripheral B cells (Hoek et al., 1995). Similar immunological profile has been shown in patients with POI and in patients with POF (van Kasteren et al., 2000), suggesting that POI is a light form of POF. It has been found a decrease in NK cells and in percentage of T-suppressor cells with a rise in T-helper/T-suppressor cell ratio, as well as an increase in B- cells and HLA-DR+ T cells. The question remains whether these changes are the cause or the consequence of the ovarian failure. Autoimmune damage of the ovary could be the primary cause of POF, whereas in natural menopause autoimmunity is a result of hormone dysfunction (Chernyshov et al.,

The variations of cellular immunity function and its relationship with AOA (Huang et al., 1996) and with anti-granulosa cells autoantibodies (Vangelov et al., 2009) have been investigated in patients with ovarian failure. The patients with ovarian failure have increased AOA, CD4+ T-cells and CD4+/CD8+ ratio. It has been shown also, that AOA positive rate in POF patients is associated with higher CD4+/CD8+ ratio (Huang et al.,

In our study, we used our propriate granulosa cell autoantibody ELISA detection assay, and commercially available TNF-α and IFN-γ ELISA kits (eBioscience, USA) to estimate the abundance of granulosa cell autoantibodies, and the concentrations of TNF-α and IFN-γ in FFls, as well as DNA-DAPI staining for the assessment of granulosa cells apoptotic index in ovarian failure patients. The obtained results reveled several findings: 1) significantly higher levels of IFN-γ, TNF-α and apoptotic cells in women with positive autoantibody ELISA test, and 2) significant correlation of anti- granulosa autoantibodies, and apoptotic index, as well as significant correlation of IFN-γ with TNF-α and apoptotic index. Moreover, we have reported (Vangelov et al., 2008, 2009), that cellular immunity (TNF-α, IFN-γ) and humoral immunity (anti-granulosa cell autoantibodies) to granulosa autoantigens can be concurrently produced in patients with ovarian failure, and we suggested that pathogenesis of ovarian failure may be associated with immune factors and apoptosis of granulosa cells. In this scenario the programmed cell dead (apoptosis) of granulosa cells could be very important player. The ovulatory process represents the classical sequence of an acute sterile inflammation under surveillance of innate immune function (Medzhitov, 2010, Rock et al 2010). The cell damage is associated with the release of danger signals-alarmins (Bianchi 2007). In the preovulatory follicle main source of danger signals are damaged granulosa cells (Spanel-Borowski 2011) and the danger signals could be recognized by CK+(cytokeratin-positive) granulosa cells via TLR-4 or other pattern-recognition receptors (PRRs) (Serke et al., 2009, 2010). The ovarian innate immune function will be activated to orchestrate the ovulatory process (Spanel-Borowski, 2011). CK+ cells are assumed to switch off the CK genes in the corpus luteum (CL) stage of secretion and regression to become granulosa-like cells. The granulosa-like cells are thought to be mature DC in the CL (Spanel-Borowski, 2011). However, the low T cell number in the preovulatory follicle wall is signature for a limited interaction with the adaptive immunity. A disturbed conversation between innate and adaptive immunity is reflected by altered T cell profiles in the FFls of patients with idiopathic infertility and in ovaries with POF due to autoimmune damage (Vujovic 2009, Spanel-Borowski 2011). Because DCs process antigens and train naive T cells to become helper and suppressor cells (Banchereau & Steinman, 1998, Turvey & Broide, 2010), DCs are players between innate and adaptive immunity. The pathway of innate immunity is well balanced to achieve the quick recovery of tissue integrity, but an

2001).

1996).

