According to World Health Organizations, Infertility is referring as a global health issue affecting millions of people of reproductive age worldwide. Infertility may occur due to male factors, female factors, a combination of male and female factors or may be unexplained WHO, [13]. Environmental and lifestyle factors such as smoking, excessive alcohol intake, obesity and exposure to environmental pollutants have been associated with lower fertility rates [10,12]. 

The pregnancy is a state characterized by hyper coagulation with decline of fibrinolytic activity, which caused by an increase of Plasminogen Activator Inhibitor-1 (PAI-1). During implantation, PAI-1 is expressed in extravillous interstitial trophoblasts and vascular trophoblasts. However, PAI-1 is responsible for degradation Extra Cellular Matrix (ECM), then inhibition of trophoblasts invasion. PAI-1 represents a biomarker for reproductive diseases and may be a treatment target in the future [15,2]. 

Follicular Fluid (FF) is a crucial external environment for the growth and development of oocytes. An in-depth analysis of specific components found in follicular fluid from women undergoing assisted reproductive therapy can advance our understanding of intracellular signal transduction and help us find potential oocyte health biomarkers Xingxing et al. [14]

Ovulation is a time-coordinated tightly regulated proteolytic event. PAI-1 is a major of physiological inhibitor, primarily produced by granulosa cells in FF of human pre ovulatory follicles, allowing localized degradation and regulating ovulation. While ovulation process is rendered redundant in IVF cycles; nevertheless, FF PAI-1 concentration may influence maturity of oocyte within the follicle Chimote, [5]. Any pathological disturbance in PAI-1 levels may lead to several pregnancy complications Pihusch et al. [8]. Expression of Plasminogen Activator Inhibitor-1 in serum and follicular fluid has critical role in embryo implantation and successful pregnancy outcome, which plays an essential role in regulating fibrinolysis during pregnancy period by blocks or inhibits the action of other protein called plasminogen activators, tissue type plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA) Ye et al. [15].

The study included Sixty-nine infertile female mean age was (33.03±5.97) years enrolled in Assisted Reproductive Technology (ART) programs to enter ICSI program in High Institute for Infertility Diagnosis and Assisted Reproductive Technologies, Al-Nahrain University (Baghdad/Iraq), during a period from October 2022 till February 2023. All infertile female has an inclusion criterion according to infertility causes such as: an ovulatory cycles and blocked tubes, mild endometriosis and normal ovarian reserve. While, exclusion criteria of female's patients involved the uterine pathology such as leiomyoma, fibroids, adenomyosis, congenital uterine anomalies, Müllerin malformations, polyps and poor ovarian reserve. 

Samples Collection

The (5ml) blood sample collected from all infertile female, from cubital veins and (5ml) of follicular fluid were collected at the day of oocyte retrieval from each female in all infertile groups. Finally, poured into clean gel plain tube for serological test and allowed to clot at room temperature, then centrifugation at speed of 3000rpm for 10 minute (serum) and 2000rpm for 10 minutes for follicular fluid, placed in Eppendorf tube stored at (-20 Cº) and use for measurement of PAI-1 levels. Every participant female was interviewed and information data including age, duration of infertility (years), body mass index (kg/m²), type of infertility, causes of infertility and hormonal assay, virology assay, biochemistry assay, complete blood samples, ovulation detection, husband’s seminal fluid analysis and ultrasonography for females. pregnancy test done for all female after 14 day of embryo transfers.

ELISA Assay

 Measurement the concentration of plasminogen activator inhibitor type 1 (PAI-1) (ng/mL), serum and follicular fluid levels by using Enzyme-linked immunosorbent assay Elisa kit (Diagnostic/ USA).

Baseline Characteristics of Females PAI-1 Levels in Serum and Follicular Fluid Enrolled in this Study

 As shown in Table (1). Serum PAI-1 levels range (3.47–16.62 ng/mL) and the range of follicular fluid PAI-1 levels (1.09–16.88 ng/mL).

The Embryos Characteristics and Fertilization Rate (FR%) in Females in this Study 

Table (2) shows the range grade I embryos G1 (0–14), (2.81±1.72) which is higher than G2(0-9) and G3(0-7) that there shows the mean grade 1, 2 and 3 embryos between infertile female's groups.

Pregnancy Rate (PR) in Females Enrolled in the Present Study 

 The pregnancy outcome at the end of the study was based on biochemical pregnancy test and the results showed that 23 females out of 69 succeeded in having positive pregnancy test making (Pregnancy Rate 33.3 %) while 46 females were not pregnant as shown in Figure (1).

