In this editorial,  Retrovirology's  choice for best basic science "retrovirus paper of the year" and a perspective on challenges and responsibilities facing HIV-1 and HTLV-I research are presented.Retrovirology's

Progress The beginning of a year provides an occasion to look back upon progress made over the past 52 weeks. With the end of 2004,  Retrovirology  concluded its first calendar year of publishing. In actual fact,  Retrovirology  launched as an Open Access journal the final week of February 2004 and has been publishing continuously for a little more than 10 months. Over that period, with the wonderful efforts from my 6 very capable Associate Editors (Monsef Benkirane, Ben Berkhout, Masa Fujii, Mike Lairmore, Andrew Lever, and Mark Wainberg), the journal has thrived. The goal that we set for  Retrovirology  is to provide a visible forum for retrovirologists so that their works can be read by all in a free and openly accessible manner. What this means is that if you are a human immunodeficiency virus (HIV)-researcher and you had published a paper in  Retrovirology , a graduate student in Sri Lanka updating his/her research protocol, an AIDS activist in South Africa looking for the latest information, and even your long-lost high school sweetheart wondering what you have been doing all these years, can all find your work (i.e. through a simple  Google  or  PubMed  search) and read your most recent findings. Perhaps more relevant to the enterprise of scientific communication is that numerous academic peers in Eastern Europe, Asia, South America, Africa and elsewhere do not have funds which would permit them to read  Cell ,  Science  or  Nature . Hence, while some can read your research in  Cell ,  Science , or  Nature , all colleagues, rich or poor alike, can read your  Retrovirology  paper. Are they reading  Retrovirology ? You bet! Our monitored statistics tell us that in 2004, the most highly accessed review article [ 1 ] published in  Retrovirology  was read by over 3,500 individuals while a comparably popular original research article [ 2 ] was read more than 2,400 times. Readers also read  Retrovirology  articles with great immediacy. Thus, a recent paper by Rana and colleagues [ 3 ] appeared in  Retrovirology  on December 27 th , 2004; and already by December 31 st , 2004, a short 4 days later, that study had been read 389 times. Just as readers are quick to read our papers, I am equally pleased by our unmatched speed in publishing authors'works. In 2004, based on all papers we published in  Retrovirology , the time from submission to publication averaged 40 days. From my personal experience of publishing in other virological journals, this duration is 3 to 4 times faster than our best competitors. Different journals/magazines recognize "Molecule of the Year", "Breakthrough of the Year", or even "Person of the Year". With this editorial,  Retrovirology  will initiate the annual recognition of the best basic science "retrovirus paper of the year". The Associate Editors and I decided that in 2004, the best basic science retrovirus paper was the work from Joseph Sodroski and colleagues describing the HIV-1 restrictive property of the tripartite motif 5 (TRIM5) protein [ 4 ]. Thus, these researchers characterized in primates a restriction factor, similar to the Friend virus susceptibility factor-1 (Fv1) in mice, which counters the ability of infecting retrovirus to establish a proviral form in target cells. In coming years, I anticipate that  Retrovirology  Editors will find it fitting to recognize a  Retrovirology  paper as the "best basic science retrovirus paper" of the preceding year.
Progress
The beginning of a year provides an occasion to look back upon progress made over the past 52 weeks. With the end of 2004,  Retrovirology  concluded its first calendar year of publishing. In actual fact,  Retrovirology  launched as an Open Access journal the final week of February 2004 and has been publishing continuously for a little more than 10 months. Over that period, with the wonderful efforts from my 6 very capable Associate Editors (Monsef Benkirane, Ben Berkhout, Masa Fujii, Mike Lairmore, Andrew Lever, and Mark Wainberg), the journal has thrived.RetrovirologyRetrovirologyThe goal that we set for  Retrovirology  is to provide a visible forum for retrovirologists so that their works can be read by all in a free and openly accessible manner. What this means is that if you are a human immunodeficiency virus (HIV)-researcher and you had published a paper in  Retrovirology , a graduate student in Sri Lanka updating his/her research protocol, an AIDS activist in South Africa looking for the latest information, and even your long-lost high school sweetheart wondering what you have been doing all these years, can all find your work (i.e. through a simple  Google  or  PubMed  search) and read your most recent findings. Perhaps more relevant to the enterprise of scientific communication is that numerous academic peers in Eastern Europe, Asia, South America, Africa and elsewhere do not have funds which would permit them to read  Cell ,  Science  or  Nature . Hence, while some can read your research in  Cell ,  Science , or  Nature , all colleagues, rich or poor alike, can read your  Retrovirology  paper.RetrovirologyRetrovirologyGooglePubMedCellScienceNatureCellScienceNatureRetrovirologyAre they reading  Retrovirology ? You bet! Our monitored statistics tell us that in 2004, the most highly accessed review article [ 1 ] published in  Retrovirology  was read by over 3,500 individuals while a comparably popular original research article [ 2 ] was read more than 2,400 times. Readers also read  Retrovirology  articles with great immediacy. Thus, a recent paper by Rana and colleagues [ 3 ] appeared in  Retrovirology  on December 27 th , 2004; and already by December 31 st , 2004, a short 4 days later, that study had been read 389 times. Just as readers are quick to read our papers, I am equally pleased by our unmatched speed in publishing authors'works. In 2004, based on all papers we published in  Retrovirology , the time from submission to publication averaged 40 days. From my personal experience of publishing in other virological journals, this duration is 3 to 4 times faster than our best competitors.RetrovirologyRetrovirologyRetrovirologyRetrovirologyRetrovirologyDifferent journals/magazines recognize "Molecule of the Year", "Breakthrough of the Year", or even "Person of the Year". With this editorial,  Retrovirology  will initiate the annual recognition of the best basic science "retrovirus paper of the year". The Associate Editors and I decided that in 2004, the best basic science retrovirus paper was the work from Joseph Sodroski and colleagues describing the HIV-1 restrictive property of the tripartite motif 5 (TRIM5) protein [ 4 ]. Thus, these researchers characterized in primates a restriction factor, similar to the Friend virus susceptibility factor-1 (Fv1) in mice, which counters the ability of infecting retrovirus to establish a proviral form in target cells. In coming years, I anticipate that  Retrovirology  Editors will find it fitting to recognize a  Retrovirology  paper as the "best basic science retrovirus paper" of the preceding year.RetrovirologyRetrovirologyRetrovirologyChallenges and responsibilities I explain to my postdoctoral fellows that challenges are those issues which you think others should solve, while responsibilities are items that you think you should tackle. As a retrovirologist depending on how you regard yourself, pressing problems are either others' challenges or your responsibilities. I study two retroviruses, HIV-1 and human T-cell leukaemia virus type 1 (HTLV-I). The start of a new year offers me a chance to review briefly my personal bias on the important research question that confronts HIV-1 and HTLV-I, respectively. For HIV-1, the "holy grail" remains the development of an effective vaccine against the virus. As we enter 2005, mortality from AIDS is staggering. It is estimated that in 2004, 3.5 million individuals perished worldwide from AIDS; or nearly 10,000 AIDS deaths each day. We can view this number in another way. The recent tsunami in South Asia is estimated to have caused 150,000 fatalities. AIDS in 2004 is then the equivalent of 23 tsunamis. Imagine, unrelentingly tsunami-like casualties every fortnight from people dying from HIV-1! While it is laudable that the World Health Organization has a goal to treat three million HIV-1 positive individuals globally using anti-retroviral (ARV) medicine over the next five years, that approach will unlikely address the full magnitude of the AIDS problem, especially in developing nations. On the other hand, 100 million infants (even those in remote regions of the world) receive basic vaccinations each year. This fact suggests that when an AIDS vaccine does become available, that vaccine could be logistically and practically effective. Separately, statistics from the World Cancer Report for the year 2000 show that 5.3 million men and 4.7 million women developed malignancy, and altogether 6.2 million persons died from cancer worldwide. The American Cancer Society estimates that approximately 553,000 individuals succumb to cancer in the United States each year. I see HTLV-I, the etiological agent for adult T-cell leukaemia (ATL), as perhaps the best retroviral system for retrovirologists to study human cancer. Substantive progress has indeed been made in our understanding as to how HTLV-I transforms cells in tissue culture [ 5 ]. What remains needed is the development of a good non-human primate model to investigate the genesis of adult T-cell leukaemia by the virus  in vivo .