component of the innate immune system. NK cells have a suppressive action on B cells, and therefore, the impaired NK cell activity has a role in some types of autoimmunity. NK cell abnormalities have been reported in women with POF (Hoek et al., 1995) and in women with POI (van Kasteren et al., 2000), as well as in murine post-thymectomy autoimmune oophoritis (Maity et al 1997), all demonstrating a decreased NK cell number and/or activity. Similarly NK cells suppression is observed in neonatal thymectomy induced mice autoimmune oophoritis (Maity et al., 1997), while treatment by the NK cell-stimulating factor (IL-12) before thymectomy preserves the NK cell activity and ameliorates the associated autoimmune oophoritis. NK cells produce the signature Th1 cytokine IFN-γ. The increased periphery NK cell activity and number during pregnancy might explain the apparent Th1 bias (Ntrivalas et al., 2001) that is also associated with multiple observations of recurrent pregnancy losses (Piccinni et al., 1998, Prado-Drayer et al., 2008). These findings suggested an important role for the innate immune system. The immune activation might cause pregnancy failure by inhibiting the reproductive endocrine system. In this way, for example, the inflammatory cytokines are thought to inhibit both the gonadotropin production at hypothalamus and pituitary gland level (Rivest & Rivier 1995), and the progesterone synthesis in corpus luteum of the ovary as well as to promote the luteal regression (Davis & Rueda 2002). Monocytes (Mo) and the monocyte-derived dendritic cells (DCs) play a prominent role in the initial stages of autoimmune reactions against endocrine organs. The accumulation of monocytes/DCs and the clustering of DCs in endocrine organs is one of the first sign of an autoimmune endocrine disorder. Mo start to replace the resident Macrophages (Ma) and DCs under normal state and in response to inflammation signals, they move in the tissue vicinities and proliferate and differentiate into Ma and DCs. The main function of DCs is to act as antigen presenting cells, being messengers between the innate and adaptive immunity. Immature DCs are found in the bloodstream, but once activated, they migrate to the lymph nodes where they interact with T- and B -cells to initiate and shape the adaptive immune response. Dysfunction of Mo and DCs has been found in patients with ovarian failure (Hoek et al., 1993) long enough - the Mo have abnormal polarization and DCs have abnormal interaction with T -cells, refractive to estrogen substitution correction. Alteration of T cell subsets and T cell mediated injury is likely to play an important role in pathogenesis of autoimmune ovarian failure as evidenced by human studies and animal models of autoimmune oophoritis (Hoek et al., 1995, Mignot et al., 1989b, van Kasteren et al., 2000, Chernyshov et al., 2001). The hypoestrogenic hormone status in ovarian failure patients could be responsible for the elevated numbers of activated blood T cells. It has been demonstrated that the estrogen substitution lowered the number of activated peripheral T cells in ovarian failure patients, although not to completely normal levels (Hoek et al 1995). Despite the fact that the data on the numbers of T cells vary between the reported studies, a consistent pattern of an increased number of activated (MHC II+ or IL-2R+) T cells have been demonstrated in the majority of the studies (Hoek et al., 1997). Elevation in the number of peripheral blood B cells has been reported in patients with ovarian failure (Ho et al., 1988), and in ovarian failure patients during initial period of disease (Chernyshov et al., 2001), and in patients with primary ovarian insufficiency (van Kasteren et al., 2000), and in murine autoimmune ovarian failure (Ivanova et al., 1984), and relationship of the raised numbers of B cells to the presence of various autoantibodies has been also demonstrated (Ho et al., 1988). The elevated numbers of peripheral blood B cells is thought to be a sign of activation of the humoral immune system, which is crucial for autoantibody production. This can be very important because estrogen substitution in

component of the innate immune system. NK cells have a suppressive action on B cells, and therefore, the impaired NK cell activity has a role in some types of autoimmunity. NK cell abnormalities have been reported in women with POF (Hoek et al., 1995) and in women with POI (van Kasteren et al., 2000), as well as in murine post-thymectomy autoimmune oophoritis (Maity et al 1997), all demonstrating a decreased NK cell number and/or activity. Similarly NK cells suppression is observed in neonatal thymectomy induced mice autoimmune oophoritis (Maity et al., 1997), while treatment by the NK cell-stimulating factor (IL-12) before thymectomy preserves the NK cell activity and ameliorates the associated autoimmune oophoritis. NK cells produce the signature Th1 cytokine IFN-γ. The increased periphery NK cell activity and number during pregnancy might explain the apparent Th1 bias (Ntrivalas et al., 2001) that is also associated with multiple observations of recurrent pregnancy losses (Piccinni et al., 1998, Prado-Drayer et al., 2008). These findings suggested an important role for the innate immune system. The immune activation might cause pregnancy failure by inhibiting the reproductive endocrine system. In this way, for example, the inflammatory cytokines are thought to inhibit both the gonadotropin production at hypothalamus and pituitary gland level (Rivest & Rivier 1995), and the progesterone synthesis in corpus luteum of the ovary as well as to promote the luteal regression (Davis & Rueda 2002). Monocytes (Mo) and the monocyte-derived dendritic cells (DCs) play a prominent role in the initial stages of autoimmune reactions against endocrine organs. The accumulation of monocytes/DCs and the clustering of DCs in endocrine organs is one of the first sign of an autoimmune endocrine disorder. Mo start to replace the resident Macrophages (Ma) and DCs under normal state and in response to inflammation signals, they move in the tissue vicinities and proliferate and differentiate into Ma and DCs. The main function of DCs is to act as antigen presenting cells, being messengers between the innate and adaptive immunity. Immature DCs are found in the bloodstream, but once activated, they migrate to the lymph nodes where they interact with T- and B -cells to initiate and shape the adaptive immune response. Dysfunction of Mo and DCs has been found in patients with ovarian failure (Hoek et al., 1993) long enough - the Mo have abnormal polarization and DCs have abnormal interaction with T -cells, refractive to estrogen substitution correction. Alteration of T cell subsets and T cell mediated injury is likely to play an important role in pathogenesis of autoimmune ovarian failure as evidenced by human studies and animal models of autoimmune oophoritis (Hoek et al., 1995, Mignot et al., 1989b, van Kasteren et al., 2000, Chernyshov et al., 2001). The hypoestrogenic hormone status in ovarian failure patients could be responsible for the elevated numbers of activated blood T cells. It has been demonstrated that the estrogen substitution lowered the number of activated peripheral T cells in ovarian failure patients, although not to completely normal levels (Hoek et al 1995). Despite the fact that the data on the numbers of T cells vary between the reported studies, a consistent pattern of an increased number of activated (MHC II+ or IL-2R+) T cells have been demonstrated in the majority of the studies (Hoek et al., 1997). Elevation in the number of peripheral blood B cells has been reported in patients with ovarian failure (Ho et al., 1988), and in ovarian failure patients during initial period of disease (Chernyshov et al., 2001), and in patients with primary ovarian insufficiency (van Kasteren et al., 2000), and in murine autoimmune ovarian failure (Ivanova et al., 1984), and relationship of the raised numbers of B cells to the presence of various autoantibodies has been also demonstrated (Ho et al., 1988). The elevated numbers of peripheral blood B cells is thought to be a sign of activation of the humoral immune system, which is crucial for autoantibody production. This can be very important because estrogen substitution in patients with ovarian failure did not lower the elevated number of peripheral B cells (Hoek et al., 1995). Similar immunological profile has been shown in patients with POI and in patients with POF (van Kasteren et al., 2000), suggesting that POI is a light form of POF. It has been found a decrease in NK cells and in percentage of T-suppressor cells with a rise in T-helper/T-suppressor cell ratio, as well as an increase in B- cells and HLA-DR+ T cells. The question remains whether these changes are the cause or the consequence of the ovarian failure. Autoimmune damage of the ovary could be the primary cause of POF, whereas in natural menopause autoimmunity is a result of hormone dysfunction (Chernyshov et al., 2001).

