investigations and Treatments of Recurrent Pregnancy Loss

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Evidence-based investigations and treatments of

recurrent pregnancy lossOle B. Christiansen, M.D., Ph.D.,a Anne-Marie Nybo Andersen, Ph.D.,b Ernesto Bosch, M.D.,c

Salim Daya, M.B.,d Peter J. Delves, Ph.D.,e Thomas V. Hviid, Ph.D.,f William H. Kutteh, Ph.D.,g

Susan M. Laird, Ph.D.,h Tin-Chiu Li, Ph.D.,i and Katrin van der Ven, M.D.j

a Fertility Clinic 4071 and fDepartment of Clinical Biochemistry, Rigshospitalet, Copenhagen; b Institute of Social Medicine,

University of Copenhagen, Copenhagen, Denmark; c Instituto Valenciano de Infertilidad, Valencia, Spain; d Department of

Obstetrics and Gynecology, McMaster University, Hamilton, Ontario, Canada; e Department of Immunology and Molecular

Pathology, Division of Infection and Immunity, University College London; h Division of Biomedical Sciences, Sheffield

Hallam University, Sheffield; i Biomedical Research Unit, Jessop Hospital for Women, Sheffield, United Kingdom;g Division

of Reproductive Endocrinology and Immunology, Department of Obstetrics and Gynecology, University of Tennessee,

Memphis, Tennessee; and j IVF Clinic, Department of Obstetrics and Gynaecology, University of Bonn, Bonn, Germany

Objective: To give an overview of currently used investigations and treatments offered to women with recurrentpregnancy loss (RPL) and, from an evidence-based point of view, to evaluate the usefulness of these interven-

tions.

Design: Ten experts on epidemiologic, genetic, anatomic, endocrinologic, thrombophilic, immunologic, andimmunogenetic aspects of RPL discussed methodologic problems threatening the validity of research in RPL

during and after an international workshop on the evidence-based management of RPL.

Conclusion(s): Most RPL patients have several risk factors for miscarriage, and an extensive investigation for allmajor factors should always be undertaken. There is an urgent need for agreement concerning the thresholds for

detecting what is normal and abnormal, irrespective of whether laboratory tests or uterine abnormalities are

concerned. A series of lifestyle factors should be reported in future studies of RPL because they might modify

the effect of laboratory or anatomic risk factors. More and larger randomized controlled trials, including trials of

surgical procedures, are urgently needed, and to achieve this objective multiple centers have to collaborate.

Current meta-analyses evaluating the efficacy of treatments of RPL are generally pooling very heterogeneous

patient populations and treatments. It is recommended that future meta-analyses look at subsets of patients andtreatment protocols that are more combinable. (Fertil Steril 2005;83:82139. 2005 by American Society for

Reproductive Medicine.)

Key Words: Abortion, anticardiolipin, HLA-G, recurrent miscarriage, uterine fibroids, recurrent pregnancy loss

There is no doubt that the introduction of a series of new

assisted reproduction technologies (ART) has recently

greatly improved the treatment options available for infertile

couples. Most of these couples will now be offered treat-

ments that are generally accepted and often evidence-based.

Unfortunately, the situation is much less clear regarding

couples with recurrent pregnancy loss (RPL), who are often

treated as second-class infertility patients in the health care

system. Accurate prevalence figures are not available, but it

has been estimated that 2%5% of women have RPL, de-

fined as three or more consecutive losses of intrauterine

pregnancies before the 28th gestational week (1, 2).

Although the array of diagnostic tests and possible thera-

peutic interventions in the management of RPL have grown

significantly, in 1992 a U.S. study (3)showed that the totallive birth rate had not increased in a cohort of RPL patients

from 1987 to 1991 as compared with a similar group from

1968 to 1977.

The motivation for organizing a workshop under the aus-

pices of the European Society for Human Reproduction and

Embryology with the title Evidence-Based Investigations

and Treatments of Recurrent Pregnancy Loss was the fact

that many of the large number of diagnostic tests that have

become available for the investigation of patients with RPL

are probably of doubtful value and need proper evaluation

and standardization. Furthermore, many treatments also need

Received August 23, 2004; revised and accepted December 14, 2004.

The workshop inspiring this article was supported by grants from the

European Society for Human Reproduction and Embryology and the

Danish National Research Agency.

