The procoagulant status. Hypercoagulability as a risk factor of primary and secondary infertility.

Thrombophilia is a disorder that makes patients susceptible to intravascular thrombosis that may increase the risk of developing a pregnancy on a known pathology. The female patient diagnosed with hypoplastic uterus and hereditary thrombophilia had a favorable evolution under properly administered anticoagulant treatment. The homozygous status for the C677T mutation may lead to an increase in plasma homocysteine levels, especially in pregnant women, being an associated risk factor for thrombosis. The risk of developing intravascular thrombosis requires primary prevention measures by adding D-dimers in the early diagnostic algorithm, being the most accurate marker of hypercoagulability and endogenous fibrinolysis. The corroboration of the hypercoagulability status with the results of genotyping, the frequencies of the minor/major alleles studied, single mononucleotide polymorphisms (SNPs) and the establishment of preventive therapy, aims to prevent intravascular thrombosis and thromboembolic phenomena.

⧉ Introduction

Today, infertility and recurrent miscarriage are serious problems with growing clinical concern. The efforts of modern medicine are concerted to reduce the level of infertility worldwide. It is estimated that approximately a third of the causes of infertility are due to female factors [1].

The presence of a hereditary abnormality in the coagulation–fibrinolysis system associated with an additional risk factor such as pregnancy, surgery, trauma, smoking, venous stasis, atherosclerosis, or consumption of contraceptive pills predisposes to the onset of the thrombotic process [2,3,4].

In recent decades, we have found many data on the association between a hypercoagulable condition and its causes and one of the adverse outcomes of pregnancy – recurrent pregnancy loss (RPL).

Early studies focused on the association between thrombophilia and RPL have highlighted the role of reduced clotting inhibitors in RPL. Subsequently, studies have highlighted a pathogenic role of the gene variant associated with hypercoagulable status in the occurrence of RPL.

During pregnancy, there are various changes in the hemostatic balance of women with a tendency to thrombophilia, a necessary change for the hemostatic challenge of childbirth [5].

Thrombophilia is a disease caused by various defects, in which the combination of procoagulant defects or acquired factors play an important role [6].

Inherited thrombophilia is a genetic condition to suffer various clinical thrombotic events, with frequent recurrences without apparent cause. This entity is defined as a primary risk factor for abnormal pregnancy due to different genes: G1619A (Factor V Leiden), R2 H1299R (Factor V Leiden polymorphism), A1298C [Methylenetetrahydrofolate reductase (MTHFR) enzyme mutation], C677T (MTHFR polymorphism), V34L (Factor XIII polymorphism), G20210A (mutation of the prothrombin gene), a/b L33P (ribosomal polymorphism of MTHFR enzyme) and 4G/5G [plasminogen activator inhibitor-1 (PAI-1)] [7].

Although many studies have shown an association between thrombophilia and embryonic/fetal loss, there are several other studies that have not shown such an association [6].

On the other hand, acquired thrombophilic abnormalities as acquired C protein, S protein, antiphospholipid syndrome, antithrombin III deficiency, drugs induced thrombophilia are a well-known cause of RPL. All should be considered for a screening.

These data should be considered, as recent studies have suggested the role of extensive thromboprophylaxis in females with RPL, which should only be addressed in case of increased risk of venous thromboembolism and known thrombophilia. However, the increase in D-dimer has often been associated with subclinical thrombophilia, due to its high predictive negative value in case of suspected thrombosis [8].

Aim

This presentation aims to educate patients in preventing the thrombosis process stirred by cause of stasis, venous parietal lesions, and hypercoagulability phenomena in the deep and placental venous system, as well as in the prevention of embolic potential, which can endanger the evolution of the pregnancy due to an existing pathology. In this case, our patient diagnosed with uterine hypoplasia, having three previous miscarriages, manages to bring to maturity the fourth pregnancy after performing the thrombophilia profile and following appropriate anticoagulant treatment throughout the pregnancy.

