Infertility is a common problem affecting between one-fifth and one-sixth of couples of reproductive age. Although it has many known causes, 15–30% of couples are still classified as having unexplained infertility (UI) because the underlying cause(s) is not found despite an extensive and meticulous infertility workup 1.
The treatment for UI is, therefore, by definition, empiric because it does not address a specific defect or functional impairment. The principal treatments for UI include expectant observation, ovulation induction with timed intercourse, clomiphene citrate and intrauterine insemination, controlled ovarian hyperstimulation with intrauterine insemination, and in-vitro fertilization (IVF) 2.
Recently, a hypothesis has been raised that the same factors associated with the occurrence of recurrent pregnancy loss may also affect the early phase of the embryo implantation process 3.
The possibility of alterations in the hemostatic mechanism of a thrombophilic nature at the implantation site, affecting trophoblast invasion and hampering implantation of the embryo, should, therefore, be taken into consideration as a possible subtle cause of infertility 4.
Thrombophilia may be congenital or acquired. Congenital factors include protein C deficiency, protein S deficiency, antithrombin III (ATIII) deficiency, the presence of factor V Leiden (FVL), a mutation in the 20210A allele of the prothrombin gene, and a mutation in the methylenetetrahydrofolate reductase (MTHFR) enzyme gene. Acquired factors include lupus anticoagulant (LA) and anticardiolipin (ACL) antibodies. In general, thrombophilia should be considered a multifactorial disorder and not as an expression of a single genetic abnormality 5.
There are limited data in the literature associating thrombophilia and UI; however, most studies focus on the potential role of thrombophilia in recurrent implantation failure following IVF/ICSI 6–9. Thus, this study was designed to evaluate the potential role of both inherited and acquired thrombophilia in infertile women with UI.
Materials and methods
This is a prospective comparative observational study, in which 140 women were enrolled from the infertility and outpatient clinics of Tanta University Hospital during the period from November 2011 to March 2013.
The study group included 70 women with UI. Inclusion criteria were women partner age less than 38 years with regular menstrual cycles, normal ovarian function, BMI less than 32, normal uterine cavity and fallopian tubes as documented by ultrasound, hysterosalpingography, and/or laparoscopy, normal hormonal profile including follicle stimulating hormone, luteinizing hormone, thyroid-stimulating hormone, testosterone, and prolactin levels, absence of autoimmune or endocrine disorders, and no consanguinity. The male partner had normal semen parameters according to the WHO criteria.
Women with endometriosis, hydrosalpinx, abnormal uterine cavity on the hysterosalpingograms, and a history of thromboembolic disease and those who were receiving hormonal treatment were not included in the study group.
The control group included 70 age-matched women who conceived spontaneously with at least one uneventful pregnancy and no history of miscarriage, autoimmune disorders, or endocrine diseases.
All women were investigated for the presence of inherited (FVL mutation and deficiencies in proteins S and C and ATIII) or acquired (LA and ACL) thrombophilic factors.
The study was approved by the ethics committee of the university and the purposes and procedures of the study were carefully explained before an informed consent was obtained from those willing to participate. As estrogen levels may alter protein S and protein C, and given that the latter levels can be modified after meals, all the blood tests were performed under fasting conditions while the women were not pregnant, at least 3 months after the last pregnancy, and in the absence of any recent or concurrent hormonal therapy.
We defined isolated thrombophilia as the presence of a specific thrombophilic marker and combined thrombophilia as the presence in the same patient of at least two thrombophilic markers.
Functional protein C levels were measured using a chromogenic assay (Diagnostica Stago, Paris, France). Protein C deficiency was diagnosed when levels were less than 60% (normal range, 60–140%). Functional protein S levels were measured using a clotting assay. Levels of less than 50% (normal range, 50–160%) were considered to indicate deficiency. Antithrombin levels were measured using a chromogenic assay (Diagnostica Stago), and deficiency was diagnosed when functional AT levels were less than 80% (normal range, 80–120%). Immunoglobulin M (IgM) and IgG ACL were assayed using a validated enzyme-linked immunosorbent assay calibrated against international standards, as described elsewhere. ACL antibodies were reported in international units (positive when >10 IU/ml). Screening for LA was performed by the kaolin cephalin clotting time utilizing sensitive reagents and by the dilute Russell’s viper venom time with a neutralization procedure using frozen–thawed platelets. Results for LA were expressed as positive or negative.