Reprint requests: Ole B. Christiansen, M.D., Ph.D., Fertility Clinic 4071,

Rigshospitalet, Blegdamsvej 9, Copenhagen DK-2100, Denmark. (FAX:

45-35-45-49-46; E-mail: [email protected]).

MODERN TRENDS

Edward E. Wallach, M.D.

Associate Editor

8210015-0282/05/$30.00 Fertility and SterilityVol. 83, No. 4, April 2005doi:10.1016/j.fertnstert.2004.12.018 Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc.


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proper evaluation concerning their therapeutic value and

possible risks.

One reason for the slow progress in RPL research might

be related to the fact that RPL is a complex area in which

information from many disciplines, such as gynecology,

genetics, epidemiology, occupational medicine, immunol-

ogy, hematology, and endocrinology, are to be integrated to

ensure that the research is valid. This collaboration among

many disciplines unfortunately has only been establishedinfrequently, with the result that most investigations in this

area have been narrowly focused, lacking an integrated ap-

proach to the subject.

A key element in this workshop was to let experts from the

different disciplines present their specific views on the

causes and treatments of RPL and engage the audience in

discussion to attempt a clarification on what is the state-of-

the-art knowledge and where consensus can be reached.

Gynecologists, obstetricians, and fertility specialists from

18 countries participated in the 3-day workshop held in

Denmark in 2002. The following review summarizes theconclusions from the discussions undertaken between the

participants during the workshop and the extensive follow-up

discussions between the authors that have continued until now.

RESEARCH METHODOLOGY AND EPIDEMIOLOGY OFRELEVANCE IN RPL STUDIES

Readers of the extensive literature about RPL often become

confused owing to the contradictory and ever-changing

views and results that are being published. Many of these

controversies are caused by the very different estimates of

the frequencies of RPL risk factors in patients and controls,

of the effect of these risk factors on pregnancy outcome, and

of the efficacy of various treatments. An important reason for

the controversy in this area is the apparent lack of appreci-

ation of the many methodologic pitfalls threatening valid

research in the area of RPL. Following is a review of some

of the pitfalls inherent in this area of research with different

study designs.

CaseControl Studies

In case control studies, flaws can occur during the sampling

of cases and controls. Patients can be incorrectly sampled

because of an incorrect RPL diagnosis or ascertainment bias.Furthermore, flaws can occur in relation to the tests carried out.

Incorrect RPL Diagnosis. Women can be diagnosed errone-ously as having RPL owing to faulty recall of the pregnancy

history, classification of biochemical pregnancies as miscar-

riages, and the investigators failure to adhere to the gener-

ally accepted criteria for RPL.

An example of information (recall) bias is that, owing to

their increased attention on pregnancy and miscarriage,

women with only two previously confirmed miscarriages

might be more prone to interpret and report delayed men-

struations in the past as early miscarriages. More miscar-

riages might also be reported owing to the womans wish to

be offered investigations and treatment. Only 71% of mis-

carriages reported by non-RPL women in a questionnaire

could be verified in hospital records (4).

Biochemical pregnancies (pregnancies documented only

by a positive urine or serum hCG test) constitute a consid-

erable proportion of some RPL patients previous pregnancy

history. Some of these pregnancies might be spontaneousresorbed ectopic pregnancies or very early implantation fail-

ures due to genetically abnormal embryos, according to

currently available tools. The etiologies of recurrent bio-

chemical pregnancies might thus be different from those of

clinical pregnancy losses. Thus, inclusion of patients with a

large proportion of biochemical pregnancies in clinical stud-

ies of RPL would be expected to diminish the estimate of a

maternal risk factor in casecontrol studies and the treatment

effect in controlled clinical trials.

Many studies have included women with only two previ-

ous miscarriages, which often might be a chance phenome-

non caused by de novo fetal chromosome abnormalities

rather than a recurrent maternal factor (5). Including women

with only two early miscarriages in the study will in most

cases dilute the estimate of the risk factor (in casecontrol

and cohort studies) or the treatment effect in controlled

clinical trials. This is supported by findings that the fre-

quency of many immunologic risk factors (6, 7) and the

possible effect of immunotherapy increases (8)and the fre-

quency of chromosomally abnormal abortions declines (9)

with the number of previous pregnancy losses.