The variations of cellular immunity function and its relationship with AOA (Huang et al., 1996) and with anti-granulosa cells autoantibodies (Vangelov et al., 2009) have been investigated in patients with ovarian failure. The patients with ovarian failure have increased AOA, CD4+ T-cells and CD4+/CD8+ ratio. It has been shown also, that AOA positive rate in POF patients is associated with higher CD4+/CD8+ ratio (Huang et al., 1996).

In our study, we used our propriate granulosa cell autoantibody ELISA detection assay, and commercially available TNF-α and IFN-γ ELISA kits (eBioscience, USA) to estimate the abundance of granulosa cell autoantibodies, and the concentrations of TNF-α and IFN-γ in FFls, as well as DNA-DAPI staining for the assessment of granulosa cells apoptotic index in ovarian failure patients. The obtained results reveled several findings: 1) significantly higher levels of IFN-γ, TNF-α and apoptotic cells in women with positive autoantibody ELISA test, and 2) significant correlation of anti- granulosa autoantibodies, and apoptotic index, as well as significant correlation of IFN-γ with TNF-α and apoptotic index. Moreover, we have reported (Vangelov et al., 2008, 2009), that cellular immunity (TNF-α, IFN-γ) and humoral immunity (anti-granulosa cell autoantibodies) to granulosa autoantigens can be concurrently produced in patients with ovarian failure, and we suggested that pathogenesis of ovarian failure may be associated with immune factors and apoptosis of granulosa cells. In this scenario the programmed cell dead (apoptosis) of granulosa cells could be very important player. The ovulatory process represents the classical sequence of an acute sterile inflammation under surveillance of innate immune function (Medzhitov, 2010, Rock et al 2010). The cell damage is associated with the release of danger signals-alarmins (Bianchi 2007). In the preovulatory follicle main source of danger signals are damaged granulosa cells (Spanel-Borowski 2011) and the danger signals could be recognized by CK+(cytokeratin-positive) granulosa cells via TLR-4 or other pattern-recognition receptors (PRRs) (Serke et al., 2009, 2010). The ovarian innate immune function will be activated to orchestrate the ovulatory process (Spanel-Borowski, 2011). CK+ cells are assumed to switch off the CK genes in the corpus luteum (CL) stage of secretion and regression to become granulosa-like cells. The granulosa-like cells are thought to be mature DC in the CL (Spanel-Borowski, 2011). However, the low T cell number in the preovulatory follicle wall is signature for a limited interaction with the adaptive immunity. A disturbed conversation between innate and adaptive immunity is reflected by altered T cell profiles in the FFls of patients with idiopathic infertility and in ovaries with POF due to autoimmune damage (Vujovic 2009, Spanel-Borowski 2011). Because DCs process antigens and train naive T cells to become helper and suppressor cells (Banchereau & Steinman, 1998, Turvey & Broide, 2010), DCs are players between innate and adaptive immunity. The pathway of innate immunity is well balanced to achieve the quick recovery of tissue integrity, but an

Ovarian Biomarkers in Infertility 109

granulosa cells and altogether were associated with poor COH/IVF outcome. Moreover, earlier Cianci et al., 1996 reported association of elevated concentration of TNF-α with reduced fertilization rate in women with immunological infertility and concluded, that the elevated TNF-α concentration in the human follicle may negatively influence both ovulation

