Hyperhomocysteinemia (HHCY) has been underlined as an emerging risk factor for several diseases such as arterial and/or venous thrombosis 1, adverse pregnancy outcome 23, congenital malformations 4 and vascular dementia 56. Inherited and acquired conditions have been involved to explain pathophysiology of HHCY such as gene polymorphisms [i.e. cystationin beta synthase (CBS) or methylenetetrahydrofolate reductase (MTHFR)] 78 and folate and/or vitamin B6/B12 deficiencies due to disregulation of their normal metabolism and/or low dietary intake 91011. Because HHCY has frequently been associated to clinical vascular thrombosis, homocysteine metabolism is investigated, together with other thrombophilic conditions such as inherited and/or acquired thrombophilia, in patients with early onset of vascular thrombosis 1. However, in this field inherited and/or acquired thrombophilia are identified as well known risk factors for adverse pregnancy outcome, in particular in case of early recurrent pregnancy loss (RPL). Literature already underlined a strong relationship between inherited deficiency of protein C, protein S or antithrombin and RPL 12. Moreover, gene variants related to thrombophilia as factor V Leiden and\or prothrombin A20210G have been reported to be associated to RPL 131415. Moreover, acquired conditions such as antiphopspholipid syndrome or increased plasma levels of clotting factor VIII have been associated to RPL 16171819. Furthermore, increasing evidence is now available also for the association of HHCY and RPL 23.

Previous studies failed to include or exclude other causes of miscarriages, when alteration of haemostasis with a trend toward hypercoagulable state were considered. On the other hand, few emerging data are now available for the role of inherited and/or acquired thrombophilia in women with in vitro fertilisation failure 2021, while data about the association of female sterility and alteration of haemostasis are lacking, in particular if the homocysteine metabolism is considered.

The aim of our study was therefore to investigate the role of homocysteine metabolism in patients with unexplained female sterility or secondary sterility due to RPL.

Whole blood samples were collected from all selected subjects in the study by venipuncture from antecubital vein in order to screen possible involvement of alteration of homocysteine metabolism. All subjects were assayed for plasma homocysteine (t-Hcy), vitamin B12 and folic acid levels and MTHFR C677T gene polymorphism.

Fasting levels of homocysteinemia were higher both in patients with UFS (i.e. 21.05 ± 8.78 μM) and with RPL (19.20 ± 6.14 μM) compared to control subjects (7.85 ± 3.31 μM); differences were both statistically significant (p: <0.01) (Table 1 and 2). Fasting homocysteine was slightly increased in patients with unexplained sterility compared to patients with RPL, but this difference did not reach statistical significance (Table 1 and 2).

Moreover, serum folic acid levels were lower both in women with UFS (6.70 ± 4.50 ng/ml) and by RPL (6.10 ± 2.81 ng/ml) compared to control subjects (20.10 ± 9.44 ng/ml) and also these differences reached statistical significance (p: <0.01) (Table 1 and 2). Interestingly, in this case we found also a significant statistical difference in folate concentrations in women with unexplained sterility compared to women with RPL (p: 0.007) (Table 1 and 2).

Serum vitamin B12 levels were comparable in women with UFS (648 ± 162 pg/dl), in women with RPL (608 ± 154 pg/dl) and in control subjects (664 ± 175 pg/dl) (Table 1 and 2) not reaching significant statistical differences (Table 2).

MTHFR C677T gene polymorphism was searched for in all subjects with UFS, RPL and in controls. Five/20 patients with UFS and 3/20 patients with RPL showed heterozigosity for MTHFR C677T compared to 9/20 of control group (Table 2).

MTHFR C677T homozigosity was present in 15/20 patients with UFS and 17/20 patients with RPL compared to 3/20 subjects of control group (Table 1); differences were significant both for sterility and RPL when compared to control group (p: <0.01 UFS vs control group and RPL vs control group) (Table 2). No differences were found between UFS and RPL (p: 0.69, ns) (Table 2). None of the subjects with UFS or RPL were wild type for MTFHR C677T gene polymorphism vs 8\20 subjects of control group.

