Unfavorably altered fibrin clot properties in women with unexplained infertility: association with pregnancy outcomes.

Women with unexplained infertility (UI) display unfavorably modified fibrin clot properties. The presence of reduced fibrin clot permeability and clot susceptibility to lysis before ovarian stimulation (OS) and intrauterine insemination (IUI) in women with negative pregnancy outcome suggests the contribution of fibrin clot properties to treatment outcome. This might be of importance in treatment plan which OS agent should be prescribed to improve the clinical management of women with UI.

The final analysis included 70 women with UI, diagnosed after 14.1 ± 5.8 months of regular, unprotected intercourse, along with 70 well-matched controls (Table  1 ). There were no differences with regard to comorbidities and hormonal profile between the two studied groups (Table  1 ). Women from the UI group had decreased thrombin generation reflected by lower ETP (−17.8%) and slightly prolonged lag time (+ 10.0%) along with reduced K S (−20.8%) and longer CLT (+ 17.5%) in contrary to the controls. In women with UI, ETP was associated with peak thrombin ( r  = 0.57, p  = 0.02), whereas CLT was negatively correlated both with K S ( r  = −0.42, p  = 0.001) and TSH ( r  = −0.56, p  = 0.04). Table 1 Characteristic and basic laboratory investigations along with coagulation, and fibrin clot properties in women with unexplained infertility (UI) and without infertility (controls) Variables UI ( n  = 70) Controls ( n  = 70) p Age, years 30.7 ± 3.5 31.0 ± 4.2 0.69 BMI, kg/m 2 24.7 ± 3.0 24.9 ± 4.0 0.82 AMH, ng/ml 3.6 [2.3–5.3] 3.0 [2.1–6.4] 0.49 Comorbidities, n (%)  Hypertension 4 (5.7) 3 (4.3) 0.76  Autoimmune diseases 9 (12.9) 12 (17.1) 0.40 Hormonal profile  TSH, uIU/mL 1.5 [1.0–2.1] 1.6 [1.0–2.1] 0.43  FSH, IU/mL 6.2 [4.9–7.2] 5.6 [4.7–6.4] 0.24  LH, IU/mL 7.3 [6.3–10.2] 7.6 [5.9–10.3] 0.83  Estradiol, pmol/L 171.2 [117.5–241.8] 162.0 [114.6–224.4] 0.70 Coagulation and fibrin clot parameters  ETP, nM × min 1399.7 [1270.6–1609.8] 1703.5 [1507.5–1885.9] 0.0007  Lag time, min 3.3 ± 0.8 3.0 ± 0.6 0.007  Peak thrombin, nM 232.3 [176.4–298.5] 263.5 [220.5–293.4] 0.27  K S , × 10 −9 cm 2 6.1 [5.1–8.4] 7.7 [6.6–9.1] 0.003  CLT, min 91.1 [81.8–103.3] 77.5 [70.0–83.0]  < 0.0001 Values are given as mean ± SD, median (interquartile range), or percentage BMI body mass index, AMH anti-Müllerian hormone, OS ovarian stimulation, PRL prolactin, TSH thyroid-stimulating hormone, FSH follicle-stimulating hormone, LH luteinizing hormone, ETP endogenous thrombin potential, K S permeability coefficient, CLT clot lysis time Characteristic and basic laboratory investigations along with coagulation, and fibrin clot properties in women with unexplained infertility (UI) and without infertility (controls) Values are given as mean ± SD, median (interquartile range), or percentage BMI body mass index, AMH anti-Müllerian hormone, OS ovarian stimulation, PRL prolactin, TSH thyroid-stimulating hormone, FSH follicle-stimulating hormone, LH luteinizing hormone, ETP endogenous thrombin potential, K S permeability coefficient, CLT clot lysis time After 2.5 ± 0.5 cycles, 27.1% of women ( n  = 19) had positive pregnancy outcomes. There were no differences between pregnant and non-pregnant women with UI regarding age, BMI, comorbidities, and hormonal profile (Table  2 ). However, women with UI and positive pregnancy outcomes showed higher