Ascertainment Bias. Ascertainment bias occurs when pa-

tients referred to clinics with special interests are deliber-ately or unconsciously selected because of that clinical fea-

ture on which the clinics interest is focused. Such patients

therefore are not representative of the general RPL popula-

tion. For example, RPL patients investigated in clinics with

expertise in coagulation and antiphospholipid antibodies

might comprise an excess of women with antiphospholipid

antibodies (10)because referring centers preferentially refer

patients who in addition to obstetric problems also have

suffered thromboembolic episodes or expressed lupus-like

symptoms.

Sampling of Controls. In casecontrol studies, the quality ofthe control group is just as important as that of the case

group. Sampling of controls is subject to confounding, mis-

match with regard to pregnancy-related variables, and ascer-

tainment bias. Age is typically an important confounding

factor because it is associated with both the risk of develop-

ing RPL and the occurrence of many serologic abnormalities

(e.g., autoantibodies).

Blood parameters are often investigated at different stages

of pregnancies in patients and controls but still compared by

statistical methods. Many immunologic (1114) and coagu-

lation factors change during pregnancy and after a pregnancy

822 Christiansen et al. Management of recurrent pregnancy loss Vol. 83, No. 4, April 2005


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that progressed beyond the first trimester. Therefore, unreli-

able results can be produced if blood samples from pregnant

RPL patients are compared with samples from women who

are not pregnant or in a different stage of pregnancy.

Ascertainment bias often occurs when controls are sam-

pled. Normal women are difficult to sample; therefore,

individuals consulting the hospital for reasons other than

RPL are often used as controls. Ascertainment of controls by

this method is often biased or subject to confounding. Thecontrols might differ from the RPL patients by the occur-

rence of a disorder that can affect the variable being evalu-

ated, or they might differ by social class or by potential

hazardous exposures (tobacco, alcohol, caffeine, medicine).

Testing Procedures. Testing is often done during and im-mediately after miscarriage. Miscarriage might induce an

inflammatory reaction and endocrine changes that locally or

systemically can affect immunologic variables, such as cy-

tokines (15, 16) or antibodies. Abnormal findings after mis-

carriage therefore might be the result of miscarriage rather

than its cause (17). It is therefore important to be careful in

establishing causality from abnormal findings in samples

taken during and just after miscarriage. The best evidence for

causality comes from samples taken remote from miscarriage.

In many studies, numerous statistical comparisons are

carried out without information being given concerning the

prior hypothesis being tested. Defining statistical signifi-

cance as a P value .05 will result in every 20th statistical

comparison being significant by chance. Without a clearly

defined prior hypothesis, statistically significant post hoc

findings should not be overstated. Multiple statistical testing

should use appropriate multiple comparison methods to

avoid incorrect inferences from being drawn.

Cohort Studies: Differential Misclassification

Differential misclassification is an uneven intensity of mon-

itoring of exposure to risk factors or outcomes in the differ-

ent cohortsa problem often reported in occupational med-

icine.

Biased intensity of monitoring is also a problem in re-

search in human reproduction. For example, owing to the

standard procedures in most ART clinics of measuring se-

rum

-hCG 14 days after ET, more biochemical pregnancieswill be registered in cohorts of ART patients compared with

women who conceive spontaneously.

An example of the impact of biased intensity of monitor-

ing of pregnancy outcomes comes from the comparison of

two cohorts of RPL patients who were found suitable and

agreed to participate in placebo-controlled trials of treatment

with intravenous Ig in Sweden (18)and Denmark (19). In the

first trial, the patients were included in the trial in the next

pregnancy when fetal heart action could be demonstrated by

ultrasound in gestational weeks 68. In the second trial, the

patients had to call the trial center as soon as a pregnancy test

was positive, and they were included immediately in the trial

within 5 days after the missed menstrual period. The two

cohorts behaved very differently. In the Swedish study 51%

of the patients were classified as not achieving pregnancy,

and only 7% had pre-embryonal miscarriages before week 6.

In the Danish trial only 15% did not report pregnancy in the

study period, and 22% of the pregnancy losses happened

before week 6, prior to the possibility of demonstrating fetal

echoes by ultrasound. This very big difference in the fre-

quencies of nonconception and pre-embryonal pregnanciesbetween the two trials probably reflects the different inten-

sity of monitoring pregnancy outcomes due to the different

times of inclusion.

Treatment Trials

Unfortunately, there is a paucity of good-quality treatment

trials in RPL, and this limits the ability to make confident

recommendations about care in pregnancy. There are many

issues that have to be addressed in therapeutic trials before

inferences can be made that are reliable. Some of these

issues will be discussed below.Design Issues. The importance of secure randomization,concealment of treatment allocation, adequate sample size,

blinding, completeness of follow-up, stratification for impor-

tant covariates, and adequate assessment of outcome are well

known to clinical trial methodology and do not require

in-depth discussion in this review. It is only by ensuring that

a study is valid that one can make confident inferences from

the results obtained.