Challenges and responsibilities
I explain to my postdoctoral fellows that challenges are those issues which you think others should solve, while responsibilities are items that you think you should tackle. As a retrovirologist depending on how you regard yourself, pressing problems are either others' challenges or your responsibilities. I study two retroviruses, HIV-1 and human T-cell leukaemia virus type 1 (HTLV-I). The start of a new year offers me a chance to review briefly my personal bias on the important research question that confronts HIV-1 and HTLV-I, respectively.For HIV-1, the "holy grail" remains the development of an effective vaccine against the virus. As we enter 2005, mortality from AIDS is staggering. It is estimated that in 2004, 3.5 million individuals perished worldwide from AIDS; or nearly 10,000 AIDS deaths each day. We can view this number in another way. The recent tsunami in South Asia is estimated to have caused 150,000 fatalities. AIDS in 2004 is then the equivalent of 23 tsunamis. Imagine, unrelentingly tsunami-like casualties every fortnight from people dying from HIV-1! While it is laudable that the World Health Organization has a goal to treat three million HIV-1 positive individuals globally using anti-retroviral (ARV) medicine over the next five years, that approach will unlikely address the full magnitude of the AIDS problem, especially in developing nations. On the other hand, 100 million infants (even those in remote regions of the world) receive basic vaccinations each year. This fact suggests that when an AIDS vaccine does become available, that vaccine could be logistically and practically effective.Separately, statistics from the World Cancer Report for the year 2000 show that 5.3 million men and 4.7 million women developed malignancy, and altogether 6.2 million persons died from cancer worldwide. The American Cancer Society estimates that approximately 553,000 individuals succumb to cancer in the United States each year. I see HTLV-I, the etiological agent for adult T-cell leukaemia (ATL), as perhaps the best retroviral system for retrovirologists to study human cancer. Substantive progress has indeed been made in our understanding as to how HTLV-I transforms cells in tissue culture [ 5 ]. What remains needed is the development of a good non-human primate model to investigate the genesis of adult T-cell leukaemia by the virus  in vivo .in vivoOpportunities and limitations at mid-career I first started studying viruses in the fall of 1977 at age 19 as a MD-PhD student at the Johns Hopkins school of medicine. For me personally, 2004 marked over a quarter-century of virus-research. At age 46, the unbridled youthful optimism of 19 is now tempered by realizations of physical and career limitations (i.e. some very interesting research problems are going to take longer to resolve than the remaining span of my scientific and physical endeavor). Nonetheless, I am optimistic and hopeful that, despite enormous odds, the opportunity to see a successful AIDS vaccine will come before I leave retrovirology research in 20 or some years. Regarding  Retrovirology , I am also optimistic that I started this project at an age that provides ample time to develop this journal into a premier research forum. Let me conclude this writing by thanking all authors, reviewers, Editorial board members, and our wonderful staff at Biomed Central who have contributed to  Retrovirology's  progress in our first year.
Opportunities and limitations at mid-career
I first started studying viruses in the fall of 1977 at age 19 as a MD-PhD student at the Johns Hopkins school of medicine. For me personally, 2004 marked over a quarter-century of virus-research. At age 46, the unbridled youthful optimism of 19 is now tempered by realizations of physical and career limitations (i.e. some very interesting research problems are going to take longer to resolve than the remaining span of my scientific and physical endeavor). Nonetheless, I am optimistic and hopeful that, despite enormous odds, the opportunity to see a successful AIDS vaccine will come before I leave retrovirology research in 20 or some years. Regarding  Retrovirology , I am also optimistic that I started this project at an age that provides ample time to develop this journal into a premier research forum.RetrovirologyLet me conclude this writing by thanking all authors, reviewers, Editorial board members, and our wonderful staff at Biomed Central who have contributed to  Retrovirology's  progress in our first year.Retrovirology's


Haematuria is a classical symptom of urological disease often signifying a primary bladder cancer. Rarely, however, the presence of blood in the urine can be due to secondary spread of tumours into the bladder from distant sites. Notably this has been reported to occur in breast cancer, malignant melanoma and gastric cancers. Haematuria due to spread from a primary oesophageal cancer to the bladder has never been reported. We present a case of haematuria confirmed histologically to be due to metastases from a primary oesophageal tumour. Oesophageal cancer is capable of spread to all three neighbouring compartments (abdomen, chest and neck) and therefore has the potential to spread to unusual sites. Clinicians should always carefully regard haematuria in a patient previously treated for cancer and retain a high index of suspicion for distant metastases as being the cause.

Background Haematuria is a commonly encountered symptom. It often represents the presence of serious disease such as a malignancy within the bladder. The majority of bladder tumours tend to be primary, and histologically these are usually transitional cell carcinomas. We present a case of haematuria which occurred due to metastases from a primary oesophageal carcinoma diagnosed 2 years prior and treated curatively.
Background
Haematuria is a commonly encountered symptom. It often represents the presence of serious disease such as a malignancy within the bladder. The majority of bladder tumours tend to be primary, and histologically these are usually transitional cell carcinomas. We present a case of haematuria which occurred due to metastases from a primary oesophageal carcinoma diagnosed 2 years prior and treated curatively.Case presentation A 45-year old male presented to our unit with acute onset macroscopic haematuria. His past medical history was significant in that he had been diagnosed with adenocarcinoma of the distal oesophagus 2 years prior and had undergone curative resection after neo-adjuvant chemotherapy. At that time the tumour was found to be poorly differentiated with evidence of local nodal spread. He had been reviewed regularly by the oncologists and remained asymptomatic until the onset of frank haematuria. He subsequently underwent cystoscopy, which revealed a solid bladder tumour on the right lateral wall, which was treated with trans-urethral resection. Pathological examination confirmed a poorly differentiated mucus-secreting adenocarcinoma, identical histologically to the original oesophageal tumour (Figures  1  and  2 ). Figure 1 Original oesophageal adenocarcinoma (H & E stain). Figure 2 Metastatic tumour to the bladder (H & E stain). Both light micrographs demonstrate extensive infiltration by a poorly differentiated adenocarcinoma with the same histopathological features. A diagnosis of metastatic oesophageal adenocarcinoma to the bladder was made. A CT scan did not demonstrate any pelvic tumour outside the bladder and therefore metastasis by the trans-coelomic route was essentially excluded, indicating haematogenous spread of the primary oesophageal carcinoma. The patient was referred for further oncological therapy but unfortunately died 4 months later from disseminated carcinoma.