K S (+ 67.9%) along with shorter CLT (−15.9%), indicating a less compact fibrin network (Fig.  2 A) with no differences in CAT variables (all p  > 0.05) when compared to non-pregnant women with UI (Table  2 ) with more compact fibrin networks (Fig.  2 B). We observed high accuracy for CLT in the prediction of pregnancy (AUC = 0.80; Fig.  3 ). Table 2 Characteristic and basic laboratory investigations along with coagulation, and fibrin clot properties in women with unexplained infertility ( UI ) and positive along with negative pregnancy outcomes Variables UI with pregnancy ( n  = 19) UI without pregnancy ( n  = 51) p Age, years 30.1 ± 4.4 31.0 ± 3.0 0.40 BMI, kg/m 2 25.5 ± 3.1 24.4 ± 2.9 0.18 Duration of infertility, months 14.1 ± 5.2 14.3 ± 5.7 0.60 AMH, ng/mL 3.6 [2.7–5.5] 3.5 [2.2–5.3] 0.78 Hormonal profile  TSH, uIU/mL 1.6 [1.0–1.9] 1.3 [1.0–2.1] 0.70  FSH, IU/mL 5.3 [4.0–6.6] 6.4 [5.4–7.3] 0.06  LH, IU/mL 7.1 [5.9–9.3] 7.4 [6.4–10.5] 0.43  Estradiol, pmol/L 174.0 [126.5–221.3] 170.4 [108.4–246.0] 0.81 Coagulation and fibrin clot parameters  ETP, nM × min 1346.4 [1211.6–1465.5] 1436.5 [1278.3–1640.4] 0.24  Lag time, min 3.1 ± 0.6 3.4 ± 0.9 0.16  Peak thrombin, nM 241.8 [193.8–298.1] 231.8 [173.3–298.2] 0.72  K S , × 10 −9 cm 2 9.4 [8.6–11.0] 5.6 [4.8–6.4]  < 0.0001  CLT, min 81.0 [76.7–87.5] 96.3 [90.0–105.0] 0.002 Values are given as mean ± SD, median (interquartile range) Abbreviations: see Table  1 Fig. 2 Representative scanning electron microscopy (SEM) images of clots from women with unexplained infertility (UI)—pregnant ( A ) and non-pregnant ( B ) Fig. 3 ROC curve for CLT. The outcome investigated in the ROC analysis was pregnancy. Optimal cutoffs and AUC (with 95% CIs) are presented Characteristic and basic laboratory investigations along with coagulation, and fibrin clot properties in women with unexplained infertility ( UI ) and positive along with negative pregnancy outcomes Values are given as mean ± SD, median (interquartile range) Abbreviations: see Table  1 Representative scanning electron microscopy (SEM) images of clots from women with unexplained infertility (UI)—pregnant ( A ) and non-pregnant ( B ) ROC curve for CLT. The outcome investigated in the ROC analysis was pregnancy. Optimal cutoffs and AUC (with 95% CIs) are presented In multivariable logistic regression analysis including CLT, Ks, age and the length of infertility, CLT remained a significant independent predictor of pregnancy (OR = 0.70, 95% CI 0.3–0.8, p  = 0.002), indicating that shorter clot lysis was associated with increased probability of pregnancy. Ks showed a trend toward a positive association (OR = 6.36, 95% CI 0.80–1.01, p  = 0.052), whereas age and the length of infertility were not associated with pregnancy outcome (OR = 0.98, 95% CI 0.76–1.26, p  = 0.889 and OR = 0.78, 95% CI 0.54–2.41, p  = 0.741, respectively). In women with positive pregnancy outcomes ETP was associated with peak thrombin ( r  = 0.61, p  = 0.03) and CLT ( r  = 0.36, p  = 0.03), whereas CLT was associated with K S ( r  = −0.26, p  = 0.04) and peak ( r  = 0.27, p  = 0.02). Women with UI and negative pregnancy outcomes showed associations between ETP and peak thrombin ( r  = 0.52, p  = 0.02) along with an inverse association of ETP and K S ( r  = −0.31, p  = 0.01). In this group CLT was negatively correlated with K S ( r  = −0.31, p  = 0.01) together with TSH ( r  = −0.33, p  = 0.02) and positively with FSH ( r  = 0.37, p  = 0.001).