Results showing variables in patients with unexplained female sterility, RPL and control group are summarised in Table 1 and 2.

Since 1990 several studies underlined a pathogenic role for inherited thrombophilia in women with RPL 2425. A pathogenic role was identified for inherited deficiency of protein C, protein S and antithrombin and for inherited gene polymorphism of factor V Leiden and A20210G of prothrombin, and also for acquired thrombophilia, in particular antiphospholipid syndrome 1213141516171819.

Also a potential pathogenic role of HHCY has been recently suggested, because the association of HHCY and thrombosis, but not univocal data are available. Increasing evidences are available for the relationship between HHCY and MTHFR C677T gene polymorphism and unexplained recurrent pregnancy loss. Several reports, in fact, described an association between early RPL and HHCY and/or MTHFR C677T gene polymorphism 232627. A different point of view on the association between HHCY and RPL has been reported only by Makino et al. 28.

Only a few studies are available on the association between sterility and HHCY and they are focused only on women with in vitro fertilization failures. Moreover, available data seem to be in contrast: Martinelli et al. did not find an association between inherited thrombophilia and patients with in vitro fertilization failure 29, while Azem et al. and Qublan et al. underlined a possible role of inherited thrombophilia and IVF failure 2021. However, both studies were not based on a possible association of homocysteine metabolism and IVF failure but only on the C677T gene polymorphism of MTHFR.

In the present study we evaluated not only homocysteinemia and MTHFR C677T gene polymorphism but also other common variables associated with homocysteine metabolism such as folate and vitamin B12 serum levels both in women showing RPL and women with UFS.

We found that women with RPL and UFS showed HHCY compared to control group (Table 1 and 2). We did not find differences in homocysteine levels between women with RPL and women with UFS. Although data concerning HHCY and RPL seem to be in agreement with those already reported 232627, data focusing the association between of HHCY and UFS are innovative because rarely described so far.

Data concerning MTHFR C677T gene polymorphism seem to support this hypothesis because we found a high frequency of homozigosity of TT genotype not only in women with RPL but also in women with UFS (Table 1). TT genotype of MTHFR C677T gene polymorphism has been already underlined in pathophysiology of RPL by several Authors but its role in pathophysiology of UFS is still a matter of discussion. Therefore, our data support the hypothesis concerning the involvement of homocysteine metabolism in cases of UFS because frequency of TT genotype was similar to that observed in women with RPL (Table 2). Moreover, our data may also support previous observations of an increased frequency of thrombophilia in women with infertility showing repeated IVF failures.

Serum folate and vitamin B12 levels were also tested to support this hypothesis because strongly associated to homocysteine metabolism, also in terms of a possible therapeutic support. Although, vitamin B12 levels were lower both in women with RPL and with UFS compared to control subjects, and these differences did not reach statistical significance (Table 1 and 2). On the other hand we found significantly lower folate levels both in women with RPL and UFS when compared with control subjects (Table 1 and 2). These data confirm the strict association between folate metabolism and homocysteine levels also in pathophysiology of women with infertility, in particular if referred to adverse pregnancy outcome such as early RPL. Moreover, for the first time we underlined a possible association between low serum folate and HHCY and UFS. Furthermore, from another point of view our data offer a new scenario on the possible therapeutic support with folic acid fortification both in women with RPL and UFS carrying HHCY.

In conclusion our study provides several data concerning the involvement of homocysteine metabolism in women with infertility: we confirmed a strict association between HHCY and TT genotype of MTHFR C677T in women with RPL without other causes of recurrent abortion. Yet, for the first time, we suggested also that homocysteine metabolism may be involved in pathophysiology of these cases of UFS because of the association between HHCY, low serum folate and TT genotype of MTHFR C677T. However, because of the small number of selected patients, our data should be confirmed by further studies based on larger population.