To make studies mutually comparable, it is necessary to

use the same terminology for the description of patients and

their miscarriages.Definition of Miscarriage. The term miscarriage (or abor-tion) is used to describe a pregnancy that fails to progress,

resulting in the death and expulsion of the embryo or fetus.

The generally accepted definition stipulates that the fetus or

embryo should weigh 500 g(20), a stage that corresponds

to a gestational age of20 weeks. Unfortunately, this def-

inition is not used consistently, and pregnancy losses at

higher gestational ages are also, in some studies, classified as

abortion instead of stillbirth or preterm neonatal death. Thus,

from a definition perspective, it is important to characterize

the population being studied so that comparisons across

therapeutic trials can be made more appropriately and reli-ably. Consensus on this issue is urgently required.

Effect of Male Partner. It is well known that some womenmight have RPL with one male partner and not with another.

Therefore, it is important when evaluating treatment efficacy

to ensure that the sample is homogeneous from this perspec-

tive (i.e., consecutive miscarriages with the same partner

should be stipulated as an inclusion criterion).

Subgroups of RPL. The pregnancy history in women withRPL might include pregnancies that have ended in live birth.

Thus, three different groups can be identified that should be

823Fertility and Sterility


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assessed separately because the risk of subsequent miscar-

riage among these groups varies (1): [1] the primary RPL

group consists of women with three or more consecutive

miscarriages with no pregnancy progressing beyond 20

weeks gestation, [2] the secondary RPL group consists of

women who have had three or more miscarriages following

a pregnancy that progressed beyond 20 weeks gestation,

which might have ended in live birth, stillbirth, or neonatal

death, and [3] the tertiary RPL group, which has not been

well characterized or studied, consists of women who have

had at least three miscarriages that are not consecutive but

are interspersed with pregnancies that have progressed be-

yond 20 weeks gestation (and might have ended in live

birth, stillbirth, or neonatal death).

From these three different groups, it is evident that the

study population being evaluated should be clearly specified

because the prognosis for a successful outcome undoubtedly

will be influenced by the group being selected. The current

approach of lumping all three groups together will not allow

the effect of the experimental intervention to be detected

easily. In the Cochrane meta-analysis of the effect of immu-

notherapy in RPL (21), studies that are very heterogenous

are combined in a common meta-analysis including patients

with both primary and secondary RPL. Because there are

strong indications that allogeneic lymphocyte immunization

might only have a beneficial effect in women with primary

RPL, and because intravenous Ig displays its strongest effect

in women with secondary RPL, it is important that meta-

analyses of the efficacy of the respective treatments be

undertaken in the relevant patient subgroups.

Untreated Control Group. In many trials of treatments in

RPL, pregnancy outcome in the treated group is comparedwith the outcome in a group of historical controls. Such

controls might comprise untreated patients from studies un-

dertaken decades ago in another setting. This method is

likely to produce flawed results because the ascertainment

and the pregnancy-monitoring procedures of the controls

might be very different from those of the treated patients.

Another variation of the use of historical controls is the

comparison of pregnancy outcome of the patients subjected

to the new treatment with the outcome in their previous

untreated pregnancies. This method is often used in studies

of surgical procedures to prevent RPL. However, womenwith RPL were sampled because of their previous pregnancy

losses, and some of them might have suffered them merely

by chance. They will still have a very favorable chance of

successful pregnancy without any treatment, owing to a

phenomenon called regression to the mean. In women

included in a trial because of RPL, the accumulated past

pregnancy success rate will often be 10%. Comparing this

low pretreatment success rate with an 85% posttreatment

success rate (e.g., after cerclage) will not require many

patients for obtaining statistical significance (22). However,

by using historical controls, most interventions can be

proved to be highly efficient in the treatment of RPL (23).

This apparent improvement of outcome can partly be attrib-

uted to the effect of regression to the mean.

In conclusion, proper evaluation of interventions in RPL

can only be accomplished in double-blinded, randomized

controlled trials in which the allocation groups are similar

with regard to all important prognostic determinants except

for the intervention that is tested.