**3. Intra ovarian regulators of survival and apoptosis in preovulatory follicle**

The oocyte-cumulus-granulosa cells unit comprises an intimate relation between somatic cell syncytium in conjunction with the oocyte involving gap junctions and complex of hormonal and peptide factors. Granulosa cells and cumulus cells play a critical role in oocyte maturation and fertilization by releasing and mediating signals to oocytes. On the other hand the oocyte also influences granulosa cells steroidogenesis, proliferation and Hyaluronic acid synthesis by CCs. Each participant in this unit can disturb signaling and regulation of apoptotic process. Apoptosis is self-destruction under physiological control and is closely involved with most of the reproductive processes, including follicular atresia. Apoptosis can be used to estimate a function of ovarian reserve in women undergoing IVF (Seifer et al., 1996). Many studies have provided evidence that poor oocyte, low IVF outcome and embryo quality can be associated with apoptosis. The incidence of apoptotic bodies has been used as a morphological marker for physiological cell renewal (Tilly et al., 1991) and the presence of apoptosis in human GLCs and CCs in COHS during IVF treatment, has been used as a very sensitive indicator for evaluation of oocyte quality and

The isolated GLCs (Vangelov et al., 2005) were fixed with formaldehyde. The GLCs suspensions were dropped on poly L-Lysine treated slides. CCs were isolated from individual cumulus-oocyte complexes (COCs) of the same patients, and were divided according to the oocyte maturation (immature – G.V. or MI or mature – MII oocytes). The cumulus masses were dispersed with 150 IU Hyaluronidase and after that were fixed and

Apoptosis of GLCs and CCs was assayed using DNA-DAPI staining (Dineva et al., 2006) that allows the observation of morphological changes of nuclear chromatin in GLCs and CCs. Morphological characteristics of apoptosis such as chromatin condensation and/or nuclear fragmentation were identified and counted in randomly selected fields among 800– 1000 cells and the percentage of apoptotic cells was calculated. The significant negative correlations between the rate of apoptosis in GLCs and both - the number of preovulatory follicles, and the number of retrieved and fertilized oocytes from patients in stimulated

The most convenient evaluation system for oocyte quality is based on oocyte morphology and status of oocytes–cumulus complexes. Cumulus cells surround and intercommunicate with oocytes during follicular development and after ovulation, suggesting that the incidence of apoptosis in cumulus cells could influence oocyte quality (Lee et al., 2001). In our study we found that the rate of apoptosis in GLCs is higher compared to CCs cells from

**3.1 Apoptosis of granulosa luteinized cells (GLCs) and cumulus cells (CCs) –**

**predictive marker for results of COH / IVF outcome in infertile women**

the IVF outcome (Nakahara et al., 1997; Suh et al., 2002).

and fertilization related events.

processed as GLCs.

cycles was found**.**

unbalanced action can cause chronic tissue injury and autoimmune diseases. In this aspect, we have reported the relationship between the level of apoptotic granulosa cells and the level of anti- granulosa cell autoantibodies in studied patient group with ovarian failure (Vangelov et al., 2009). Moreover, the biomolecules are susceptible to glycosylation, glycation, phosphorylation and oxidation. Oxidative stress is the major event causing structural modification of proteins with consequent appearance of neo-epitopes (Buttari et al., 2005). The role for oxidative stress in promoting the autoimmune responses have been reviewed (Kannan 2006). Autoimmune responses may be directed against self-structures altered by high-affinity ligand binding or by chemical damage due to environmental events, such as oxidative stress. Several systems that generate reactive oxygen species catalyze a variety of oxidative damage to nucleic acids, lipids, and proteins. In physiological conditions oxidative stress is well compensated, but when there is an overproduction of reactive oxygen species or a deficiency of antioxidant enzyme activity, a biological damage may occur (Puddu et al., 2009). Oxidative stress and inflammation may determine the modification of self-structures also favoring the formation of Advanced glycation end products (AGEs). Chronic oxidative stress causes an accumulation of AGFs products (Ramasamy et al., 2009). The generation of AFGs products and augmentation of proinflammatory mechanisms provide a potent feedback loop for sustained oxidant stress, ongoing the generation of AFGs products. Therefore, self-molecules modified by oxidative events can become targets of autoimmune reactions, thus sustaining the inflammatory responses. In another hand, the disturbed apoptosis execution and clearance can potentially render apoptotic cells as a source of autoantigens normally sequestered in the intracellular environment. Structural changes occurring during cell death may influence the immunogenicity of clustered self-antigens at the surface of apoptotic body (Navratil et al., 2005). In this scenario, high concentrations of self-antigens are packaged during generation of apoptotic cells; the packages also may contain altered fragments of self-antigens that have not been encountered previously by the immune system; under normal circumstances, apoptotic cells are cleared rapidly by macrophages and DCs; therefore, the apoptosis-altered self-antigens are either ignored by the immune system or tolerance to those antigens is maintained. Clearance is achieved through complex mechanisms that enable macrophages and DCs to recognize apoptotic cells as nonthreatening "self" particles, but defects in this process that cause a delay in clearance could change the appearance of apoptotic cells and cause them to be recognized as "foreign invaders," thereby stimulating an inflammatory response that, in turn, activates an immune response to self-antigens (Navratil et al., 2005).