We recruited 109 women at the Department of Gynecological Endocrinology, University Hospital. Women with UI were screened in accordance with the ESHRE criteria [ 1 ] and qualified to 3–6 cycles of OS with IUI. The inclusion criteria were: (1) age < 40 yrs old; (2) ovulatory cycles; (3) confirmed bilateral tubal patency by hysterosalpingography; (4) confirmed suitable sperm quality (per WHO 2021 guidelines). The exclusion criteria included: (1) decreased ovarian reserve test (anti-Müllerian hormone, AMH  2.5 mIU/ml), smoking, known malignancy, thrombophilia (i.e. factor V Leiden mutation), any chronic inflammatory diseases (e.g. rheumatoid arthritis) or signs of an acute infection, advanced chronic renal disease (estimated glomerular filtration rate [eGFR] < 30 mL/min), international normalized ratio (INR) more than 1.2 at the day of blood draw; (4) Endometriosis stages ≥ III according to revised American Society for Reproductive Medicine score (rASRM) (Fig.  1 ). Fig. 1 Flow chart of inclusion of women with unexplained infertility ( UI ). FVL factor V Leiden mutation; TSH thyroid-stimulating hormone; OS ovarian stimulation Flow chart of inclusion of women with unexplained infertility ( UI ). FVL factor V Leiden mutation; TSH thyroid-stimulating hormone; OS ovarian stimulation We also recruited seventy women not diagnosed with infertility, with live birth in history, and served as controls. Cases and controls were matched for age (± 1 years) and body mass index (BMI; ± 1.5 kg/m 2 ). All women with UI received letrozole orally (2.5 mg daily) between 3rd and 5th day of the menstrual cycle for 5 days. Transvaginal ultrasound (TVU) was performed at letrozole initiation—to confirm lack of any growing follicle and every second day from day 10 of the cycle to monitor both the number and size of developing follicles along with endometrial thickness. If ≥ one follicle (not more than 3 follicles) has achieved ≥ 18 mm and endometrial thickness attained ≥ 7 mm, the 250 mcg hCG (Ovitrelle, Merck Europe B. V.) s.c. was prescribed to induce ovarian ovulation. IUI was performed 24–36 h from hCG injection. All women were qualified for a maximum of 6 cycles with OS and IUI. Serum βhCG level was measured at least 14 days from hCG administration. Two weeks from positive pregnancy test (βhCG level > 10 mIU/ml) TVU was performed to confirm clinical pregnancy. The evolution of the pregnancy was not recorded. In case of a negative pregnancy test the patient was enrolled in another course of OS with letrozole. The Ethics Committee at Jagiellonian University Medical College approved the study (reference number KBET 1072.6120.220.2022), and participants provided informed, written consent in accordance with the Declaration of Helsinki. The study was registered at www.clinicaltrials.gov ( NCT04166825 ) and is reported according to the CONSORT guidelines. Venous blood samples were drawn with minimal stasis using atraumatic venipuncture, after an overnight fast and a 10-min rest, between 08.00 and 10.00 AM. Blood samples were collected from all participants once—at study entry (between the third and the fifth day of the cycle). Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E 2 ), thyroid-stimulating hormone (TSH), prolactin (PRL) were measured using an enzyme-linked fluorescent assay (Vidas, BioMerieux). For fibrin analysis, blood samples were mixed with 3.2% citrate (9:1) and centrifuged for 20 min and stored at − 80 °C to allow batch analyses. All measurements were determined at baseline and fibrin clot analysis was performed by technicians blinded to the origin of the samples. Intra- and intraassay coefficients of variation were less than 8%. Plasma fibrin clot permeability was measured as described previously [ 13 ]. Briefly, 20 mmol/L of calcium chloride and 1 U/mL of human thrombin (Sigma, St. Louis, MO) were added to 120 µl of citrated plasma. Tubes containing the