Detection of genotype mutation
All reagents used in these steps were provided by the HVD Strip Assay kit manufactured by ‘Viennalab Diagnostika GmbH’ (ViennaLab Diagnostics, GmbH Gaudenzdorfer Guertel, Vienna, Austria).
Blood DNA extraction
DNA was extracted from a 100 μl blood sample. The DNA concentration extracted was confirmed through measurement by a UV spectrophotometer; readings were taken at wave lengths of 260 and 280 nm according to the method reported by Kupferminc et al. 10. The DNA concentration extracted ranged from 20 to 30 ng DNA/μl. Multiplex PCR amplification: A multiplex PCR amplification reaction mixture was prepared for each sample using a biotinylated primer with specific sequences for the detection of FVL genotype mutations.
In a thermal cycler (G-Storm, Catcombe, Somerton, Somerset, UK), the prepared reaction components were processed under conditions of 94°C, 2 min (initial denaturation), followed by 35 cycles of 94°C, 15 s, 58°C, 30 s, and 72°C, 30 s, for denaturation, annealing, and extension steps, respectively. The amplified DNA was analyzed by gel electrophoresis. Ten microliter of each reaction mixture and 1000 bp ladder (molecular weight marker) were separated on a 3% agarose gel that contained 0.3 μg/ml of ethidium bromide. The bands of fragment lengths suspected to see at 173, 202, and 223 bp indicated successful amplification.
Reverse hybridization of amplification products
The amplified products were selectively hybridized to a strip assay that included allele-specific oligonucleotide probes (wild and mutant specific) immobilized as an array of parallel lines; instead of using a radioactive probe, the bound biotinylated sequences were detected using streptavidin–alkaline phosphatase and color substrates. In a thermoshaker (Shy line shaker DTS-2; ELMI Ltd., Riga, Latvia), 10 μl of each amplification product was used in the typing trays, with 1 ml hybridization buffer added to a test strip with orbital shaking at 45°C for 30 min at 50 rpm. Washing was performed at 45°C with shaking.
Identification of the hybridized products
Detection of specifically bound mutant and wild-type alleles was performed by a visible enzymatic color reaction. For each test strip, 1 ml conjugate solution (contains streptavidin–alkaline phosphatase) was added and incubated for 15 min at room temperature. Then, 1 ml Color Developer (contains nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate) was added and incubated for 15 min at room temperature in the dark on an orbital shaker.
The results were presented as means±SD and percentages. Comparisons of categorical variables were made between patient and control groups using the χ2-test, Student’s t-test, and the F-test. The statistics package for social sciences and Microsoft Office Excel (IBM Corporation, Armonk, New York, USA) were used for data processing and data analysis. Differences were considered statistically significant at a P value less than 0.05.
The clinical data of the study and control groups are summarized in Table 1. There were no significant differences between both groups in age and BMI. Table 2 shows the frequency of the thrombophilic factors studied in both groups. At least one inherited or acquired thrombophilic factor was detected in 42.86% (30/70) of women with UI compared with 8.57% (6/70) in the control group. This difference was statistically significant (P<0.01). A significantly higher prevalence of the FVL gene mutation was observed in women with unexplained infertility in comparison with that in fertile women (control) (14.29 vs. 1.43%; P=0.04). In women with UI, homozygosity for the FVL mutation was found in 5.71% (4/70) of patients compared with none in the control group. Similarly, 8.57% (6/70) of women in the study group were heterozygous for the FVL mutation versus 1.43% in the control group.
For the other thrombophilic factors, ACL antibodies and LA antibodies were more common in women with UI (20%) compared with 4.29% in the control group. However, these differences were not statistically significant. Also, there were no statistically significant differences between both the study and the control group, although more common in the study group, in the deficiencies of ATIII and proteins C and S. Finally, combined thrombophilia (two or more thrombophilic factors) was significantly higher in women with UI as compared with the control group (22.86 vs. 1.43%) (P<0.0001).
This study was designed to examine the influence of thrombophilia on a common and contentious problem encountered in reproductive medicine, namely, UI.