The ovary is an extremely dynamic organ in which excessive or defective follicles are rapidly and effectively eliminated throughout reproductive life. Greater than 99 % of follicles disappear, due to apoptosis of granulosa cells. The balance between signals for cell death and survival determines the destiny of the follicles. The potential use of scores for apoptosis in granulosa cells and characteristics of FFls have been discussed more recently as prognostic markers for predicting the outcome of assisted reproduction (Lefèvre et al., 2011). Follicular fluid and surrounding tissue contains various lymphocytes that synthesize different cytokines. The other sources of cytokines are ovarian somatic cells. We studed the impact of proinflamatory cytokines TNF-α and IFN-γ concentration in FFls, and apoptotic index of granulosa cells on the results of COH/IVF in women with ovarian failure (Vangelov et al., 2009). The obtained results in this study clearly showed that the elevated levels of TNF-α and IFN-γ were directly related with increased incidences of apoptotic

unbalanced action can cause chronic tissue injury and autoimmune diseases. In this aspect, we have reported the relationship between the level of apoptotic granulosa cells and the level of anti- granulosa cell autoantibodies in studied patient group with ovarian failure (Vangelov et al., 2009). Moreover, the biomolecules are susceptible to glycosylation, glycation, phosphorylation and oxidation. Oxidative stress is the major event causing structural modification of proteins with consequent appearance of neo-epitopes (Buttari et al., 2005). The role for oxidative stress in promoting the autoimmune responses have been reviewed (Kannan 2006). Autoimmune responses may be directed against self-structures altered by high-affinity ligand binding or by chemical damage due to environmental events, such as oxidative stress. Several systems that generate reactive oxygen species catalyze a variety of oxidative damage to nucleic acids, lipids, and proteins. In physiological conditions oxidative stress is well compensated, but when there is an overproduction of reactive oxygen species or a deficiency of antioxidant enzyme activity, a biological damage may occur (Puddu et al., 2009). Oxidative stress and inflammation may determine the modification of self-structures also favoring the formation of Advanced glycation end products (AGEs). Chronic oxidative stress causes an accumulation of AGFs products (Ramasamy et al., 2009). The generation of AFGs products and augmentation of proinflammatory mechanisms provide a potent feedback loop for sustained oxidant stress, ongoing the generation of AFGs products. Therefore, self-molecules modified by oxidative events can become targets of autoimmune reactions, thus sustaining the inflammatory responses. In another hand, the disturbed apoptosis execution and clearance can potentially render apoptotic cells as a source of autoantigens normally sequestered in the intracellular environment. Structural changes occurring during cell death may influence the immunogenicity of clustered self-antigens at the surface of apoptotic body (Navratil et al., 2005). In this scenario, high concentrations of self-antigens are packaged during generation of apoptotic cells; the packages also may contain altered fragments of self-antigens that have not been encountered previously by the immune system; under normal circumstances, apoptotic cells are cleared rapidly by macrophages and DCs; therefore, the apoptosis-altered self-antigens are either ignored by the immune system or tolerance to those antigens is maintained. Clearance is achieved through complex mechanisms that enable macrophages and DCs to recognize apoptotic cells as nonthreatening "self" particles, but defects in this process that cause a delay in clearance could change the appearance of apoptotic cells and cause them to be recognized as "foreign invaders," thereby stimulating an inflammatory response that, in turn, activates an immune response to self-antigens (Navratil et al., 2005). The ovary is an extremely dynamic organ in which excessive or defective follicles are rapidly and effectively eliminated throughout reproductive life. Greater than 99 % of follicles disappear, due to apoptosis of granulosa cells. The balance between signals for cell death and survival determines the destiny of the follicles. The potential use of scores for apoptosis in granulosa cells and characteristics of FFls have been discussed more recently as prognostic markers for predicting the outcome of assisted reproduction (Lefèvre et al., 2011). Follicular fluid and surrounding tissue contains various lymphocytes that synthesize different cytokines. The other sources of cytokines are ovarian somatic cells. We studed the impact of proinflamatory cytokines TNF-α and IFN-γ concentration in FFls, and apoptotic index of granulosa cells on the results of COH/IVF in women with ovarian failure (Vangelov et al., 2009). The obtained results in this study clearly showed that the elevated levels of TNF-α and IFN-γ were directly related with increased incidences of apoptotic granulosa cells and altogether were associated with poor COH/IVF outcome. Moreover, earlier Cianci et al., 1996 reported association of elevated concentration of TNF-α with reduced fertilization rate in women with immunological infertility and concluded, that the elevated TNF-α concentration in the human follicle may negatively influence both ovulation and fertilization related events.