clots were connected via plastic tubing to a reservoir of Tris buffer and the volume flowing through the gels was measured. A permeation coefficient (K s ), which indicates the pore size in fibrin networks, was assessed using a pressure-driven system and was measured from the equation: K s  = QxLxƞ/txAxΔp, where Q is the flow rate in time t; L, the length of a fibrin gel; ƞ, the viscosity of liquid (in poise); A, the cross-sectional area (in cm 2 ) and Δp, a differential pressure (in dyne/cm 2 ). The lower the K s values, the more compact the fibrin clot structure. The efficacy of fibrin clot lysis was measured as described previously [ 13 ]. Briefly, 15 µl of citrated plasma was mixed with calcium chloride, 10,000-diluted human TF (Innovin, Siemens, Marburg, Germany), 12 µM of phospholipid vesicles and 60 ng/ml of recombinant t-PA (rt-PA, Boehringer Ingelheim, Ingelheim, Germany). Clot lysis time (CLT) was defined as the time from the midpoint of the clear-to-maximum-turbid transition to the midpoint of the maximum-turbid-to-clear transition. The intraassay and interassay coefficients of variation for fibrin variables were < 8%. Plasma clots from 6 randomly selected women, including 3 with positive pregnancy outcomes and 3 with negative pregnancy outcomes, were fixed using 2.5% glutaraldehyde, washed with distilled water, dehydrated in graded water–ethanol solutions, dried by the critical point procedure and sputter coated with gold. Samples were scanned in 10 different areas (microscope JEOL JCM-6000; JEOL Ltd., Tokyo, Japan) at a magnification of 5,000x. Thrombin generation was measured using calibrated automated thrombography (CAT), as previously described [ 14 ]. The maximum concentration of thrombin formed during the time of registration was described as the thrombin peak, and the area under the curve represented endogenous thrombin potential (ETP). The study was powered to have a 90% chance of detecting a 10% difference in K s and CLT using a p value of 0.01 based on values of K s and CLT as described previously [ 15 ]. To demonstrate such a difference or greater in K s and CLT, at least 18 women were enrolled, based on the values from the previous article [ 14 ]. Categorical variables are presented as numbers and percentages. Continuous variables are expressed as mean ± standard deviation or median and interquartile range (IQR), as appropriate. The Kolmogorov–Smirnov test was used to assess conformity with a normal distribution whereas non-normally distributed data were analyzed by Mann–Whitney’s U test. Categorical variables were analyzed using either the chi 2 test or Fisher’s exact test. Pearson’s correlation coefficient (Pearson’s r) or Spearman’s rank correlation coefficient was calculated to assess the linear correlations between variables with a normal or non-normal distribution, respectively. Receiver operating characteristic (ROC) curves and AUC were used to analyze the discriminatory power of variables in respect to the presence of pregnancy. A multiple logistic regression analysis was performed with pregnancy (yes/no) as a dependent variable and CLT, Ks, age, duration of infertility as independent variables. Given the limited number of pregnancy events, the model included a restricted number of parameters to avoid overfitting. Associations between the variables were expressed as odds ratios (OR) with 95% confidence intervals. Two-sided P -values < 0.05 were considered statistically significant. Statistical analysis was performed with the STATISTICA 13.0 software (StatSoft, Poland).

The presence of reduced fibrin clot permeability and clot susceptibility to lysis before OS and IUI in women with negative pregnancy outcomes suggests a contribution of fibrin clot properties to treatment outcome. This might be important in the treatment plan which OS agent should be prescribed to improve the clinical management of women with UI.