Thrombophilia is believed to be a multiple gene disease with more than one defect, which explains why some women with thrombophilia never have a thrombotic event whereas others have complications 9.
The results of the present study are in agreement with those of Bianca et al.11, who compared the frequency of five thrombophilic gene mutations between couples with a history of UI and those with a history of recurrent pregnancy loss and found an association between them with FVL and MTHFR C677T. These findings are in agreement with those published previously among individuals with a history of not only UI 12, recurrent implantation failure 9, and recurrent pregnancy loss 13,14 but also deep vein thrombosis 15. Taken together, these observations suggest that the association of reproductive disorders (as well as cardiovascular disease) and inherited thrombophilia is manifested by the total number of thrombophilic mutations rather than specific genes involved.
The mechanism by which thrombophilic gene mutations impact the frequency of recurrent miscarriage is considered to be related to clotting of placental vessels 16, recurrent implantation failure involved in the effects of hypofibrinolysis on trophoblast migration 9, and UI leading to folic acid metabolism and/or recurrent unrecognized very early pregnancy losses because of failure of implantation 12.
The importance of angiogenesis as a crucial factor in embryo implantation and growth must be taken into consideration. As thrombophilia may lead to the development of microthrombosis at the implantation site, screening for thrombophilia in infertile women, particularly in those with implantation failure, is relevant 17.
The role of immunological causes and thrombophilia in implantation failure and infertility through mechanisms similar to recurrent miscarriages has been the focus of many recent research efforts. In agreement with the results of other studies 6–8, this study showed that at least one thrombophilic factor was detected in 42.86% of women with UI.
FVL is a genetic disorder that is characterized by an impaired anticoagulant response to activated protein C. Mutation in the FVL gene increases thrombin generation, with a four-fold to eight-fold increased risk of thrombosis in the heterozygous mutation and an 80-fold increased risk in the homozygous mutation 18. In a recent meta-analysis, a three-fold to four-fold increased risk of recurrent early pregnancy loss was found in women with the FVL mutation 19. Vascular placental insufficiency has been suggested as a potential cause of these early pregnancy losses. The results of our study showed that the prevalence of the FVL mutation was higher in women with UI (14.29%) than those in the control group (1.43%). This finding is consistent with that reported by Grandone et al.6, who found an incidence of 11.1% in women with greater than or equal to three IVF failures compared with none in the control groups. Others have failed to find such an association 7,8.
Compared with fertile women, a finding of a higher incidence of thrombophilia in women with recurrent implantation failures following repeat cycles of IVF has become increasingly common. Azem et al.8 carried out a case–control study including 45 women with implantation failure, 44 fertile women, and 15 infertile women who had, however, become pregnant at their first IVF attempt, aiming to investigate the following thrombophilic factors: prothrombin gene mutation, MTHFR gene mutation, the presence of FVL, and antithrombin, and protein C and protein S deficiency. A high frequency of thrombophilia was found in the subgroup of women with implantation failure (17.8%) compared with the group of fertile women and the group of women who became pregnant at the first IVF attempt (a frequency of 8.9% in both groups). This fact reinforces the association of this pathology with vascular impairment and a consequent difficulty in embryo implantation 8.
Vaquero et al.3 evaluated 59 women with implantation failure and 20 fertile women in a case–control study; a higher rate of acquired, but not congenital, thrombophilia was found in the infertile population.
Silva Soligo et al. 20 published a study in 2007 on the prevalence of thrombophilia in a fertile and infertile female population. This study concluded that thrombophilia was more common in the group of infertile women.
In 2008, Qublan et al. 21 carried out a case–control study to evaluate the use of low-molecular-weight heparin for the treatment of embryo implantation failure in view of the association between thrombophilia and implantation failure. Although the exact mechanism behind implantation failure and infertility remains to be fully clarified, this study suggests that maternal blood vascularization with adequate syncytiotrophoblast invasion may be affected by microthrombosis at the implantation site.