#### **3. Intra ovarian regulators of survival and apoptosis in preovulatory follicle**

#### **3.1 Apoptosis of granulosa luteinized cells (GLCs) and cumulus cells (CCs) – predictive marker for results of COH / IVF outcome in infertile women**

The oocyte-cumulus-granulosa cells unit comprises an intimate relation between somatic cell syncytium in conjunction with the oocyte involving gap junctions and complex of hormonal and peptide factors. Granulosa cells and cumulus cells play a critical role in oocyte maturation and fertilization by releasing and mediating signals to oocytes. On the other hand the oocyte also influences granulosa cells steroidogenesis, proliferation and Hyaluronic acid synthesis by CCs. Each participant in this unit can disturb signaling and regulation of apoptotic process. Apoptosis is self-destruction under physiological control and is closely involved with most of the reproductive processes, including follicular atresia. Apoptosis can be used to estimate a function of ovarian reserve in women undergoing IVF (Seifer et al., 1996). Many studies have provided evidence that poor oocyte, low IVF outcome and embryo quality can be associated with apoptosis. The incidence of apoptotic bodies has been used as a morphological marker for physiological cell renewal (Tilly et al., 1991) and the presence of apoptosis in human GLCs and CCs in COHS during IVF treatment, has been used as a very sensitive indicator for evaluation of oocyte quality and the IVF outcome (Nakahara et al., 1997; Suh et al., 2002).

The isolated GLCs (Vangelov et al., 2005) were fixed with formaldehyde. The GLCs suspensions were dropped on poly L-Lysine treated slides. CCs were isolated from individual cumulus-oocyte complexes (COCs) of the same patients, and were divided according to the oocyte maturation (immature – G.V. or MI or mature – MII oocytes). The cumulus masses were dispersed with 150 IU Hyaluronidase and after that were fixed and processed as GLCs.

Apoptosis of GLCs and CCs was assayed using DNA-DAPI staining (Dineva et al., 2006) that allows the observation of morphological changes of nuclear chromatin in GLCs and CCs. Morphological characteristics of apoptosis such as chromatin condensation and/or nuclear fragmentation were identified and counted in randomly selected fields among 800– 1000 cells and the percentage of apoptotic cells was calculated. The significant negative correlations between the rate of apoptosis in GLCs and both - the number of preovulatory follicles, and the number of retrieved and fertilized oocytes from patients in stimulated cycles was found**.**

The most convenient evaluation system for oocyte quality is based on oocyte morphology and status of oocytes–cumulus complexes. Cumulus cells surround and intercommunicate with oocytes during follicular development and after ovulation, suggesting that the incidence of apoptosis in cumulus cells could influence oocyte quality (Lee et al., 2001). In our study we found that the rate of apoptosis in GLCs is higher compared to CCs cells from

Ovarian Biomarkers in Infertility 111

represent localization of ANP during the different stages of posttranslational processing

The women included in this study were divided into two groups: women with high level of

For us the question of great interest was, whether the GLCs are the principal source of ANP in the human preovulatory follicle? For this reason, we measured the ANP concentrations in FFl obtained from the same woman that donated the GLCs specimens followed by an analysis of relationship between the follicular ANP concentration and the expression levels of immunoreactive ANP in GLCs. The concentration of ANP was measured in FFls, obtained from the same woman using a proANP (1-98) kit, with levels of immunoreactive ANP in GLCs found positively correlating to the corresponding FFls ANP concentrations. The concentration of ANP in the FFls of women with high level of immunoreactive ANP expression in GLCs was found to be significantly higher, compared to the women with low

Our earlier study showed that, in in vitro luteinized porcine granulosa cells (isolated from prepubertal porcine ovaries) supplemented with FSH and luteinizing hormone (LH), the expression of immunoreactive ANP was increased and was highest among the FSH and LH

The established positive relationship between the levels of ANP in FFls and its immunoreactive expression on GLCs together with findings of in vitro experiment show

Diverse effects of NPs are mediated by the activation of membrane bound receptors with a particulate guanylate cyclase (GC) activity to generate Cyclic Guanosine Monophosphate (cGMP). The physiological activities of ANP are mediated by a specific plasma membrane receptor, guanylate cyclase-type receptor GC-A (Chang et al., 1989). Recent studies have shown that components of cGMP signaling pathways are expressed in the ovary and that cGMP can alter granulosa cell function (La Polt et al., 2003). The cGMP/PKG signaling is a target for the prevention of mitochondrial dysfunction-mediated cell death (Chun SY et al.

The survival role of ANP for GLCs, was studied in cultured human GLCs treated with 2BucGMP (analog of cGMP, Sigma) and with human recombinant ANP (hrANP, Sigma). The higher level of ANP production (after 2Bu-cGMP treatment) and the supplementation of cultured GLCs with hrANP were associated with significant suppression of Caspase-3 activity, that is to say, with the inhibition of apoptosis in GLCs, compared to control cells. For Caspase-3 activity assay, the cultured cells were lysed and the soluble fraction of the cell

ANP can affect oocyte maturation via cGMP signaling pathways, and suppress spontaneous oocyte maturation (Zhang et al., 2005) and in this manner ANP may be the factor which determines the development of fertilizable oocytes. We analyzed the relationship between

that the GLCs are the main local source of ANP in the preovulatory human follicle.