This study is the first to show unfavorably altered fibrin clot phenotype among women with UI, reflected by reduced fibrin clot porosity and susceptibility to lysis. We also demonstrated prothrombotic fibrin clot features in women with UI and negative pregnancy outcomes after OS followed by IUI in contrast to women diagnosed with UI who achieved pregnancy. The current findings provide new insights into possible mechanisms that condition positive pregnancy outcomes in women with UI. Coagulation and fibrin clot properties have been explored in women undergoing COH for IVF. Procoagulable state is reflected by increased levels of coagulation factors, such as fibrinogen, factor V, and factor VIII, together with a reduced activity of coagulation inhibitors (protein S and antithrombin) [ 16 ] and elevated ETP [ 11 , 17 , 18 ]. Moreover, in our previous study, we observed enhanced thrombin generation capacity and decreased clot density in women undergoing COH with the GnRH antagonist (GnRH-ant) protocol, compared to the GnRH agonist one [ 19 ]. Regarding fibrin clot phenotype, several studies have shown impaired fibrinolysis reflected by COH-associated prolonged CLT [ 20 , 21 ]. However, the use of OS separately or combined with IUI is a first-line treatment in women diagnosed with infertility [ 22 ]. Data on hemostatic variables and fibrin clot properties in infertile women are scarce. We observed in women with UI compared to apparently healthy controls, lower thrombin generation potential reflected by increased lag time of thrombin generation and decreased ETP. Hugon-Rodin et al. [ 23 ] reported in infertile women, qualified for three different ovarian stimulation protocols, ETP levels (1263–1282 nM × min) comparable to values observed in our UI cohort. On the other hand, our previous study showed increased ETP and peak thrombin along with shorter lag time in women with infertility related to ovulatory disorders connected with polycystic ovary syndrome (PCOS) (Piróg et al. 2025). This can be explained by differences in patient characteristics. Women with PCOS are more often characterized by higher BMI, metabolic disorders, such as insulin resistance or unfavorably changed lipid profile, and, consequently, lead to markedly enhanced thrombin generation [ 24 , 25 ]. Moreover, in the current study no differences in CAT variables between pregnant and non-pregnant women with UI were shown. The coagulation variables recorded in the CAT assay have been studied in our previous study among women with infertility related to PCOS where no differences in coagulation variables were observed between women with positive and negative pregnancy outcomes. Anyhow, pregnant women showed higher ETP contrary to non-pregnant ones (Piróg et al. 2025, under review). Thus, variations in CAT variables underscore the complex relationship between thrombin generation and the underlying causes of infertility. It should be underlined that hyperestrogenism related to ovarian stimulation may enhance the thrombogenic effect by increasing procoagulant factor levels or activity (such as fibrinogen, von Willebrand factor, factors VIII and V), reducing the levels/activity of natural anticoagulants (antithrombin and protein S) and attenuating fibrinolytic potential [ 20 , 26 ]. These alterations suggest the presence of a prothrombotic state. Anyhow, the levels of most of these parameters remain within normal ranges, therefore, their clinical relevance is unclear. Moreover, it has been shown that an adequate thrombin concentration is necessary for blastocyst implantation, remodeling of both human endometrial stromal cells and spiral arteries, together with undisturbed trophoblast development [ 27 , 28 ]. However, enhanced thrombin generation has been reported as a predictor of IVF failure [ 27 ]. Moreover, it has been shown that the fibrinolytic system plays an important role in modulating miscarriage and implantation failure [ 27 ]. Reduced fibrinolytic activity reflected by increased plasminogen activator inhibitor-1 (PAI-1) levels along with decreased tissue plasminogen activator (tPA) activity is suggested to be responsible for recurrent implantation failure following embryo transfer [ 10 ]. Moreover, we found favorably altered fibrin clot properties reflected by higher K S and shorter CLT in pregnant women with UI when compared to women with negative pregnancy outcomes. The current results are comparable with our recent study in PCOS-related infertility showing increased fibrin clot permeability and decreased clot susceptibility to lysis in pregnant women contrary to non-pregnant ones (Piróg et al. 2024, under review). For a correct interpretation of the current results in the context of previous studies, it should be taken into account that, in our study, only baseline parameters before OS were determined. Additional studies with determination of hemostasis parameters in different timelines, especially