⧉ Patient, Materials and Methods

We present the case of the 37-year-old female patient, referred to as ‘TG’, from an urban area, 40 kg, in the medical records of the Filantropia Municipal Hospital, Craiova, Romania, diagnosed with uterine hypoplasia, having a history of three miscarriages. The patient underwent clinical evaluations and paraclinical explorations: molecular techniques [real-time polymerase chain reaction (PCR) sequencing], hematological, biochemical, immunological, bacteriological tests, the biological material studied was blood collected in containers with anticoagulant [Ethylenediaminetetraacetic acid (EDTA), respectively 3.8% Sodium Citrate], as well as without anticoagulant and endocervical secretion.

The hybridization reactions used to identify nucleic acids in the biological product were performed in two steps: denaturation of the nucleic acid in the research sample followed by hybridization of the chains obtained with known nucleic acid sequences.

Southern blot, the deoxyribonucleic acid (DNA) visualization method was performed after its separation into fragments with restriction enzymes, followed by polyacrylamide gel electrophoresis, transfer to the nitrocellulose membrane, and hybridization with labeled probe and autoradiography detection.

PCR, the highly sensitive cyclic method of amplifying specific DNA sequences, required a set of primers (short oligonucleotide sequence) and a heat-resistant DNA polymerase. Each cycle comprised three stages: denaturation of the DNA in the target sequence, hybridization of complementary primers, and extension of the primers using polymerase.

The obtained DNA sequences (amplicons) were then identified by hybridization with labeled probes or by restriction fragment length polymorphism analysis (RFLP). RFLP was based on electrophoretic analysis of nucleic acid fragments obtained by cleavage using restriction enzymes.

Hematological, biochemical, immunological, and bacteriological investigations were performed according to the working techniques corresponding to the reagent of the manufacturing company.

The pregnant woman was constantly monitored through medical imaging techniques, and at birth the placenta was sent to the Department of Pathological Anatomy for the histopathological (HP) examination.

For HP examination, classical Hematoxylin–Eosin (HE) staining was used to identify placental elements, Periodic Acid–Schiff (PAS)–Hematoxylin (PAS-H) staining to highlight basement membranes and massive fibrinogen deposits and special immunohistochemical staining with the anti-cluster of differentiation 34 (CD34) antibody was used to see the infarcted areas without vascularization (Dako, monoclonal mouse anti-human CD34 Class II, clone QBEnd 10, antigenic exposure 1:50 Citrate buffer, 1:50 dilution, labeling neoformed blood vessels).

⧉ Case presentation

The results of the paraclinical investigations indicate the homozygous genotype for the C677T mutation and the PAI-1 gene (675 4G/4G polymorphism), and the coagulation tests confirm hypercoagulability (Table 1).

Table 1

Thrombophilic, bacteriological and genetic status of patient

Investigation type	Investigation	Mean ± SD
		Negative results	Positive results
Antiphospholipid syndrome profile	Anticardiolipin antibodies IgG [GPL/mL]	3.6±0.08	–
	Anti-beta2 glycoprotein 1 antibodies IgG [U/mL]	2.4±0.07	–
	Anti-beta2 glycoprotein 1 antibodies IgM [U/mL]	1.2±0.02	–
Hereditary thrombophilia profile	Antithrombin III [%]	108±1.77	–
	C protein [%]	110±1.91	–
	S protein [%]	103±1.32	–
	Factor V Leiden mutation	Negative	–
	MTHFR gene
	C677T mutation	–	Homozygous genotype
	A1298C mutation	Negative	–
	PAI-1 gene
	675 4G/5G polymorphism	–	Risk of thrombophilia
	675 4G/4G polymorphism	–	Homozygous genotype
Bacteriology	Chlamydia, Mycoplasma hominis, Ureaplasma urealyticum	Negative	–
Genetic analysis	Karyotype	–	46,XX,9qh+

GPL: IgG phospholipid units (1 GPL unit = 1 μg of IgG antibody); IgG: Immunoglobulin G; IgM: Immunoglobulin M; MTHFR: Methylenetetrahydrofolate reductase; PAI-1: Plasminogen activator inhibitor-1; SD: Standard deviation.