Case presentation
A 45-year old male presented to our unit with acute onset macroscopic haematuria. His past medical history was significant in that he had been diagnosed with adenocarcinoma of the distal oesophagus 2 years prior and had undergone curative resection after neo-adjuvant chemotherapy. At that time the tumour was found to be poorly differentiated with evidence of local nodal spread. He had been reviewed regularly by the oncologists and remained asymptomatic until the onset of frank haematuria.He subsequently underwent cystoscopy, which revealed a solid bladder tumour on the right lateral wall, which was treated with trans-urethral resection. Pathological examination confirmed a poorly differentiated mucus-secreting adenocarcinoma, identical histologically to the original oesophageal tumour (Figures  1  and  2 ).A diagnosis of metastatic oesophageal adenocarcinoma to the bladder was made. A CT scan did not demonstrate any pelvic tumour outside the bladder and therefore metastasis by the trans-coelomic route was essentially excluded, indicating haematogenous spread of the primary oesophageal carcinoma. The patient was referred for further oncological therapy but unfortunately died 4 months later from disseminated carcinoma.Discussion Metastatic tumour spread to the bladder constitutes approximately 2% of all bladder neoplasms [ 1 ]. Gross haematuria occurs relatively infrequently in secondary tumours of the bladder as most lesions are small and infiltrate the bladder wall without causing ulceration of the mucosa [ 2 ]. Therefore most metastases to the bladder remain asymptomatic and often undiagnosed. The bladder can be the recipient of metastatic tumour spread from a potentially large variety of primary sites. Most commonly direct invasion can occur from adjacent tumours of the lower gastrointestinal tract (33% of secondary neoplasms), prostate (19%) and female genital tract (11%) [ 1 ]. Less commonly, distant metastases have been described, notably from the stomach, skin, breast and lung in descending order of frequency [ 2 - 5 ]. The management and prognosis of such tumours can differ significantly from that of primary bladder tumours since they are often indicative of late disease. Despite curative intent, surgical resection of oesophageal adenocarcinoma is associated with an overall tumour recurrence rate of 66% at 5 years [ 6 ]. The lymphatic drainage of the oesophagus is longitudinal via the submucosal plexus and not segmental. As a consequence lymph node metastases can occur relatively early in all three compartments (abdomen, chest and neck) regardless of the location of the primary tumour [ 7 ]. In autopsy studies, isolated lymph node metastases were found in approximately one half of patients with end-stage oesophageal carcinoma, with a similar proportion having combined lymph node and visceral metastases. Isolated visceral spread however is rare, accounting for only a handful of cases of primary oesophageal tumour spread [ 8 ]. Notable sites of haematogenous dissemination of primary oesophageal carcinoma to distant organs include bone, liver, skin, lungs, adrenals, brain and peritoneum in descending order of frequency [ 6 ]. A few authors, most notably in Japan, have described cases of oesophageal cancers metastasising to the kidney. These cases may present with haematuria but often are associated with flank pain [ 9 - 14 ]. Interestingly, rare synchronous primary tumours of the bladder and oesophagus have been described [ 15 , 16 ] but haematuria due to secondary spread of oesophageal cancer has never previously been reported.
Discussion
Metastatic tumour spread to the bladder constitutes approximately 2% of all bladder neoplasms [ 1 ]. Gross haematuria occurs relatively infrequently in secondary tumours of the bladder as most lesions are small and infiltrate the bladder wall without causing ulceration of the mucosa [ 2 ]. Therefore most metastases to the bladder remain asymptomatic and often undiagnosed.The bladder can be the recipient of metastatic tumour spread from a potentially large variety of primary sites. Most commonly direct invasion can occur from adjacent tumours of the lower gastrointestinal tract (33% of secondary neoplasms), prostate (19%) and female genital tract (11%) [ 1 ]. Less commonly, distant metastases have been described, notably from the stomach, skin, breast and lung in descending order of frequency [ 2 - 5 ]. The management and prognosis of such tumours can differ significantly from that of primary bladder tumours since they are often indicative of late disease.Despite curative intent, surgical resection of oesophageal adenocarcinoma is associated with an overall tumour recurrence rate of 66% at 5 years [ 6 ]. The lymphatic drainage of the oesophagus is longitudinal via the submucosal plexus and not segmental. As a consequence lymph node metastases can occur relatively early in all three compartments (abdomen, chest and neck) regardless of the location of the primary tumour [ 7 ].In autopsy studies, isolated lymph node metastases were found in approximately one half of patients with end-stage oesophageal carcinoma, with a similar proportion having combined lymph node and visceral metastases. Isolated visceral spread however is rare, accounting for only a handful of cases of primary oesophageal tumour spread [ 8 ].Notable sites of haematogenous dissemination of primary oesophageal carcinoma to distant organs include bone, liver, skin, lungs, adrenals, brain and peritoneum in descending order of frequency [ 6 ]. A few authors, most notably in Japan, have described cases of oesophageal cancers metastasising to the kidney. These cases may present with haematuria but often are associated with flank pain [ 9 - 14 ].Interestingly, rare synchronous primary tumours of the bladder and oesophagus have been described [ 15 , 16 ] but haematuria due to secondary spread of oesophageal cancer has never previously been reported.Conclusion Haematuria may be the only clinically apparent symptom of metastatic tumour spread to the bladder from a potentially large number of primary sites and therefore should be considered by all clinicians irrespective of specialty.
Conclusion
Haematuria may be the only clinically apparent symptom of metastatic tumour spread to the bladder from a potentially large number of primary sites and therefore should be considered by all clinicians irrespective of specialty.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.


Background D-dimer is considered a marker of hypercoagulable state and of endogenous fibrinolysis, so increased d-dimer is detectable in patients affected by thrombosis. Yet, several studies showed that also infertility, in particular secondary infertility due to recurrent fetal losses, has been often related to thrombotic events, in particular in women carrying thrombotic risk factors such as inherited thrombophilia (MTHFR C677T , PTHR A20210G , Factor V Leiden polimorphisms and/or inhAfter this screening we selected 39erited protein C, protein S, AT III deficiency) or acquired thrombophilia (primary antiphospholipid syndrome, acquired protein C, protein S, AT III deficiency, drugs induced thrombophilia). However, because its high predictive negative value in case of suspected thrombosis, increased d-dimer has been often associated to subclinical thrombophilia. The aim of this study is to investigate the role of d-dimer as first marker of thrombophilia in women affected by unexplained infertility and subsequently to search the cause of increased d-dimer, such as inherited and/or acquired thrombophilia.