Comparison of Serum and Follicular Fluid PAI-1 Levels between Pregnant and Non- Pregnant Female

The comparison of serum PAI-1 levels at the day of oocytes retrievals and follicular fluid PAI-1 levels at the day of oocytes retrievals between pregnant and non-pregnant females were demonstrated in Table (3). According to the findings there were significantly lower serum PAI-1 in pregnant females (5.41±0.88 vs. 6.95±0.56 & p=0.013); however, there was insignificantly lower follicular fluid PAI-1 in pregnant females (6.22±1.97 vs. 7.61±0.76 & p=0.115).

Serum Plasminogen Activator Inhibitor-1 Level in Females as a Predictor of Positive Pregnancy 

 In this study, the Receiver Operative Characteristic curve (ROC curve) showed invalid cut-off value of serum PAI-1 levels as a predictor of positive pregnancy with unacceptable low sensitivity, specificity area under the curve. The cut-off value was ≤5.52 ng/mL with low sensitivity and specificity (43.5% and 39.0% respectively) and very low area under the curve (AUC= 0.331) as presented in Table (4).

As shown in a Table (1). The serum results were in line with the normal value PAI-1 levels present in plasma at concentration (2-46 ng/mL). These findings are consistent with the findings obtained by [1,11].

Moreover, Zhang X et al. [16] agrees with this in terms of significant association between the PAI-1 levels and the risk of female infertility.

Table 1: Baseline characteristics of serum and follicular fluid PAI-1 levels 

SD: Standard Deviation; PAI-1: Plasminogen activator inhibitor

Table 2: The Embryo characteristics of infertile females enrolled in this study. 

SD: Standard deviation; G: Grade

Table 3: Comparison of serum and follicular fluid PAI-1 levels between pregnant and non-pregnant females

PAI-1: Plasminogen activator inhibitor; Ŧ: Independent sample t test; S: Significant (p≤0.05); NS: Non-significant (p>0.05)

Table 4: ROC curve characteristics of serum PAI-1 levels in females to predict positive pregnancy

Figure 1: Pie chart showing the frequency distribution of infertile females according to the results of biochemical pregnancy test (Pregnancy rate of females)

Figure 2: ROC curve representing the discriminatory ability of serum PAI-1 level to predict positive pregnancy

The Embryos Characteristics and Fertilization Rate (FR%) in Females in this Study 

In Table (2). Astbury et al. [3] agree that obtaining oocyte with high quality was an important aspect that result in embryo with high quality and because there were approximate numbers of germinal vesical, MI and MII oocytes, there was no significant difference in the mean grade 1, 2 and 3 embryos between infertile female's groups. 

Pregnancy Rate (PR) in Females Enrolled in the Present Study 

As compared to the results of pregnancy rate (33.3 %) in this study, which is very approximately to the Hassan et al.study [6], a results published in which overall rate of pregnancy after ICSI was 32.3. In this study the result showed agreement with many previous studies which confirmed that human PAI-1 levels are very closely related to embryonic development and pregnancy success rates in pregnant females Chen et al. [4]. Additionally, all females who undergo to Antagonist protocol, which helps to increase the pregnancy rate, this was confirmed by a study according to Raheem et al. [9] which concluded that antagonist protocol associated with significantly higher positive pregnancy outcome in comparison with agonist protocol because of the anti-oxidant effect attributed to antagonist use Figure (1).

Comparison of Serum and Follicular Fluid PAI-1 levels between Pregnant and Non-Pregnant Female

As shown in the Table (4). The PAI-1 measured between pregnant and non-pregnant women and the corresponding linear rhythmic rise in serum PAI-1 levels is a key determinant of transfer embryos within the cycle. Therefore, periodicity in serum PAI-1 levels is a reliable predictive indicator for estimating the likelihood of a clinical pregnancy following an ICSI cycle.

Mechanism of Plasminogen Activator Inhubitor-1 

Lower inhibitor (PAI-1) level observed at the time of is presumably necessary for active proteolysis of extracellular matrix components in order to prepare a “pocket” or “cavity” for the incoming embryo to implant. Thereafter, when the supposed ‘implantation window’ closes, persistence in proteolytic activity may cause hindrance in the development of implanted embryo. Therefore, the observed rise in PAI-1 levels at day7 and day14 may logically account for the protection of embryo and prevention of possible pathological invasion of embryo within uterus. The PAI-1 levels recorded continuous rise till the sixth week of gestation, is in tune with earlier reports of increase in serum PAI-1 levels with gestational age Mehta et al. [7]. 