Prognostic Determinants: Number of Previous Miscarriages.For many years, the mathematic estimates of miscarriage

rates were used as control rates against which the efficacy of

various therapeutic regimens introduced to prevent miscar-

riage were assessed. The reliability of these rates was chal-

lenged after evidence from a number of clinical studies

suggested that the miscarriage rate after three consecutive

miscarriages was substantially lower than had been predicted

by the earlier models (1). Nevertheless, despite the varied

methods of ascertainment, the results of the studies showed

remarkable consistency in finding an increasing risk of mis-

carriage as the number of previous miscarriages increases.

The effect of prior losses on the subsequent probability oflive birth was confirmed with the data from the placebo arm

of studies in unexplained RPL and provided a quantitative

estimate of the risk (24). It is clear from this evidence that

the number of previous miscarriages is an important covari-

ate, which has to be taken into account when planning

therapeutic trials.

The ideal trial should have stratification for the number of

previous miscarriages, with randomization between control

and experimental treatments within each stratum. To date,

such a study has not been undertaken. It is quite likely that

by stratifying the sample by number of previous miscar-riages that the effect of the experimental intervention will

become more easy to demonstrate in those women with

higher numbers of previous miscarriage than in those with

fewer previous miscarriages because the control event rate is

so much lower in the former group (8, 24).

Prognostic Determinant: Female Age. Miscarriage-pronewomen have more pregnancies and have their pregnancies

occur at a later age than successful reproducers. Because

gravidity is closely linked to female age, it is possible that

the increased risk of miscarriage with gravidity can be as-

cribed, in part, to the effect of maternal age, particularly inview of the fact that chromosomal anomalies are associated

with advancing maternal age. It is well established that the

risk of miscarriage resulting from trisomic conceptuses in-

creases with maternal age, especially after 35 years. Thus,

clinical trials of treatment efficacy must take female age into

consideration during the design of the trial by using strati-

fication for this covariate.

Issues Relating to Treatment Protocols and the Evaluationof Effect. Of crucial importance for the evaluation of theresults of a treatment trial are the timing, doses, and the

numbers of treatments given, possible termination of the trial



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prematurely after interim analyses, the possible inclusion of

several pregnancies from the same patient, and the results of

karyotypic analysis of products of conception.

There is no standardization in many of the treatment

protocols with respect to the onset of treatment. For exam-

ple, intravenous Ig has been administered preconceptionally

in some studies, whereas in other studies treatment is com-

menced only after confirmation of the pregnancy (25).

Sometimes treatment is instituted only after fetal cardiacactivity has been demonstrated, as has been observed in

some of the thrombophilia treatment trials. The likelihood of

a successful outcome without treatment once fetal cardiac

activity has been demonstrated is relatively high and will

result in efficacy studies failing to detect a sizable treatment

effect with the experimental intervention.

Another methodologic concern in efficacy trials is the

practice of limiting the number of cycles of preconceptional

treatment patients might undergo before they are withdrawn

from the study, despite the fact that they have been random-

ized into one of the two comparable groups. The overall

result becomes biased by excluding the patients who have

failed to conceive within a specified (usually short) period of

time after randomization. Women with RPL, compared with

those with sporadic miscarriage, have a longer interpreg-

nancy conception interval (i.e., length of time taken for

conception to occur in women attempting pregnancy after

the miscarriage) (26, 27). The pathologic mechanism for this

observation is not clear. One possible hypothesis is that fear

of miscarriage in a subsequent pregnancy induces significant

stress that might adversely influence the hypothalamus and

result in subtle ovulatory dysfunction (1). Thus, it is clear

that the evaluation of treatment commenced preconception-

ally will require many cycles of observation before preg-

nancy can be achieved. For this reason and for the method-

ologic reasons discussed, women enrolled into such

randomized trials should not be withdrawn just because they

fail to conceive within a specified period.

Interim analyses are sometimes carried out during a pla-

cebo-controlled trial, and the trial is stopped when a signif-

icant result for or against a treatment effect is reached.

However, if premature termination of the study after interim

analyses occurs, there is a great risk that the trial will be

concluded at a point at which the difference between the

allocation groups has reached an extreme fluctuation. Theresult at this stopping point is not likely to be typical for the

result if the study were to be conducted to the end. The trials

by Jablonowska et al. (18) and Ober et al. (28) might be

affected by this problem because the very high success rate

of the placebo group in the former study and the low success

rate of the lymphocyte-immunized group (46%) in the latter

trial might reflect a short extreme fluctuation resulting in a

decision to terminate the study after interim analysis. In

conclusion, interim analyses and premature termination of

trials should be avoided, and such trials should be clearly

identified in any meta-analyses. If interim analyses are

planned, the stopping rules should be very specific and

should only be used when there is clear evidence of benefit

or harm.