**3.3 ANP is survival factor for GLCs in preovulatory follicle**

lysate was assayed using specific substrate (Dineva et al., 2007)

**3.4 Role of ANP for folliculogenesis and results of COH/IVF**

ANP expression and women with low level of ANP expression in GLCs.

and/or export of ANP from GLCs.

level of ANP expression in GLCs.

treated GLCs (Dineva et al., 2007).

1996, Takuma et al. 2001).

the same patient**.** The higher rate of apoptosis in CCs from follicles with immature oocytes than from those with mature showed that the level of CCs apoptosis is directly related to the maturation of oocytes and support the data of Host et al., 2000.

The obtained results showed that the incidence of apoptosis in GLCs as well as in CCs could be a predictive marker for IVF outcome.

#### **3.2 Role of Atrial Natriuretic Peptide (ANP) in the ovarian immunoendorine function**

#### **3.2.1 GLCs are the main local source of ANP in the preovulatory human follicle**

Many endocrine, paracrine and autocrine factors (gonadotrophic and steroid hormones, peptide hormones, cytokines, highly- reactive free radicals) play an important role in the regulation of GLCs proliferation, differentiation and death.

The expression of natriuretic peptides (NPs) and its receptors (NPRs) has been demonstrated in the mammalian ovary (Gutkowska et al., 1993; Russinova et al., 2001).

The presence of ANP was demonstrated in the follicular fluid (Kim et al., 1987); granulosa cells (Ivanova et al., 2003) and luteal cells (Vollmar et al., 1988). ANP plays an important role in the regulation of different aspects of ovarian physiology, such as oocyte meiosis, cumulus cells expansion, spontaneous oocyte maturation, ovarian steroidogenesis and follicular atresia (Samson WK et al., 1988, Tornel et al., 1990; Gutkowska et al., 1999; Zhang et al., 2005).

The indirect immunofluorescence technique (IIF) was used to investigate the localization patterns of ANP on GLCs isolated from women undergoing IVF. The isolated GLCs were fixed on the protocols mentioned above. The GLCs suspensions were dropped on poly L-Lysine treated slides. The cells were permeabilized with Triton X-100 for 5 min and after washing the cells were incubated with cocktail of monoclonal antibodies (Mabs) against recombinant human ANP (Mabs: 6C3, 6F11, 5D3 supernatants), obtained and characterized at the Department of Molecular Immunology (Institute of Biology and Immunology of reproduction, Sofia, Bulgaria), diluted 1:1 in PBS/BSA, overnight, followed by washing with PBS-Tween 20 and then incubated with secondary antibody (Sw/am IgG labeled with FITC, Sevac, Czech Republic), diluted 1:20, for 1h. After washing in PBS-Tween 20, slides were mounted with Mowiol (Sigma®). The expression and localization of ANP were examined using fluorescence microscope (Leiz Labor-Lux, Zeiss). The immunofluorescent expression level of ANP was scored as the intensity of specific immunofluorescence and the number of positive cells from (+) to (+++) as follows: (+++) – intensive immunoreactive ANP expression in prevailing cells population; (++) - intensive immunoreactive ANP expression in most of cells observed; (+) – intensive expression of immunoreactive ANP in a minority of cells observed.

ANP expression was observed as membrane, submembrane and particular granular cytoplasm localized staining. The mature ANP is generated by proteolytic processing in two steps: generation of pro-ANP (stored in the cytoplasmic granules) from immature pre-pro ANP (Bloch et al., 1985), followed by proteolytic maturation of pro-ANP to mature bioactive ANP (Schwartz et al., 1984). The bioactive ANP gets into extracellular space and in this manner ANP acts as autocrine and/or paracrine factor that modulates different aspects of GLCs functions. We consider that the different patterns of ANP expression in GLCs

the same patient**.** The higher rate of apoptosis in CCs from follicles with immature oocytes than from those with mature showed that the level of CCs apoptosis is directly related to the

The obtained results showed that the incidence of apoptosis in GLCs as well as in CCs could

**3.2 Role of Atrial Natriuretic Peptide (ANP) in the ovarian immunoendorine function**

Many endocrine, paracrine and autocrine factors (gonadotrophic and steroid hormones, peptide hormones, cytokines, highly- reactive free radicals) play an important role in the

The expression of natriuretic peptides (NPs) and its receptors (NPRs) has been demonstrated in the mammalian ovary (Gutkowska et al., 1993; Russinova et al., 2001).