The patient is a homozygous carrier of deletion in the PAI-1 gene promoter. The genetic analysis report identifies in the mother an elongated secondary constriction on the long arm of chromosome 9, normal karyotype with 46, XX,9qh+ polymorphisms, while the paternal chromosomal map does not indicate anomalies in number or structure, 46,XY karyotype.

The evolution during the last pregnancy was spectacular by supplementing the medication with anticoagulant (Clexane 0.4 UI/mL), along with antiplatelet therapy (Aspenter 75 mg), tocolytic therapy (Magnesium Sulfate) and progestogen therapy (Arefam 200 mg). Clexane’s interference with thrombin is insignificant, as evidenced by Quick time (QT), international normalized ratio (INR) values before and after administration. The effective anti-thrombotic action was due to the overpowering inhibition of activated Factor X and the activation of the circulatory and parietal fibrinolytic system, complemented by the antiplatelet mechanism of Aspenter.

Following anticoagulant therapy, laboratory tests showed that the value of D-dimers decreased from 4.3±0.05 μg/mL to 0.48±0.01 μg/mL, QT increased from 11.0±0.15 s to 12.3±0.31 s, INR increased from 0.77±0.09 to 0.92±0.12, the sideremia increased from 116.0±3.38 μg/dL to 121.1±3.42 μg/dL, and the serum ferritin value increased from 36.34±0.55 ng/mL to 40.3±0.65 ng/mL, thus observing the importance of anticoagulant, vitamin, and antiplatelet treatment (Table 2).

Table 2

Mean ± SD values of some coagulation and biochemical parameters

Variable (mean ± SD)	Before medication	After medication: Clexane, vitamin B1, B6, Aspenter, vitamin C
D-dimers [μg/mL]	4.3±0.05	0.48±0.01
QT [s]	11.0±0.15	12.3±0.31
INR	0.77±0.09	0.92±0.12
Sideremia [μg/dL]	116.0±3.38	121.1±3.42
Serum ferritin [ng/dL]	36.34±0.55	40.3±0.65

INR: International normalized ratio; QT: Quick time; SD: Standard deviation

All biological parameters before medication signal a state of hypercoagulability.

After birth, the HP examination showed at the level of the placenta frequent stem villosities of variable sizes with fibrous stroma, sometimes zonal or circumferential fibrinoid necrosis and massive calcification zone (Figure 1, A and B), with dilated vessels, some thrombosed (Figure 2A), frequent mature intermediate villosities and of terminal type with angiomatous appearance with stasis, rare syncytial buds, dilated umbilical vein, thrombosis, lesions of endothelial continuity, middle tunic dissociated from interstitial edema (Figure 2B), areas of hemorrhagic necrosis that alternate with areas with intervillositary blood infiltrates (Figure 3A) and areas of placental infarction, fibrosed, with lack of immunolabeling of vascular endothelial cells are observed (Figure 3B).

Figure 1

(A and B) Normal and pathological placental villi with the presence of intravillositis and perivillous fibrin deposition (intense pink color) and massive calcification zone (intense blue color). HE staining: (A) ×100; PAS-H staining: (B) ×100. HE: Hematoxylin–Eosin; PAS-H: Periodic Acid Schiff–Hematoxylin

Figure 2

Normal and pathological placental villi and partially thrombosed umbilical cord blood vessel: (A) Thrombotic vessels that block the vascular lumen and intravillositis and perivillous fibrin deposition are observed; (B) The presence of microthrombosis and hemorrhagic microfocus in the middle tunic are observed. HE staining: (A) ×100; (B) ×200. HE: Hematoxylin–Eosin

Figure 3

Normal and pathological placental villi: (A) An intense extravillous hemorrhagic process is observed; (B) Normal placental villi show blood vessels with brown-immunolabeled endothelium, and infarcted villi no longer have immunolabeled blood vessels. HE staining: (A) ×100. Immunohistochemical staining with anti-CD34 antibody: (B) ×100. CD34: Cluster of differentiation 34; HE: Hematoxylin–Eosin

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⧉ Discussions

Thrombophilia includes several states of hypercoagulability, which can lead to intravascular thrombosis. The causes of thrombophilia can be inherited (hyperactivity of coagulation, deficiency of the anticoagulant system) or acquired, which carry out a procoagulant status [9,10,11].