Background
D-dimer is considered a marker of hypercoagulable state and of endogenous fibrinolysis, so increased d-dimer is detectable in patients affected by thrombosis. Yet, several studies showed that also infertility, in particular secondary infertility due to recurrent fetal losses, has been often related to thrombotic events, in particular in women carrying thrombotic risk factors such as inherited thrombophilia (MTHFR C677T , PTHR A20210G , Factor V Leiden polimorphisms and/or inhAfter this screening we selected 39erited protein C, protein S, AT III deficiency) or acquired thrombophilia (primary antiphospholipid syndrome, acquired protein C, protein S, AT III deficiency, drugs induced thrombophilia). However, because its high predictive negative value in case of suspected thrombosis, increased d-dimer has been often associated to subclinical thrombophilia. The aim of this study is to investigate the role of d-dimer as first marker of thrombophilia in women affected by unexplained infertility and subsequently to search the cause of increased d-dimer, such as inherited and/or acquired thrombophilia.Patients and Methods We selected 79 patients with unexplained primary or secondary infertility. We excluded 40 patients affected by hydrosalpinx, uterine fibroids, uterine malformations, endocrinological and immunological diseases, luteal insufficiency, cytogenetical alterations. All remaining 39 patients were tested for d-dimer and divided in two groups: the patients of group A (25 patients) showed increased plasma d-dimer, in group B were included 14 patients with normal plasma level of d-dimer. After this step all 39 patients were screened for MTHFR C677T , PTHR A20210G , Factor V Leiden polimorphisms, protein C, protein S, AT III, anticardiolipin IgM and IgG, lupus anticoagulant. In the control group were included 15 age matched women without sterility problems referred to our outpatient's section of vascular medicine for suspected deep venous thrombosis. Statistical analysis was based on χ 2  test, differences were considered to be significant if p < 0.05.
Patients and Methods
We selected 79 patients with unexplained primary or secondary infertility. We excluded 40 patients affected by hydrosalpinx, uterine fibroids, uterine malformations, endocrinological and immunological diseases, luteal insufficiency, cytogenetical alterations. All remaining 39 patients were tested for d-dimer and divided in two groups: the patients of group A (25 patients) showed increased plasma d-dimer, in group B were included 14 patients with normal plasma level of d-dimer. After this step all 39 patients were screened for MTHFR C677T , PTHR A20210G , Factor V Leiden polimorphisms, protein C, protein S, AT III, anticardiolipin IgM and IgG, lupus anticoagulant. In the control group were included 15 age matched women without sterility problems referred to our outpatient's section of vascular medicine for suspected deep venous thrombosis.Statistical analysis was based on χ 2  test, differences were considered to be significant if p < 0.05.Results D-dimer was increased in 25/39 and 20/25 showed inherited/acquired thrombophilia while patients with normal d-dimer showed inherited/acquired thrombophilia in 7/14 (p: < 0.05, s).
Results
D-dimer was increased in 25/39 and 20/25 showed inherited/acquired thrombophilia while patients with normal d-dimer showed inherited/acquired thrombophilia in 7/14 (p: < 0.05, s).Discussion D-dimer is a well known marker of hypercoagulable state, in particular its high predictive negative value in case of suspected thrombosis has been recognised by several reports. Yet, increased d-dimer has been identified also for subclinical thrombophilia besides for vascular thrombosis. Our data, in fact, for the first time suggest an interesting role of d-dimer to identify women affected by unexplained primary or secondary infertility and thrombophilia. So, probably there is a role for d-dimer in these subjects for its predictive positive value. Of course, further data on large based population are needed to confirm our results, because these findings may speed up a diagnostic screening in these patients also for a good cost/effectiveness of this test.
Discussion
D-dimer is a well known marker of hypercoagulable state, in particular its high predictive negative value in case of suspected thrombosis has been recognised by several reports. Yet, increased d-dimer has been identified also for subclinical thrombophilia besides for vascular thrombosis. Our data, in fact, for the first time suggest an interesting role of d-dimer to identify women affected by unexplained primary or secondary infertility and thrombophilia. So, probably there is a role for d-dimer in these subjects for its predictive positive value. Of course, further data on large based population are needed to confirm our results, because these findings may speed up a diagnostic screening in these patients also for a good cost/effectiveness of this test.

Introduction D-dimer is considered a marker of hypercoagulable state besides of endogenous fibrinolysis, so increased d-dimer is detectable in patients affected by arterial and/or venous thrombosis [ 1 ]. Yet, several studies showed increased d-dimer also in patients affected by subclinical thrombophilia without ongoing thrombosis [ 2 ]. Moreover, also in other clinical conditions, such as chronic inflammation as infectious disease (also as marker of disseminated intravascular coagulation if sepsis is associated) as cancer as necrosis as eldership and pregnancy we may observe an increase of plasma d-dimer [ 3 - 8 ]. So, for this reason d-dimer test is usually used in clinical management for its high predictive negative value in suspected thrombosis, particularly in deep vein thrombosis (DVT) [ 9 - 11 ]. However, several studies showed that frequently women affected by sterility, in particular secondary sterility for recurrent foetal losses, may be affected by an underlying inherited and/or acquired thrombophilia [ 12 - 20 ]. Besides, common thrombotic risk factors which include also a bad lifestyle (e.g. obesity, non regard to Mediterranean diet, sedentary life), a lot of molecular thrombotic risk factors such as inherited or acquired clotting inhibitor deficiency (i.e. protein C, protein S, antithrombin III), inherited thrombophilia (factor V Leiden, prothrombin A20210G mutation), primary or secondary hyperhomcysteinemia, primary or secondary antiphospholipid syndrome and increased plasma factor VIII levels have been identified [ 21 ]. Furthermore, these molecular alterations may be also associated in some subjects so inducing gene-gene interactions and/or gene-enviromental interactions [ 22 - 24 ]. So, because the high incidence of clotting abnormalities in these patients, according to the data of Brenner et al. [ 24 , 25 ], we investigated the role of d-dimer as first marker of thrombophilia in women affected by sterility in order to identify causes of increased d-dimer and probably of the induced sterility.