The presence of ANP was demonstrated in the follicular fluid (Kim et al., 1987); granulosa cells (Ivanova et al., 2003) and luteal cells (Vollmar et al., 1988). ANP plays an important role in the regulation of different aspects of ovarian physiology, such as oocyte meiosis, cumulus cells expansion, spontaneous oocyte maturation, ovarian steroidogenesis and follicular atresia

The indirect immunofluorescence technique (IIF) was used to investigate the localization patterns of ANP on GLCs isolated from women undergoing IVF. The isolated GLCs were fixed on the protocols mentioned above. The GLCs suspensions were dropped on poly L-Lysine treated slides. The cells were permeabilized with Triton X-100 for 5 min and after washing the cells were incubated with cocktail of monoclonal antibodies (Mabs) against recombinant human ANP (Mabs: 6C3, 6F11, 5D3 supernatants), obtained and characterized at the Department of Molecular Immunology (Institute of Biology and Immunology of reproduction, Sofia, Bulgaria), diluted 1:1 in PBS/BSA, overnight, followed by washing with PBS-Tween 20 and then incubated with secondary antibody (Sw/am IgG labeled with FITC, Sevac, Czech Republic), diluted 1:20, for 1h. After washing in PBS-Tween 20, slides were mounted with Mowiol (Sigma®). The expression and localization of ANP were examined using fluorescence microscope (Leiz Labor-Lux, Zeiss). The immunofluorescent expression level of ANP was scored as the intensity of specific immunofluorescence and the number of positive cells from (+) to (+++) as follows: (+++) – intensive immunoreactive ANP expression in prevailing cells population; (++) - intensive immunoreactive ANP expression in most of cells observed; (+) – intensive expression of immunoreactive ANP in a minority of

ANP expression was observed as membrane, submembrane and particular granular cytoplasm localized staining. The mature ANP is generated by proteolytic processing in two steps: generation of pro-ANP (stored in the cytoplasmic granules) from immature pre-pro ANP (Bloch et al., 1985), followed by proteolytic maturation of pro-ANP to mature bioactive ANP (Schwartz et al., 1984). The bioactive ANP gets into extracellular space and in this manner ANP acts as autocrine and/or paracrine factor that modulates different aspects of GLCs functions. We consider that the different patterns of ANP expression in GLCs

(Samson WK et al., 1988, Tornel et al., 1990; Gutkowska et al., 1999; Zhang et al., 2005).

**3.2.1 GLCs are the main local source of ANP in the preovulatory human follicle**

maturation of oocytes and support the data of Host et al., 2000.

regulation of GLCs proliferation, differentiation and death.

be a predictive marker for IVF outcome.

cells observed.

represent localization of ANP during the different stages of posttranslational processing and/or export of ANP from GLCs.

The women included in this study were divided into two groups: women with high level of ANP expression and women with low level of ANP expression in GLCs.

For us the question of great interest was, whether the GLCs are the principal source of ANP in the human preovulatory follicle? For this reason, we measured the ANP concentrations in FFl obtained from the same woman that donated the GLCs specimens followed by an analysis of relationship between the follicular ANP concentration and the expression levels of immunoreactive ANP in GLCs. The concentration of ANP was measured in FFls, obtained from the same woman using a proANP (1-98) kit, with levels of immunoreactive ANP in GLCs found positively correlating to the corresponding FFls ANP concentrations. The concentration of ANP in the FFls of women with high level of immunoreactive ANP expression in GLCs was found to be significantly higher, compared to the women with low level of ANP expression in GLCs.

Our earlier study showed that, in in vitro luteinized porcine granulosa cells (isolated from prepubertal porcine ovaries) supplemented with FSH and luteinizing hormone (LH), the expression of immunoreactive ANP was increased and was highest among the FSH and LH treated GLCs (Dineva et al., 2007).

The established positive relationship between the levels of ANP in FFls and its immunoreactive expression on GLCs together with findings of in vitro experiment show that the GLCs are the main local source of ANP in the preovulatory human follicle.

#### **3.3 ANP is survival factor for GLCs in preovulatory follicle**

Diverse effects of NPs are mediated by the activation of membrane bound receptors with a particulate guanylate cyclase (GC) activity to generate Cyclic Guanosine Monophosphate (cGMP). The physiological activities of ANP are mediated by a specific plasma membrane receptor, guanylate cyclase-type receptor GC-A (Chang et al., 1989). Recent studies have shown that components of cGMP signaling pathways are expressed in the ovary and that cGMP can alter granulosa cell function (La Polt et al., 2003). The cGMP/PKG signaling is a target for the prevention of mitochondrial dysfunction-mediated cell death (Chun SY et al. 1996, Takuma et al. 2001).

The survival role of ANP for GLCs, was studied in cultured human GLCs treated with 2BucGMP (analog of cGMP, Sigma) and with human recombinant ANP (hrANP, Sigma). The higher level of ANP production (after 2Bu-cGMP treatment) and the supplementation of cultured GLCs with hrANP were associated with significant suppression of Caspase-3 activity, that is to say, with the inhibition of apoptosis in GLCs, compared to control cells. For Caspase-3 activity assay, the cultured cells were lysed and the soluble fraction of the cell lysate was assayed using specific substrate (Dineva et al., 2007)

#### **3.4 Role of ANP for folliculogenesis and results of COH/IVF**

ANP can affect oocyte maturation via cGMP signaling pathways, and suppress spontaneous oocyte maturation (Zhang et al., 2005) and in this manner ANP may be the factor which determines the development of fertilizable oocytes. We analyzed the relationship between

Ovarian Biomarkers in Infertility 113

and obtained results by us showed that ANP is an important intra ovarian regulator of inflammatory response and is a potential modulator of immune environment within