Antiphospholipid syndrome is one of the causes of thrombophilia. Among the manifestations of this syndrome, increased titers of antibodies against associated plasma proteins and anionic membrane phospholipids, cause venous or arterial thrombosis or other complications that can lead to pregnancy termination [12,13].

In the case of our patient, the paraclinical examinations refuted the antiphospholipid syndrome profile, so it is excluded as the cause of thrombophilia, antibodies specific to the investigation of the antiphospholipid syndrome profile [anticardiolipin antibodies immunoglobulin G (IgG), anti-beta2 glycoprotein 1 antibodies IgG, anti-beta2 glycoprotein 1 antibodies immunoglobulin M (IgM)], being within normal limits.

In an individual suffering from hereditary trauma, even surgery constitutes an associated risk factor for thrombosis. D-dimer dosing has a negative predictive value for deep vein thrombosis of the lower limbs and pulmonary thromboembolism in the sense that obtaining a negative result in patients suspected of these diseases excludes their presence in a percentage of over 90%.

There are several conditions in which it has been observed increase in D-dimer without ongoing thrombosis as well infections, chronic inflammation, malignancy, necrosis, acute coronary syndromes. We performed this analysis starting from the role of dimer d in the diagnostic screening of women who associated thrombophilia and sterility [8, 14].

Ever since Brenner et al. (1999) mentioned that over 40% of women with RPL have the main cause of thrombophilia, the medical world was concerned about the pathogenetic role of thrombophilia inherited in this category of women [15].

Five years ago, it was mentioned that inherited thrombophilia presents different patterns in different ethnic groups, suggesting that there is no difference between patients with two abortions and patients with three or more abortions [16]. Our patient diagnosed with inherited thrombophilia had three abortions.

One of the risk factors for RPL is thrombophilia gene polymorphism, and this theory is proved in our patient. Probably, Caucasian race females with RPL risk have this thrombophilic genes polymorphism, as it is mentioned in a few studies.

The Barut et al. study was conducted between 2012 and 2016 to decipher, in Turkish women, the importance of mutations in genetic polymorphisms for the impact of thrombophilia (homozygous/heterozygous) on RPL. In the peripheral blood samples of these patients, several genetic mutations were found – of prothrombin G20210A, Factor V Leiden H1299R, MTHFR A1298C, MTHFR C677T, PAI-1 4G/4G and PAI-1 4G/5G [17]. Factor V Leiden was not found in the patient studied, although it is thought to be responsible for more than three-quarters of the inherited activated C protein resistance, more precisely it is the most common inherited thrombotic risk factor associated with RPL [17].

A study published in 2018 concluded that both MTHFR C677T and Factor V Leiden polymorphisms were significantly associated with RPL, in contrast to the lack of significant association between PAI-1 4G/5G polymorphisms and prothrombin G20210A in Bosnian women [18].

Similar results were mentioned by other authors who stated that prothrombin A20210G and/or Factor V Leiden are two genetic variants commonly associated with RPL [4, 19]. These gene cues were not highlighted in our patient.

The C677T homozygous mutation of the MTHFR gene may promote thrombosis [20]. Thromboembolism can generate cardiorespiratory failure in an organism, with a genetic pathology and an increased risk of thrombosis, which is why anticoagulant treatment and monitoring of the patient using coagulation tests have been recommended [21].