Introduction
D-dimer is considered a marker of hypercoagulable state besides of endogenous fibrinolysis, so increased d-dimer is detectable in patients affected by arterial and/or venous thrombosis [ 1 ]. Yet, several studies showed increased d-dimer also in patients affected by subclinical thrombophilia without ongoing thrombosis [ 2 ]. Moreover, also in other clinical conditions, such as chronic inflammation as infectious disease (also as marker of disseminated intravascular coagulation if sepsis is associated) as cancer as necrosis as eldership and pregnancy we may observe an increase of plasma d-dimer [ 3 - 8 ]. So, for this reason d-dimer test is usually used in clinical management for its high predictive negative value in suspected thrombosis, particularly in deep vein thrombosis (DVT) [ 9 - 11 ]. However, several studies showed that frequently women affected by sterility, in particular secondary sterility for recurrent foetal losses, may be affected by an underlying inherited and/or acquired thrombophilia [ 12 - 20 ]. Besides, common thrombotic risk factors which include also a bad lifestyle (e.g. obesity, non regard to Mediterranean diet, sedentary life), a lot of molecular thrombotic risk factors such as inherited or acquired clotting inhibitor deficiency (i.e. protein C, protein S, antithrombin III), inherited thrombophilia (factor V Leiden, prothrombin A20210G mutation), primary or secondary hyperhomcysteinemia, primary or secondary antiphospholipid syndrome and increased plasma factor VIII levels have been identified [ 21 ]. Furthermore, these molecular alterations may be also associated in some subjects so inducing gene-gene interactions and/or gene-enviromental interactions [ 22 - 24 ]. So, because the high incidence of clotting abnormalities in these patients, according to the data of Brenner et al. [ 24 , 25 ], we investigated the role of d-dimer as first marker of thrombophilia in women affected by sterility in order to identify causes of increased d-dimer and probably of the induced sterility.Patients and Methods We selected 79 women affected by primary or secondary sterility (due to three or more fetal losses) referred to our sterility center. We excluded 40 patients affected by hydrosalpinx, uterine fibroids, uterine malformations, luteal insufficiency, anovulation, cytogenetical alterations, infectious diseases, endocrinological diseases (ie diabetes, subpituitarism), and by immunological diseases (inherited and/or acquired immunodeficiency, rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, vasculitis). After this screening we selected 39 patients (12 affected by primary sterility and 27 by secondary sterility due to recurrent foetal loss). These 39 patients were tested for d-dimer. D-dimer was measured by several methods [ 26 ]; d-dimer were tested randomly in various periods of the ovarian menstrual cycle in 31 patients, in one patient during menstrual bleeding and in seven patients during hormonal therapy in order to obtain controlled ovarian hyperstimulation (COH). Following d-dimer examination patients were divided in two different groups: group A including 25 patients with increased d-dimer levels and group B including 14 patients with normal d-dimer levels. As control group we selected 15 age-matched women, without sterility problem in their anamnesis, referred to our outpatient's section of vascular medicine for suspected deep venous thrombosis. Subsequently d-dimer evaluation, in order to identify a possible inherited and/or acquired thrombophilia, all patients were screened for methilene-tetra-hydro-folate-reductase C677T gene polimorphism (MTHFR C677T ), Factor V Leiden gene polimorphism (FVL), prothrombin A20210G gene polimorphism (PTHR A20210G ), protein S deficiency, protein C deficiency, antithrombin III deficiency (AT III), lupus anticoagulant, IgM and/or IgG anticardiolipin autoantibodies [ 22 , 27 ]. Moreover, all patients showing increased d-dimer were tested also for β-human-corionic-gonadotropin (β-HCG) to exclude early pregnancy, and lower limb ultrasound vascular examination associated to compression ultrasonography (CUS) to exclude a lower limb deep venous thrombosis (DVT); both conditions, in fact, are well known conditions associated to increased d-dimer [ 4 , 9 - 11 ]. Furthermore, patients with increased d-dimer (group A, 25 patients) and patients with normal d-dimer (group B, 14 patients) were compared also for possible differences in molecular markers of inherited and/or acquired thrombophilia. Statistical analysis was based on χ 2  test, differences were considered to be significant if p < 0.05.
Patients and Methods
We selected 79 women affected by primary or secondary sterility (due to three or more fetal losses) referred to our sterility center. We excluded 40 patients affected by hydrosalpinx, uterine fibroids, uterine malformations, luteal insufficiency, anovulation, cytogenetical alterations, infectious diseases, endocrinological diseases (ie diabetes, subpituitarism), and by immunological diseases (inherited and/or acquired immunodeficiency, rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, vasculitis).After this screening we selected 39 patients (12 affected by primary sterility and 27 by secondary sterility due to recurrent foetal loss). These 39 patients were tested for d-dimer. D-dimer was measured by several methods [ 26 ]; d-dimer were tested randomly in various periods of the ovarian menstrual cycle in 31 patients, in one patient during menstrual bleeding and in seven patients during hormonal therapy in order to obtain controlled ovarian hyperstimulation (COH). Following d-dimer examination patients were divided in two different groups: group A including 25 patients with increased d-dimer levels and group B including 14 patients with normal d-dimer levels. As control group we selected 15 age-matched women, without sterility problem in their anamnesis, referred to our outpatient's section of vascular medicine for suspected deep venous thrombosis.Subsequently d-dimer evaluation, in order to identify a possible inherited and/or acquired thrombophilia, all patients were screened for methilene-tetra-hydro-folate-reductase C677T gene polimorphism (MTHFR C677T ), Factor V Leiden gene polimorphism (FVL), prothrombin A20210G gene polimorphism (PTHR A20210G ), protein S deficiency, protein C deficiency, antithrombin III deficiency (AT III), lupus anticoagulant, IgM and/or IgG anticardiolipin autoantibodies [ 22 , 27 ]. Moreover, all patients showing increased d-dimer were tested also for β-human-corionic-gonadotropin (β-HCG) to exclude early pregnancy, and lower limb ultrasound vascular examination associated to compression ultrasonography (CUS) to exclude a lower limb deep venous thrombosis (DVT); both conditions, in fact, are well known conditions associated to increased d-dimer [ 4 , 9 - 11 ].Furthermore, patients with increased d-dimer (group A, 25 patients) and patients with normal d-dimer (group B, 14 patients) were compared also for possible differences in molecular markers of inherited and/or acquired thrombophilia.Statistical analysis was based on χ 2  test, differences were considered to be significant if p < 0.05.Results We found thrombophilia in group A, 80%, and in group B, 50%, so thrombophilia rate in all 39 selected was 65% if we consider together group A (i.e. women affected by sterility and showing increased d-dimer) and group B (i.e. women affected by sterility with normal d-dimer levels) (table 1, 'see  additional file 1 '). Twenty patients of group A (80%) affected by sterility with increased d-dimer levels, showed inherited and/or acquired thrombophilia [(six MTHFR C677T  homozygosity, four FVL heterozygosity, five PTHR A20210G  heterozygosity, three inherited Protein S deficiency, two showing combined defects (one MTHFR C677T  homozygosity associated to protein S deficiency and one MTHFR C677T  homozygosity associated to FVL heterozygosity), none protein C deficiency or AT III deficiency, none positive for the presence of lupus anticoagulant, none with increased anticardiolipin autoantibodies IgM and/or increased anticardiolipin autoantibodies IgG)] (table 2, 'see  additional file 2 '). Remaining five women of the group A did not show molecular thrombophilia, but in their anamnesis we found some possible correlation with an acquired thrombophilia: controlled ovarian hyperstimulation in one patient, monthlies in one patient, early pregnancy in one patient, miscarriage in one patient, none apparent cause in 1 patient; among them two of these five patients were heterozygous for MTHFR C677T . Furthermore, two patients of group A carrying inherited thrombophilia for the presence of heterozygous FVL and increased d-dimer revealed previous DVT with following pulmonary embolism in their anamnesis. Data of patients of group A are summarised in table 2 ('see  additional file 2 '). Seven patients of group B (50%) showed inherited and/or acquired thrombophilia (one MTHFR C677T  homozygosity, one FVL heterozygosity, five PTHR A20210G  heterozygosity, none inherited Protein S deficiency, protein C deficiency, AT III deficiency, none presence of lupus anticoagulant, none with increased anticardiolipin autoantibodies IgM and/or IgG), as reported in table 2 ('see  additional file 2 '). All remaining seven patients of group B showed all heterozygosity for MTHFR C677T . Moreover, none patients of group B revealed previous DVT and/or pulmonary embolism. Five patients of group C (i.e. control group) (33.3%) showed increased d-dimer as molecular markers of ongoing proximal DVT confirmed by ultrasound vascular examination associated to CUS; moreover, all five patients revealed an underlying inherited and/or acquired thrombophilia (three MTHFR C677T  homozygosity, one protein S deficiency, one combined thrombophilia: FVL heterozygosity associated to protein S deficiency). Data of patients of group C are summarised in table 2 ('see  additional file 2 '). In all groups positivity for anticardiolipin antibodies or lupus anticoagulant mimicking a primary antiphospholipid syndrome (APS) was not discovered. So, as showed in table 3 ('see  additional file 3 '), increased d-dimer is frequently associated with thrombophilia in women affected by sterility, while this association is less present in patients with normal d-dimer, and this difference reaches statistical significance (p < 0.05); furthermore thrombophilia is more frequent in group A than in control group (i.e. group C) and also this difference reaches statistical significance (p < 0.05); finally, thrombophilia in group B is more frequent than in control group (i.e. group C), but this difference does not reach statistical significance (p: 0.08, ns).