In some case series and controlled trials, several pregnan-

cies from the same patients were included. The commonly

used statistical methods, such as the2 test andt-test, require

that all measurements are independent. Pregnancy outcomes

in the same women are not independent variables; therefore,

inclusions of several pregnancies from the same womenrequire suitable sophisticated tests.

The possibility that a miscarriage after treatment is the

result of aneuploidy must be investigated, particularly in

efficacy trials. Without this information it is impossible to

ascertain whether the pregnancy loss is the result of treat-

ment failure or a de novo chromosomal anomaly. The mag-

nitude of the size of the treatment effect will be affected

without correction for the aneuploidy factor. The improve-

ment in ultrasonography technology now provides images

with better resolution, thereby allowing the diagnosis of

pregnancy failure to be made much earlier, a process that isassisted with hormone assays. Thus, it is possible to have

access to viable and noninfected fetal and trophoblast tissue

that can be submitted for karyotypic analyses in a way that

does not jeopardize the cell culture. Furthermore, improved

techniques in cytogenetics have permitted more accurate and

reliable assessments of the products of conception. Given

these improvements in our diagnostic ability, it is even more

important that every effort be made to study the products of

conception in every case of miscarriage in therapeutic trials

so that a more valid evaluation can be made regarding the

efficacy of the experimental treatment.

Intention-to-treat analysis is a necessary first step in the

analysis of randomized controlled trials, but an explanatory

analysis is an important second step. In this step we can

exclude aneuploid pregnancies. Calculations of statistical

power and sample size can incorporate an estimated aneu-

ploidy rate, but this is generally not done. It might be ideal,

but it will increase the sample size.

Summary. The most important methodologic issues consid-ered to affect and potentially flaw studies of RPL are listed

inTable 1.Apart from the obvious study design issues, it is

important to select the sample appropriately by clearly de-

fining the population through strict inclusion criteria. Intreatment trials, stratification for relevant covariates (espe-

cially number of previous miscarriages) must be undertaken

ad hoc. Onset of treatment (preconceptionally or postimplan-

tation) should be supported by a biological rationale. Suffi-

cient time should be provided for the intervention to have

been adequately tested. Postrandomization withdrawals

should be avoided. The outcome should be clearly docu-

mented, and every effort should be made to submit products

of conception for karyotypic analysis. Only through such a

comprehensive approach can the resulting evidence be used

appropriately to direct care of couples with RPL.



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CHROMOSOMAL ABNORMALITIES IN RPL

Only 4.7% of couples with two or more abortions include a

carrier of a balanced reciprocal or Robertsonian structural

abnormality (29), but chromosomal analysis of RPL couples

must still be considered a part of the routine investigation

(Table 2).

It is well documented that fetal de novo chromosomal

abnormalities are a major cause of sporadic first-trimester

spontaneous abortions, and some cases of RPL thus might be

caused by repeated fetal chromosome abnormality. Cytoge-netic evaluations of specimens of sporadic abortions have

revealed an overall incidence of chromosomal abnormalities

of 50%70% (3034). The most common cause of sponta-

neous abortions is de novo numerical abnormalities, in par-

ticular autosomal trisomies for chromosomes 13, 14, 15, 16,

21, and 22, followed by monosomy X (3436). On the other

hand, autosomal monosomies are rarely found in spontane-

ous abortions and considered to be responsible for preclini-

cal abortions (30, 31, 34). This mechanism of natural selection

might also operate during preimplantation embryogenesis, with

a progressive loss of abnormal embryos at specific stages in

early development through developmental arrest and degener-ation of abnormal embryos.

TABLE 1

Methodologic factors to evaluate in studies of recurrent pregnancy loss.