at the end of OS and pregnancy confirmation are needed to elucidate the mechanism beyond this effect. However, our findings of altered fibrin clot properties in pregnant women with UI compared to those with negative pregnancy outcomes further underscore the association between coagulation and reproductive success. The separate issue is that different OS agents can be used. In the current study, women were assigned to OS with letrozole, a third-generation aromatase inhibitor that interferes with estrogenic feedback at the pituitary due to inhibition of estrogen biosynthesis and, consequently, stimulates FSH production. Other agents for OS include exogenous gonadotropins (Gn) and clomiphene citrate (CC). While letrozole and CC are taken orally, Gn are injected subcutaneously. Multiple systemic reviews have compared the pregnancy outcomes of all three OS agents among women with UI [ 4 , 7 , 29 ]. OS with Gn increased the chance of live birth (32% for Gn, 22% for CC and 19% for letrozole) and reduced the time to conception compared to oral OS agents, at the cost of a higher multiple pregnancy rate (32% of all Gn-related pregnancies were multiple gestations) and increased treatment costs [ 30 ]. Estradiol levels in COH for IVF, where Gn are also used, increase to 2500–3000 pg/ml [ 9 ], whereas in our recent study we observed, associated with letrozole, an increase in estradiol level to 898.29 pg/ml (Piróg et al. 2024). It might be speculated that use of different OS agents may favorably modify fibrin clot properties and, therefore, increase pregnancy outcomes. Additional studies are needed to elucidate this hypothesis. The current study has several limitations. First, it is a single-center study and the studied population is relatively low, anyhow, this study was adequately powered and it is hypothesis generating. Nevertheless, the presented associations do not necessarily mean the cause-effect relationship. Therefore, our results should be interpreted with caution. Second, blood collection and, therefore, determination of CAT variables and fibrin clot properties were performed only once—at study entry and not during OS or after confirmation of pregnancy. Therefore, we cannot exclude variability of certain fibrin parameters over time, as well as their impact on pregnancy outcomes. Further studies are needed to determine changes in fibrin clot properties in different timelines. Finally, the significant associations presented in our research do not necessarily mean the cause-effect relationship.

Unexplained infertility (UI) is diagnosed when no abnormalities in the female and male reproductive systems are identified [ 1 ]. Despite the improvement of the diagnostic investigations, infertility remains unexplained in approximately 30% of couples [ 2 ]. The diagnosis is made in the case of absence of conception despite 12 months of unprotected intercourse after the exclusion of anovulation, tubal pathology, poor sperm quality or any known cause of infertility [ 3 ]. As a diagnosis of exclusion, UI treatment is empirical [ 4 ]. Both the European Society of Reproductive Medicine [ 1 ] and the National Institute for Health and Care Excellence (NICE) [ 5 ] guidelines insist that couples should obtain information about their chance of natural conception and should not be exposed to unnecessary risks or ineffective treatment. Expectant management can be effective in good-prognosis couples, whereas assisted reproductive technologies (ART) are recommended if the chances of conception with treatment are higher than those without treatment [ 5 , 6 ]. Intrauterine insemination (IUI) with ovarian stimulation (OS) is recommended as a first-line treatment for couples with UI [ 1 , 7 ]. OS is associated mostly with monofollicular development, which may result in enhanced fertility and a reduced risk of ovarian hyperstimulation syndrome and multiple births, as compared with control ovarian hyperstimulation (COH) for in vitro fertilization (IVF) [ 8 ]. Moreover, it has been shown that COH-related supraphysiological estrogen concentration, may unfavorably modify fibrin clot properties and enhance thrombin generation, which subsequently decreases pregnancy outcomes [ 9 ]. It has been suggested that the undisturbed blood coagulation with subsequent fibrinolysis are significant parts of ovarian remodeling during ovulation [ 10 ]. Moreover, a suitable thrombin generation is necessary for embryonic implantation and proper development of the fetus [ 11 ]. However, hypercoagulability along with decreased fibrinolytic activity may restrict trophoblast invasion and embryonic development and have been reported as predictors of implantation failure [ 12 ]. Given the clinical significance of UI, we determined the fibrin clot phenotype together with thrombin generation among women with UI and investigated their associations with positive and negative pregnancy outcomes after OS followed by IUI.