Results
We found thrombophilia in group A, 80%, and in group B, 50%, so thrombophilia rate in all 39 selected was 65% if we consider together group A (i.e. women affected by sterility and showing increased d-dimer) and group B (i.e. women affected by sterility with normal d-dimer levels) (table 1, 'see  additional file 1 ').Twenty patients of group A (80%) affected by sterility with increased d-dimer levels, showed inherited and/or acquired thrombophilia [(six MTHFR C677T  homozygosity, four FVL heterozygosity, five PTHR A20210G  heterozygosity, three inherited Protein S deficiency, two showing combined defects (one MTHFR C677T  homozygosity associated to protein S deficiency and one MTHFR C677T  homozygosity associated to FVL heterozygosity), none protein C deficiency or AT III deficiency, none positive for the presence of lupus anticoagulant, none with increased anticardiolipin autoantibodies IgM and/or increased anticardiolipin autoantibodies IgG)] (table 2, 'see  additional file 2 '). Remaining five women of the group A did not show molecular thrombophilia, but in their anamnesis we found some possible correlation with an acquired thrombophilia: controlled ovarian hyperstimulation in one patient, monthlies in one patient, early pregnancy in one patient, miscarriage in one patient, none apparent cause in 1 patient; among them two of these five patients were heterozygous for MTHFR C677T . Furthermore, two patients of group A carrying inherited thrombophilia for the presence of heterozygous FVL and increased d-dimer revealed previous DVT with following pulmonary embolism in their anamnesis. Data of patients of group A are summarised in table 2 ('see  additional file 2 ').Seven patients of group B (50%) showed inherited and/or acquired thrombophilia (one MTHFR C677T  homozygosity, one FVL heterozygosity, five PTHR A20210G  heterozygosity, none inherited Protein S deficiency, protein C deficiency, AT III deficiency, none presence of lupus anticoagulant, none with increased anticardiolipin autoantibodies IgM and/or IgG), as reported in table 2 ('see  additional file 2 '). All remaining seven patients of group B showed all heterozygosity for MTHFR C677T . Moreover, none patients of group B revealed previous DVT and/or pulmonary embolism.Five patients of group C (i.e. control group) (33.3%) showed increased d-dimer as molecular markers of ongoing proximal DVT confirmed by ultrasound vascular examination associated to CUS; moreover, all five patients revealed an underlying inherited and/or acquired thrombophilia (three MTHFR C677T  homozygosity, one protein S deficiency, one combined thrombophilia: FVL heterozygosity associated to protein S deficiency). Data of patients of group C are summarised in table 2 ('see  additional file 2 ').In all groups positivity for anticardiolipin antibodies or lupus anticoagulant mimicking a primary antiphospholipid syndrome (APS) was not discovered.So, as showed in table 3 ('see  additional file 3 '), increased d-dimer is frequently associated with thrombophilia in women affected by sterility, while this association is less present in patients with normal d-dimer, and this difference reaches statistical significance (p < 0.05); furthermore thrombophilia is more frequent in group A than in control group (i.e. group C) and also this difference reaches statistical significance (p < 0.05); finally, thrombophilia in group B is more frequent than in control group (i.e. group C), but this difference does not reach statistical significance (p: 0.08, ns).Discussion In this report for the first time the role of d-dimer was investigated in diagnostic screening of patients affected by sterility and this is a really innovative data available in this clinical setting. D-dimer is a fibrin degradation product which usually is extensively screened in patients with suspected thrombosis and/or pulmonary embolism [ 9 ]. An increased plasma d-dimer might have a predictive positive value for DVT and/or pulmonary embolism, but because increased d-dimer has been observed also in several conditions not associated with ongoing thrombosis (malignancy, chronic inflammation, infections, acute coronary syndromes, necrosis, eldership) [ 3 - 9 ] the really interesting role of d-dimer in this clinical setting is for its high negative predictive value as reported by Bounameaux et al. in a series of patients with suspected pulmonary embolism [ 9 ]. However, increased d-dimer has been observed also in subjects affected by thrombophilia (i.e. inherited thrombophilia and/or acquired thrombophilia) showing hypercoagulable state without ongoing thrombosis as reported by Arkel et al. and Humphries et al. [ 2 , 23 ]. So, our data showed that patients of group A, carrying increased d-dimer, has been extensively screened for inherited and/or acquired thrombophilia and 80% of them revealed a well known molecular condition associated to hypercoagulable state which may explain increased d-dimer levels (table 2, 'see  additional file 2 '). Moreover, this our clinical and laboratory screening reaches statistical significance compared to group B and group C (table 3, 'see  additional file 3 '). Furthermore, five patients with increased d-dimer did not reveal inherited and/or acquired thrombophilia, but a thorough anamnesis and a clinical evaluation permitted to identify other causes of increased d-dimer in four of these patients: one patient showed early pregnancy (confirmed by β-HCG measurements and following ultrasound scan), a known condition associated to hypercoagulability and increased d-dimer [ 4 , 28 , 29 ], one patient revealed an early abortion, confirmed by following decrease of β-HCG, and increased dimer levels might be related to uteroplacental thrombosis and/or necrosis [ 30 ], one patient was ongoing to controlled ovarian stimulation and this condition may be associated to alteration of haemostasis with a trend toward thrombophilia [ 31 , 32 ] and one patient showed ongoing monthlies, a condition associated to wound healing which involves also clotting factors and might explain increased d-dimer [ 33 ]; remaining one patient showed increased d-dimer for unknown causes probably related to not well studied thrombophilia [ 34 ] or idiopathic thrombophilia and/or other conditions although we excluded in our selection criteria several other diseases associated to increased d-dimer. So for the first time we showed an interesting and relevant role of d-dimer in the screening of sterility causes, particular an underlying thrombophilia may be suspected in pathophysiology of sterility if plasma d-dimer is increased. However, also an evaluation of other conditions associated to increased d-dimer (e.g. chronic inflammation, immunopathological diseases, infectious diseases, cancer, necrosis, eldership, pregnancy, controlled ovarian stimulation, monthlies) should be performed in order to avoid a misinterpretation. Also group B, with normal d-dimer levels, showed an increased rate of thrombophilia (50%, table 1, 'see  additional file 1 '), so confirming one more time the clear relationship between thrombophilia and sterility, even if these data did not reach statistical significance compared to group C (table 3, 'see  additional file 3 '). Yet, patients of group B, although affected by thrombophilia and sterility did not show increased d-dimer. This finding might be explained by several causes and a laboratory mistake cannot be excluded; furthermore, these patients of group B may show also transient and/or silent thrombophilia which may trigger a hypercoagulable state if associated to other causes (i.e. acquired conditions associated to thrombophilia) during their natural history and our evaluation of d-dimer might be done during a not-hypercoagulable transient state. An extensive screening of causes of increased d-dimer in our population was also performed. The association between thrombophilia and sterility due to recurrent foetal loss is well known as reported by several reports [ 12 - 20 ] and also by our data. However, recently an