Factor to evaluate Effect on study outcome

Definition of RPL as 2 miscarriages Decreases difference in frequency of factor

studied in CCS or treatment effect in RCT

Ascertainment bias Increases prevalence of factor studied

Selection of controls Increases difference in prevalence of factor

studied in CCS or treatment effect in RCT

Advanced age of patients Increases recurrence of RPL

Uneven monitoring of two cohorts studied Increases difference in prevalence of factor

Historical controls Increases treatment effect in treatment trials

Poor characterization of miscarriage and

subgroups of RPL

Renders comparisons between studies and

meta-analyses difficult

Premature termination after interim analysis Decreases or increases treatment effect in RCT

Inclusion after detection of fetal heart action Decreases treatment effect in RCT

No exclusion of aneuploid abortuses Decreases treatment effect in RCT

Note:RPL recurrent pregnancy loss; CCS casecontrol studies; RCT randomized control trials.

Christiansen. Management of recurrent pregnancy loss. Fertil Steril 2005.

TABLE 2

Potential etiologic factors in the causation of recurrent pregnancy loss.

Factor Association with RPL Causation of RPL

Parental genetics Definite Definite

Uterine abormalities Definite Probable

Uncontrolled thyroid disease Probable Probable

Uncontrolled diabetes Probable ProbablePolycystic ovary syndrome Definite Probable

Antithyroid antibodies Doubtful Doubtful

Antiphospholipid antibodies Definite Probable

Factor V Leiden mutation Definite Probable

Th1 cytokine bias Probable Probable

Increased NK cell cytotoxicity Probable Probable

Maternal HLA alleles Probable Probable

Parental HLA sharing Doubtful Doubtful

Note:RPL recurrent pregnancy loss; Th1 T helper 1; NK natural killer; HLA human leukocyte antigen.




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With IVF and preimplantational genetic diagnosis (PGD),

the in vitro developmental ability of human embryos at these

stages can be observed, enabling us to learn about their

behavior and perhaps about the mechanisms involved in the

genetic causes of RPL (37). A study involving PGD on

embryos from 106 couples with RPL classified women into

two groups according to their age: 37 years or 37 years.

A control group of 28 couples undergoing PGD because of

the risk of sex-linked diseases, and without other infertility

problems, was included to compare clinical results and theincidence of abnormalities for each chromosome with the

RPL group. The fluorescence in situ hybridization and PGD

protocols have been previously described (37, 38).

In the RPL group, all embryos were diagnosed as abnor-

mal in 33 cycles, and no ET was performed, and in the other

95 cycles, at least one embryo was transferred, resulting in

31 pregnancies (32.6%), with an implantation rate of 25.3%

and a miscarriage rate of 12.9%. In the control group, 35

cycles were included and ET was performed in 31. Nine

clinical pregnancies were achieved, and no miscarriages

were observed.

In the RPL group, 532 of 764 embryos were abnormal

(69.6%), whereas in the control group 97 of 215 embryos

(45.1%) were abnormal (P.0001). Results were also com-

pared separately in the two age subgroups and showed an

increased incidence in chromosomally abnormal embryos in

both (more evident, however, in younger patients [P.0001]

than in the aged women [P.046]). All the abortion speci-

mens were cytogenetically analyzed: in three of the cases, a

normal male karyotype was identified, and in one case a

trisomy for chromosome 15 was observed.

These results confirm preliminary findings that coupleswith RPL seem to produce chromosomally abnormal em-

bryos in significantly higher percentages than those not hav-

ing this reproductive problem (38, 39). Moreover, in 25.8%

of the cases, the incidence of chromosomal aberrations af-

fects all the embryos, and the percentage of abnormal em-

bryos is quite similar in subsequent attempts. Thus, the use

of PGD in these couples, after appropriate workup has ruled

out other cases of RPL, might be advisable not only as a

therapeutic but also as a diagnostic tool, as long as an

acceptable implantation rate per embryo replaced is reached,

providing evidence that PGD would not damage the em-

bryos.

An open question is why the frequency of chromosomal

abnormality was not increased with increased age in the RPL

patients. In controls, the frequency of chromosomal abnor-

malities increased, as expected, with increased age. It is

possible that the basic frequency of chromosomal abnormal-

ities was so high in RPL patients that no age differences

could be detected.

It cannot be ruled out that the high frequency of chromo-

somally abnormal embryos in the study might be a result of

the IVF procedure. The gonadotropin-stimulated superovu-

lation might stimulate a cohort of abnormal eggs that would

not be ovulated in a spontaneous cycle. However, this would

be difficult to prove because it will be almost impossible to

get eggs from normal cycles.

It can be claimed that PGD in RPL is not cost effective.