The homozygous status for the C677T mutation may lead to increases in plasma homocysteine levels, especially in patients with low folate and B vitamins. Hyperhomocysteinemia (HHCY) is a risk factor for arterial, venous thrombosis and miscarriage, hence balancing the homocysteine levels through adequate vitamin replacement and regular control of homocysteinemia is recommended. Associated risk factors, such as immobilization, trauma, surgery, pregnancy, smoking, obesity, use of oral contraceptives may lead to a marked predisposition to thrombosis. The role of this mutation as a risk factor for coronary heart disease, myocardial infarction and preeclampsia is obvious [22].

The pathogenetic role of HHCY in RPL has been studied worldwide. The results published in the literature on the relationship between HHCY, MTHFR C677T gene polymorphism and RPL are not in fact unambiguous, the possible explanation being due to the ethnicity of the patients [15, 23].

Thus, recently developed techniques in genetic screening, such as next-generation sequencing, are a better choice to detect variants of genetic risk for thrombosis in ethnic groups [24].

Not to be overlooked is the fact that folic acid supplementation during pregnancy may lead to a transient reduction in homocysteine concentration [15].

Sterility conditioning by MTHFR gene polymorphism and homocysteine-associated metabolism it is also possible through a vicious lifestyle, with a diet low in vitamin B12 and folic acid [8]. The role of MTHFR C677T polymorphism in pregnancy complications as preeclampsia, placental infarcts, fetal growth restriction was confirmed in several other studies [25].

The homozygous status for PAI-1 gene promoter deletion (4G/4G mutant status) promotes preeclampsia in pregnant women. Deletion of guanosine in position 675 of the PAI-1 gene leads to an increase in the level of PAI-1 in the blood, being a risk factor for thromboembolic disorder.

The carrier status of the 4G allele is an element of risk for thromboembolic disorder and is also correlated with a high blood pressure and increased risk of myocardial infarction, and the transmission of the genetic defect is autosomal recessive, so a family check-up is recommended. Congenital diseases, in general, aggravate the clinical-biological evolution of the pregnant woman and the embryo [26].

Provisional to the studies cited in medical literature, there is no direct correlation between gene polymorphisms involved in folate metabolism, the process of fibrinolysis and the risk of miscarriage [27,28].

Although it is certainly established that the women’s predisposition to RPL correlates with the presence of thrombophilic polymorphisms, future research is needed to determine the timing of anticoagulant treatment in people with these mutations [18], as we demonstrated in our patient management – the clinical result can be improved by applying an antithrombotic treatment during pregnancy.

Anticoagulant treatment is indicated when there is a history of at least two miscarriages/two pregnancies stopped evolving without another determining cause or when associative thrombotic phenomena occur [29]. The patient diagnosed with uterine hypoplasia and thrombophilia, under correctly administered anticoagulant treatment and monitoring had a favorable evolution and gave birth to a male fetus at 38–39 weeks of gestation. Learning the habits of maintaining good health, by avoiding additional risk factors, is an important element in maintaining general health.

⧉ Conclusions

The severity of the consequences induced by the presence of intravascular thrombosis, justifies primary prevention measures, corroborated with the need to add D-dimers in the early diagnostic algorithm, being considered the most accurate marker of hypercoagulability and endogenous fibrinolysis. The correlation of the hypercoagulability status highlighted by coagulation tests with the results of genotyping, the frequencies of minor/major alleles studied, single mononucleotide polymorphisms (SNPs) and the application of preventive therapy, aim to prevent intravascular thrombosis with very serious consequences. D-dimer dosing should be included in screening tests for women of childbearing potential to diagnose subclinical thrombophilia and reduce the risk of unexplained primary or secondary infertility. The recommendation of the extended thrombophilic profile in the preconceptional period aims at decreasing fetal and perinatal mortality and morbidity as well as increasing the probability of obtaining, at the end of pregnancy, a full-term, eutrophic newborn, with possibilities for normal, physical, and mental development.

Conflict of interest

The authors declare that they have no conflict of interests.