In a prospective study carried out by Bellver et al.22, 119 women were evaluated. Thirty-two White women included in the control group were egg donors with no endocrine or autoimmune disorders, with normal karyotype, and no history of obstetric pathology. A second group included 31 women with infertility of no apparent cause, whereas a third group included 26 women with implantation failure and a fourth group included 30 women who had a history of recurrent pregnancy loss. The group of women with implantation failure and the recurrent pregnancy loss group had been diagnosed as normal before implantation. The following factors were investigated in these four groups: protein C, protein S, ATIII, LA, activated protein C resistance, IgG and IgM ACL antibodies, homocysteine, FVL, prothrombin mutation, MTHFR mutation, thyroid-stimulating hormone, free thyroxine, antithyroid peroxidase antibody, and antithyroglobulin antibody. In the group of women with implantation failure, a higher prevalence of activated protein C resistance and LA was found, in addition to the presence of more than one thrombophilic factor. Thyroid autoimmunity was more common in the group of women with implantation failure and in the group with infertility of no apparent cause. The authors suggest an association between thrombophilia and implantation failure, but do not recommend screening for all infertile women 22.
In 2009, a case–control study was carried out comparing a group of 51 women with implantation failure with 50 fertile women in terms of three hereditary thrombophilic factors: the presence of FVL, MTHFR mutation, and prothrombin mutation. No statistically significant difference was found in the frequency of thrombophilic factors between both groups. Nevertheless, a finding of at least one thrombophilic factor (62.7%) was more common in the group of women with implantation failure versus the control group (53.9%), although not statistically significant; the authors suggest that the difference may become significant if the sample size were larger 23.
Casadei et al.24 published a case–control study that included 100 women with UI and 200 fertile women. The following hereditary factors were investigated: FVL (G1691A), prothrombin gene mutation (G20210A), and MTHFR enzyme mutation (C677T). This study found no difference in the frequencies of thrombophilic factors between the two populations evaluated 24.
Also, in 2010, Sharif et al.25 carried out a prospective cohort study and analyzed 273 cases of implantation failure after IVF/ICSI. Serial ultrasound examinations, hysteroscopy, and research of hereditary and acquired thrombophilias were performed. Eighty-four women had tested positive for thrombophilia (63 hereditary thrombophilia and 21 acquired thrombophilias). This study confirms the value of microthrombosis formation at the implantation site preventing trophoblastic invasion and subsequent embryo implantation 25.
Deficiencies in the natural anticoagulant proteins C and S and ATIII occur much less frequently. Several studies, with conflicting results, have evaluated the potential role of these deficiencies in recurrent early pregnancy losses 26,27. This might be related to the rarity of these deficiencies and the smaller-size studies. Only one study has examined the prevalence and the role of these deficiencies in women with repeated IVF failure 8. Azem et al.8 found no significant association between these deficiencies and repeated IVF failures, which is consistent with the findings of our study.
APLAs are a nonorgan-specific antibodies directed against the membrane phospholipids or phospholipid-binding plasma proteins 28. The importance of these antibodies as an etiological factor of implantation failure after IVF-embryo transfer is now well established 29–31. It has been suggested that APLAs reduce the levels of Annexin V, which is a potent anticoagulant, leading to thrombosis and possible implantation failure or early pregnancy loss 32,33.
Applying the same mechanism, this might also be the case in women with UI. Repeated implantation failures with unrecognized pregnancy losses could clarify part of the gray area of one of the frustrating problems, for both the patient and the clinician, in reproductive medicine, namely, UI.
In our study, 20% (14/70) of women with UI were positive for APLAs compared with 4.29% (3/70) in the control group, but this difference was not statistically significant. These results are in agreement with previous reports 29–31. Birkenfeld et al.29 evaluated 56 women with implantation failure and 14 women who successfully conceived after IVF-embryo transfer, and found that 32% of women with implantation failure versus 10% in the control group were positive for APLAs (P=0.02). Moreover, Kaider et al.30 , investigating 42 women with IVF failure and 42 women who had conceived successfully after IVF-embryo transfer, found that 26.2% of women in the study group compared with 4.8% in the control group were positive for APLAs (P=0.01). These authors recommended testing for the presence of APLAs in all patients undergoing IVF before initiating treatment 30. Coulam et al.31, in a larger size study, found that 69 women of 312 (22%) with implantation failure after IVF treatment were positive for APLAs compared with five women of 100 (5%) in the fertile control group (P<0.01).