association between primary sterility and thrombophilia has been underlined such as also between thrombophilia and repeated in vitro fertilisation failures [ 35 , 36 ]. A clear relationship between thrombophilia and recurrent foetal loss has been reported for inherited deficiency of clotting inhibitors (i.e. protein C deficiency, protein S deficiency, AT III deficiency) [ 20 , 36 ], but we did not find in our population this strong association (only four cases of protein S deficiency, one of these associated to MTHFR C677T  homozygosity, and none case of protein C deficiency and/or AT III deficiency). However, this aspect seems to be in agreement with other reports in which other thrombophilic conditions were more frequent than clotting inhibitor deficiencies (e.g. FVL, MTHFR C677T  homozygosity, antiphospholipid syndrome and so on) [ 12 - 20 ]. FVL gene polymorphism has been frequently found in women affected by recurrent fetal loss, although the frequency of FVL differs in each study [ 15 , 24 ]. These differences could be related, besides to ethnic background, also to different inclusion criteria of investigated patients. However, FVL is associated to sterility also in our study (four cases in group A and one case in group B; table 2, 'see  additional file 2 '). An increased MTHFR C677T  homozygosity has been found in our study population (six cases in group A and one case in group B), so confirming a clear role of homocysteine metabolism and of the related hypercoagulable state in sterility pathophysiology [ 38 - 40 ]. Of course, MTHFR gene polymorphism and related homocysteine metabolism may influence sterility also through folic acid and vitamin B12 deficiency due to uncorrected diet and/or lifestyle [ 41 ]. We found also an increased frequency of PTHR A20210G  in women affected by sterility (five cases in group A and five cases in group B), and these data seem to be different from data reported by Pickering et al [ 42 ] and Deitcher et al [ 43 ] and in agreement with data reported by Brenner et al [ 24 , 25 ]. As we previously underlined, these differences could be related to inclusion criteria established by Investigators of each study and also to an ethnic background; this gene polymorphism, in fact, is more frequent in Southern Europe than in Northern Europe [ 44 , 45 ]. A really interesting data is the absence of APS from our study population and this data differs from data of the Literature. A possible explanation could be offered by different selection criteria: we exclude, in fact, women with immunopathological diseases (e.g. rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, vasculitis), so excluding the most common causes of secondary APS and so searching only primary APS that is more rare than primary [ 46 ]. In conclusion, in this investigation both groups of women affected by sterility, group A and B, showed increased incidence of thrombophilia compared to control group (group A vs group C, p: < 0.05, s; group B vs group C, p: 0.08, ns; table 3, 'see  additional file 3 '), so confirming, one more time the relevant role of thrombophilia in pathophysiology of sterility. So, the first relevant data we offer in this study is the role of d-dimer in the screening of sterility causes in order to early suspect an underlying thrombophilia; this screening, as also showed by our data, is in agreement with an elevated frequency of thrombophilia in women affected by sterility (80 % in group A, 50% in group B, 65% if we consider together group A and B). Of course, although several Authors already reported the association between thrombophilia and recurrent foetal loss we may testify that probably the role of thrombophilia is an underestimated problem if we consider all sterility conditions because usually thrombophilia is screened only for repeated foetal loss and not screened in any case of unexplained sterility as in this study. So, based on our data further studies on large population are needed not only to confirm our results but also to focus a possible different prognosis of these groups, in particular to sterility prognosis.
Discussion
In this report for the first time the role of d-dimer was investigated in diagnostic screening of patients affected by sterility and this is a really innovative data available in this clinical setting.D-dimer is a fibrin degradation product which usually is extensively screened in patients with suspected thrombosis and/or pulmonary embolism [ 9 ]. An increased plasma d-dimer might have a predictive positive value for DVT and/or pulmonary embolism, but because increased d-dimer has been observed also in several conditions not associated with ongoing thrombosis (malignancy, chronic inflammation, infections, acute coronary syndromes, necrosis, eldership) [ 3 - 9 ] the really interesting role of d-dimer in this clinical setting is for its high negative predictive value as reported by Bounameaux et al. in a series of patients with suspected pulmonary embolism [ 9 ]. However, increased d-dimer has been observed also in subjects affected by thrombophilia (i.e. inherited thrombophilia and/or acquired thrombophilia) showing hypercoagulable state without ongoing thrombosis as reported by Arkel et al. and Humphries et al. [ 2 , 23 ].So, our data showed that patients of group A, carrying increased d-dimer, has been extensively screened for inherited and/or acquired thrombophilia and 80% of them revealed a well known molecular condition associated to hypercoagulable state which may explain increased d-dimer levels (table 2, 'see  additional file 2 '). Moreover, this our clinical and laboratory screening reaches statistical significance compared to group B and group C (table 3, 'see  additional file 3 '). Furthermore, five patients with increased d-dimer did not reveal inherited and/or acquired thrombophilia, but a thorough anamnesis and a clinical evaluation permitted to identify other causes of increased d-dimer in four of these patients: one patient showed early pregnancy (confirmed by β-HCG measurements and following ultrasound scan), a known condition associated to hypercoagulability and increased d-dimer [ 4 , 28 , 29 ], one patient revealed an early abortion, confirmed by following decrease of β-HCG, and increased dimer levels might be related to uteroplacental thrombosis and/or necrosis [ 30 ], one patient was ongoing to controlled ovarian stimulation and this condition may be associated to alteration of haemostasis with a trend toward thrombophilia [ 31 , 32 ] and one patient showed ongoing monthlies, a condition associated to wound healing which involves also clotting factors and might explain increased d-dimer [ 33 ]; remaining one patient showed increased d-dimer for unknown causes probably related to not well studied thrombophilia [ 34 ] or idiopathic thrombophilia and/or other conditions although we excluded in our selection criteria several other diseases associated to increased d-dimer.So for the first time we showed an interesting and relevant role of d-dimer in the screening of sterility causes, particular an underlying thrombophilia may be suspected in pathophysiology of sterility if plasma d-dimer is increased. However, also an evaluation of other conditions associated to increased d-dimer (e.g. chronic inflammation, immunopathological diseases, infectious diseases, cancer, necrosis, eldership, pregnancy, controlled ovarian stimulation, monthlies) should be performed in order to avoid a misinterpretation.Also group B, with normal d-dimer levels, showed an increased rate of thrombophilia (50%, table 1, 'see  additional file 1 '), so confirming one more time the clear relationship between thrombophilia and sterility, even if these data did not reach statistical significance compared to group C (table 3, 'see  additional file 3 '). Yet, patients of group B, although affected by thrombophilia and sterility did not show increased d-dimer. This finding might be explained by several causes and a laboratory mistake cannot be excluded; furthermore, these patients of group B may show