The PAI-1 levels role through the trophoblast invasion when the fetal side of the human placenta mainly includes cytotrophoblasts and syncytiotrophoblasts, the sycytiotrophoblasts are differentiated and fused by cytotrophoblasts causing invasion of the maternal side and differentiate into extravillous interstitial trophoblasts, intermediate trophoblasts and endovascular trophoblasts. Among them, extravillous interstitial trophoblasts and endovascular trophoblasts express plasminogen activator inhibitor type 1 (PAI-1). Furthermore, cells from the maternal side take part in trophoblast invasion, such as endometrial stromal cells, decidual cells, macrophages and endothelial cells. Extravillous trophoblast invasion in early pregnancy is precisely controlled by many factors expressed by trophoblasts and maternal cells, where PAI-1 is the main anti-invasive factor. PAI-1 prevents trophoblast invasion by inhibiting extracellular matrix degradation, which leads to fibrin accumulation in the maternal side. PAI-1 can remodel maternal uterine spiral arteries Ye et al. [15].

The lower of PAI-1 level in blood serum at the retrieval day is a promising parameter to predict pregnancy of females undergoing ICSI cycles. Also, the follicular fluid PAI-1 levels at the retrieval day was insignificant in pregnant females. 

Concentration of PAI-1 in serum and follicular fluid at the retrieval day are not correlated with the embryo grading.

1. Abbassi-Ghanavati, M., et al. "Pregnancy and laboratory studies: A reference table for clinicians." Obstetrics & Gynecology, vol. 114, no. 6, 2009, pp. 1326–1331.

2. Agersnap, I., et al. "The role of plasminogen activator inhibitor type 1 (PAI-1) in placenta-mediated pregnancy complications: A systematic review." Seminars in Thrombosis and Hemostasis, vol. 48, no. 5, 2022, pp. 607–624.

3. Astbury, P., et al. "The presence of immature GV− stage oocytes during IVF/ICSI Is a marker of poor oocyte quality: A pilot study." Medical Sciences, vol. 8, no. 1, 2020, p. 4.

4. Chen, H., et al. "Association between plasminogen activator inhibitor‐1 gene polymorphisms and recurrent pregnancy loss: A systematic review and meta‐analysis." American Journal of Reproductive Immunology, vol. 73, no. 4, 2015, pp. 292–300.

5. Chimote, N.M. and B. Chimote. "P-695 Anti-Muellerian Hormone (AMH) and Plasminogen Activator Inhibitor (PAI-1) Are Robust Follicular-Fluid (FF) biomarkers predictive of follicle maturity and oocyte quality in women undergoing IVF cycles." Human Reproduction, vol. 37, suppl. 1, 2022, pp. deac107–644.

6. Hassan, N.A., et al. "Correlation among obesity, oocyte characteristics, embryo characteristics and maternal plasma folate level in a sample of iraqi women undergoing Intracytoplasmic Sperm Injection (ICSI)." Iraqi Journal of Embryos and Infertility Researches, vol. 10, no. 1, 2020, pp. 1–19.

7. Mehta, B.N., et al. "Periodicity in the levels of serum plasminogen activator inhibitor-1 is a robust prognostic factor for embryo implantation and clinical pregnancy in ongoing IVF cycles." Journal of Human Reproductive Sciences, vol. 7, no. 3, 2014, p. 198.

8. Pihusch, M., et al. "Plasminogen activator inhibitor-1: A review plasminogen-aktivator-inhibitor-1." Laboratoriums Medizin, vol. 29, no. 6, 2005, pp. 403–411.

9. Raheem, F.A., et al. "Assessment of oxidative stress changes in serum and follicular fluid in relevance to gnrh rival protocol in Iraqi infertile ladies undergoing ICSI." European Journal of Medical and Health Sciences, vol. 1, no. 5, 2019.

10. Silvestris, E., et al. "Obesity as disruptor of the female fertility." Reproductive Biology and Endocrinology, vol. 16, 2018, pp. 1–13.

11. Tjärnlund-Wolf, A., et al. "Plasminogen activator inhibitor-1 and thrombotic cerebrovascular diseases." Stroke, vol. 43, no. 10, 2012, pp. 2833–2839.

12. Van Heertum, K. and B. Rossi. "Alcohol and fertility: How much is too much?" Fertility Research and Practice, vol. 3, no. 1, 2017, pp. 1–7.

13. World Health Organization. World Health Organization Classification of Anovulation. WHO, 2022.

14. Zhang, X et al. "Study on follicular fluid metabolomics components at different ages based on lipid metabolism." Reproductive Biology and Endocrinology, vol. 18, no. 42, 2020.

15. Ye, Y., et al. "Role of plasminogen activator inhibitor type 1 in pathologies of female reproductive diseases." International Journal of Molecular Sciences, vol. 18, no. 8, 2017, p. 1651.

16. Zhang, X., et al. "Association between plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism and risk of Alzheimer's disease, Metabolic Syndrome and Female Infertility: A Meta-Analysis." Medicine, vol. 99, no. 50, 2020.