The results of our study showed that the combination of two or more thrombophilic factors was significantly higher in women with UI compared with the controls. These findings are in agreement with those reported by Coulam et al.13 , who observed that 74% of women with recurrent implantation failure after IVF-embryo transfer had three or more gene mutations compared with 20% of healthy fertile controls.
The results of our study indicate that thrombophilia may play a significant negative role in patients with UI. Thrombophilia seems to be more frequent than expected among cases with UI, and could impair fertility in this subgroup of infertile women. Further studies with larger numbers in this field and effective therapies to improve the reproductive performance of the affected couples should be developed.
Infertile women, who have tested positive for thrombophilia and who achieve their goal of becoming pregnant, merit particular attention during prenatal care. Obstetric care must be rigorous as thrombophilia is known to be associated with an increased risk of complications during pregnancy such as preeclampsia, intrauterine growth restriction, placental abruption, preterm labor, and recurrent pregnancy loss, in addition to ischemic events during pregnancy.
Even in those who did not conceive spontaneously or preferred an IVF/ICSI trial directly, the diagnosis of thrombophilia with its possible role in implantation failure in ICSI and planning for appropriate management will definitely add to the success of the ICSI trial.
Conflicts of interest
There are no conflicts of interest.
1. Boivin J, Bunting L, Collins JA, Nygren KG. International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care. Hum Reprod 2007; 22:1506–1512.
2. Practice Committee of the American Society for Reproductive Medicine. Effectiveness and treatment for unexplained infertility
. Fertil Steril 2006; 865 SupplS111–S114.
3. Vaquero E, Lazzarin N, Caserta D, Valensise H, Baldi M, Moscarini M, Arduini D. Diagnostic evaluation of women experiencing repeated in vitro fertilization failure. Eur J Obstet Gynecol Reprod Biol 2006; 125:79–84.
4. Reitsma PH, Rosendaal FR. Past and future of genetic research in thrombosis. J Thromb Haemost 2007; 5Suppl 1264–269.
5. Buchholz T, Thaler CJ. Inherited thrombophilia: impact on human reproduction. Am J Reprod Immunol 2003; 50:20–32.
6. Grandone E, Colaizzo D, Lo Bue A, Checola MG, Cittadini E, Margaglione M. Inherited thrombophilia and in vitro fertilization implantation failure. Fertil Steril 2001; 76:201–202.
7. Martinelli I, Taioli E, Ragni G, Levi-Setti P, Passamonti SM, Battaglioli T, et al.. Embryo implantation after assisted reproductive procedures and maternal thrombophilia. Haematologica 2003; 88:789–793.
8. Azem F, Many A, Yovel I, Amit A, Lessing JB, Kupferminc MJ. Increased rates of thrombophilia in women with repeated IVF failures. Hum Reprod 2004; 19:368–370.
9. Coulam CB, Jeyendran RS, Fishel LA, Roussev R. Multiple thrombophilic gene mutations are risk factors for implantation failure. Reprod Biomed Online 2006; 12:322–327.
10. Kupferminc MJ, Eldor A, Steinman N, Many A, Bar-Am A, Jaffa A, et al.. Increased frequency of genetic thrombophilia in women with complications of pregnancy. N Engl J Med 1999; 340:9–13.
11. Bianca S, Barrano B, Cutuli N, Indaco L, Cataliotti A, Milana G, et al.. Unexplained infertility
and inherited thrombophilia. Fertil Steril 2009; 92:e4.
12. Coulam CB, Jeyendran RS. Thrombophilic gene polymorphisms are risk factors for unexplained infertility
. Fertil Steril 2009; 914 Suppl1516–1517.
13. Coulam CB, Jeyendran RS, Fishel LA, Roussev R. Multiple thrombophilic gene mutations rather than specific gene mutations are risk factors for recurrent miscarriage. Am J Reprod Immunol 2006; 55:360–368.
14. Goodman CS, Coulam CB, Jeyendran RS, Acosta VA, Roussev R. Which thrombophilic gene mutations are risk factors for recurrent pregnancy loss? Am J Reprod Immunol 2006; 56:230–236.