also transient and/or silent thrombophilia which may trigger a hypercoagulable state if associated to other causes (i.e. acquired conditions associated to thrombophilia) during their natural history and our evaluation of d-dimer might be done during a not-hypercoagulable transient state.An extensive screening of causes of increased d-dimer in our population was also performed. The association between thrombophilia and sterility due to recurrent foetal loss is well known as reported by several reports [ 12 - 20 ] and also by our data. However, recently an association between primary sterility and thrombophilia has been underlined such as also between thrombophilia and repeated in vitro fertilisation failures [ 35 , 36 ].A clear relationship between thrombophilia and recurrent foetal loss has been reported for inherited deficiency of clotting inhibitors (i.e. protein C deficiency, protein S deficiency, AT III deficiency) [ 20 , 36 ], but we did not find in our population this strong association (only four cases of protein S deficiency, one of these associated to MTHFR C677T  homozygosity, and none case of protein C deficiency and/or AT III deficiency). However, this aspect seems to be in agreement with other reports in which other thrombophilic conditions were more frequent than clotting inhibitor deficiencies (e.g. FVL, MTHFR C677T  homozygosity, antiphospholipid syndrome and so on) [ 12 - 20 ].FVL gene polymorphism has been frequently found in women affected by recurrent fetal loss, although the frequency of FVL differs in each study [ 15 , 24 ]. These differences could be related, besides to ethnic background, also to different inclusion criteria of investigated patients. However, FVL is associated to sterility also in our study (four cases in group A and one case in group B; table 2, 'see  additional file 2 ').An increased MTHFR C677T  homozygosity has been found in our study population (six cases in group A and one case in group B), so confirming a clear role of homocysteine metabolism and of the related hypercoagulable state in sterility pathophysiology [ 38 - 40 ]. Of course, MTHFR gene polymorphism and related homocysteine metabolism may influence sterility also through folic acid and vitamin B12 deficiency due to uncorrected diet and/or lifestyle [ 41 ].We found also an increased frequency of PTHR A20210G  in women affected by sterility (five cases in group A and five cases in group B), and these data seem to be different from data reported by Pickering et al [ 42 ] and Deitcher et al [ 43 ] and in agreement with data reported by Brenner et al [ 24 , 25 ]. As we previously underlined, these differences could be related to inclusion criteria established by Investigators of each study and also to an ethnic background; this gene polymorphism, in fact, is more frequent in Southern Europe than in Northern Europe [ 44 , 45 ].A really interesting data is the absence of APS from our study population and this data differs from data of the Literature. A possible explanation could be offered by different selection criteria: we exclude, in fact, women with immunopathological diseases (e.g. rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, vasculitis), so excluding the most common causes of secondary APS and so searching only primary APS that is more rare than primary [ 46 ].In conclusion, in this investigation both groups of women affected by sterility, group A and B, showed increased incidence of thrombophilia compared to control group (group A vs group C, p: < 0.05, s; group B vs group C, p: 0.08, ns; table 3, 'see  additional file 3 '), so confirming, one more time the relevant role of thrombophilia in pathophysiology of sterility. So, the first relevant data we offer in this study is the role of d-dimer in the screening of sterility causes in order to early suspect an underlying thrombophilia; this screening, as also showed by our data, is in agreement with an elevated frequency of thrombophilia in women affected by sterility (80 % in group A, 50% in group B, 65% if we consider together group A and B). Of course, although several Authors already reported the association between thrombophilia and recurrent foetal loss we may testify that probably the role of thrombophilia is an underestimated problem if we consider all sterility conditions because usually thrombophilia is screened only for repeated foetal loss and not screened in any case of unexplained sterility as in this study.So, based on our data further studies on large population are needed not only to confirm our results but also to focus a possible different prognosis of these groups, in particular to sterility prognosis.Conclusion Our data demonstrated a clear role of thrombophilia in patients affected by sterility, but suggesting a clear diagnostic role of increased d-dimer in a lot of these patients. This diagnostic screening of thrombophilia in women affected by sterility, based on the d-dimer levels, may also represent a really speed method to suspect thrombophilia in these subject and has also a good cost/benefit ratio, although other causes of increased d-dimer should be always considered. In a second step, if increased d-dimer levels are present causes of hypercoagulable state may be investigated (i.e. inherited thrombophilia and/or acquired thrombophilia). This approach may play a role not only in differential diagnosis of sterility but also in the early diagnosis of sterility due to thrombophilia. After the first step in which d-dimer may be evaluated, causes of increased d-dimer should be subsequently identified in order to start a possible antithrombotic treatment soon. Nevertheless thrombophilia may be present in few cases also in subjects with normal d-dimer, it should be investigated always if other causes of sterility are not present. So, we strongly suggest to test d-dimer in patients affected by sterility as first step of a possible underlying thrombophilia in order to early identify the cause of thrombophilia and its prompt treatment but other data should be confirmed by further investigations on large based population.
Conclusion
Our data demonstrated a clear role of thrombophilia in patients affected by sterility, but suggesting a clear diagnostic role of increased d-dimer in a lot of these patients. This diagnostic screening of thrombophilia in women affected by sterility, based on the d-dimer levels, may also represent a really speed method to suspect thrombophilia in these subject and has also a good cost/benefit ratio, although other causes of increased d-dimer should be always considered. In a second step, if increased d-dimer levels are present causes of hypercoagulable state may be investigated (i.e. inherited thrombophilia and/or acquired thrombophilia). This approach may play a role not only in differential diagnosis of sterility but also in the early diagnosis of sterility due to thrombophilia. After the first step in which d-dimer may be evaluated, causes of increased d-dimer should be subsequently identified in order to start a possible antithrombotic treatment soon.Nevertheless thrombophilia may be present in few cases also in subjects with normal d-dimer, it should be investigated always if other causes of sterility are not present.So, we strongly suggest to test d-dimer in patients affected by sterility as first step of a possible underlying thrombophilia in order to early identify the cause of thrombophilia and its prompt treatment but other data should be confirmed by further investigations on large based population.Supplementary Material Additional File 1 Thrombophilia frequency in studied groups Click here for file Additional File 2 Thrombophilia in studied groups Click here for file Additional File 3 statistical analysis according with χ 2  method Click here for file
Supplementary Material
Additional File 1
Thrombophilia frequency in studied groupsClick here for fileAdditional File 2
Thrombophilia in studied groupsClick here for fileAdditional File 3
statistical analysis according with χ 2  methodClick here for file