15. Coulam CB, Wallis D, Weinstein J, Dasgupta DS, Jeyendran RS. Comparison of thrombophilic gene mutations among patients experiencing recurrent miscarriage and deep vein thrombosis. Am J Reprod Immunol 2008; 60:426–431.
16. Many A, Schreiber L, Rosner S, Lessing JB, Eldor A, Kupferminc MJ. Pathologic features of the placenta in women with severe pregnancy complications and thrombophilia. Obstet Gynecol 2001; 98:1041–1044.
17. Nelen WLDM, Bulten J, Steegers EAP, Blom HJ, Hanselaar AGJM, Eskes TKAB. Maternal homocysteine and chorionic vascularization in recurrent early pregnancy loss. Hum Reprod 2000; 15:954–960.
18. Kujovich JL. Thrombophilia and pregnancy complications. Am J Obstet Gynecol 2004; 191:412–424.
19. Rey E, Kahn SR, David M, Shrier I. Thrombophilic disorders and fetal loss: a meta-analysis. Lancet 2003; 361:901–908.
20. Silva Soligo ADG, Barini R, De Carvalho ECC, Annichino-Bizzacchi J. Prevalence of thrombophilic factors in infertile women. Revista Brasileira de Ginecologia e Obstetricia 2007; 29:235–240.
21. Qublan H, Amarin Z, Dabbas M, Farraj A-E, Beni-Merei Z, Al-Akash H, et al.. Low-molecular-weight heparin in the treatment of recurrent IVF-ET failure and thrombophilia: a prospective randomized placebo-controlled trial. Hum Fertil 2008; 11:246–253.
22. Bellver J, Soares SR, Álvarez C, Muñoz E, Ramírez A, Rubio C, et al.. The role of thrombophilia and thyroid autoimmunity in unexplained infertility
, implantation failure and recurrent spontaneous abortion. Hum Reprod 2008; 23:278–284.
23. Simur A, Özdemir S, Acar H, Çolakoǧlu MC, Görkemli H, Balci O, Nergis S. Repeated in vitro fertilization failure and its relation with thrombophilia. Gynecol Obstet Invest 2009; 67:109–112.
24. Casadei L, Puca F, Privitera L, Zamaro V, Emidi E. Inherited thrombophilia in infertile women: implication in unexplained infertility
. Fertil Steril 2010; 94:755–757.
25. Sharif KW, Ghunaim S. Management of 273 cases of recurrent implantation failure: results of a combined evidence-based protocol. Reprod Biomed Online 2010; 21:373–380.
26. Sanson B-J, Friederich PW, Simioni P, Zanardi S, Huisman MV, Girolami A, et al.. The risk of abortion and stillbirth in antithrombin-, protein C-, and protein S-deficient women. Thromb Haemost 1996; 75:387–388.
27. Raziel A, Kornberg Y, Friedler S, Schachter M, Sela BA, Ron-El R. Hypercoagulable thrombophilic defects and hyperhomocysteinemia in patients with recurrent pregnancy loss. Am J Reprod Immunol 2001; 45:65–71.
28. Galli M, Comfurius P, Maassen C, Hemker HC, De Baets MH, Van Breda-Vriesman PJC, et al.. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990; 335:1544–1547.
29. Birkenfeld A, Mukaida T, Minichiello L, Jackson M, Kase NG, Yemini M. Incidence of autoimmune antibodies in failed embryo transfer cycles. Am J Reprod Immunol 1994; 312–365–68.
30. Kaider BD, Price DE, Roussev RG, Coulam CB. Antiphospholipid antibody prevalence in patients with IVF failure. Am J Reprod Immunol 1996; 35:388–393.
31. Coulam CB, Kaider BD, Kaider AS, Janowicz P, Roussev RG. Antiphospholipid antibodies associated with implantation failure after IVF/ET. J Assist Reprod Genet 1997; 14:603–608.
32. Lockwood CJ, Rand JH. The immunobiology and obstetrical consequences of antiphospholipid antibodies. Obstet Gynecol Surv 1994; 49:432–441.
33. Matsubayashi H, Arai T, Izumi S-I, Sugi T, McIntyre JA, Makino T. Anti-annexin V antibodies in patients with early pregnancy loss or implantation failures. Fertil Steril 2001; 76:694–699.