After four previous miscarriages, the spontaneous birth rate

in RPL patients is still 50%; although the selection of em-

bryos by PGD might decrease the miscarriage rate, only 33%

become pregnant after each PGD cycle. This might be ac-ceptable in countries with high birth rates, where the women

are willing to attempt many pregnancies. In countries with

low birth rates, such as Spain, PGD for improving the

prognosis in RPL might be cost effective.

NONCHROMOSOMAL GENETICS OF RPL

Few studies have investigated the genetics of RPL in fami-

lies of RPL couples with normal chromosomes. Three rele-

vant studies have been published, all of which found that the

RPL prevalence in first-degree relatives of women with

unexplained RPL was significantly higher than in controls

(4042). In all the studies, the risk of RPL in first-degree

relatives was approximately six times higher than the risk in

the background population. It can be calculated (43)that the

observed RPL prevalence in first-degree relatives is in clos-

est agreement with a polygenic mode of inheritance with

RPL, occurring when a patient has inherited a sufficient

number of risk factors to exceed a threshold for disease

development (Fig. 1). There is evidence that one of the

factors determining the increased risk of RPL among sisters

of RPL patients is linked to the human leukocyte antigen

(HLA) system (44), but now a series of other relevantcongenital defects in coagulation or immune function have

been discovered that might explain the increased miscarriage

risk among relatives.

UTERINE ANATOMIC ANOMALIES IN RPL

Several uterine factors have often been reported as associ-

ated with RPL, although there is not always sufficient proof

of a causative role.

Congenital Uterine AnomalyOf the various congenital structural uterine anomalies, the

septate uterus is the most common. There is sufficient ob-

servational data to suggest that a septate uterus is associated

with an increased risk of miscarriage due to impairment of

implantation (45). The septum might be removed hystero-

scopically with the use of scissors or electrical or laser

energy. Uncontrolled studies have reported a better preg-

nancy performance after surgery than before surgery in the

same patients, but no prospective trial has been carried out in

which appropriate randomization to surgery and no surgery

was undertaken.



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Ashermans Syndrome/Endometrial Fibrosis

Ashermans syndrome is an acquired condition, which is due

to the presence of posttraumatic intrauterine adhesions partly

or completely obliterating the uterine cavity. Endometrial

responsiveness to the steroid hormones is reduced in areas

affected by intrauterine adhesions or fibrosis. The prognosis

depends on the extent of endometrial damage. Although the

adhesions might be divided hysteroscopically, endometrialfunction might be permanently damaged in the presence of

extensive, dense fibrosis, which is associated with a poor

prognosis.

Uterine Fibroid

There is convincing observational data from several IVF

series (4651) to suggest that reproductive outcome is sig-

nificantly compromised by submucosal fibroids, modestly

compromised by intramural fibroids, and possibly compro-

mised by subserosal fibroids. There is an association be-

tween advanced age and uterine fibroids, thus age might be

a confounding factor when the real impact of fibroids on

subsequent reproductive performance is to be assessed. From

nonrandomized studies, there seems to be some evidence to

suggest that removal of submucosal fibroids reduces the

miscarriage rate and some evidence that removal of intra-

mural fibroids also reduces miscarriage rates (52). For gy-

necologists who are not very experienced in laparoscopic

surgery, it is advised to remove fibroids by laparotomybecause the uterine scar should be carefully and appropri-

ately closed to avoid adhesions and bleeding. However, it is

important to bear in mind that no prospective study has been

carried out in which random allocation to surgical removal

of fibroids or no surgery was done.

SYSTEMIC AND LOCALIZED ENDOCRINEABNORMALITIES IN RPL

Several endocrinologic factors might be associated with

RPL.

FIGURE 1

Figures are illustrating the traditional mono-etiological thinking (pie) and the recommended multifactorial

thinking (column) regarding the pathogenesis of recurrent pregnancy loss. NK-cells natural killer cells;

Th1 cytokine T helper cell type 1 cytokines; MBL mannan-binding lectin; HLA human leukocyte

antigen.




9/19

Thyroid Disease

For some years, hyper- or hypothyroidism has been thought

to be associated with RPL. Current evidence seems to sug-

gest that treated thyroid dysfunction is not associated with

RPL (53). A recent study suggested that 2% of women with

a midtrimester loss were found to be hypothyroid (54).

Because measuring TSH is a sensitive and inexpensive tool

to assess thyroid function, it should be considered in the

evaluation of RPL for women with any signs or symptoms of

thyroid dysfunction.

Diabetes Mellitus

Current evidence suggests that well-controlled diabetes mel-

litus is not associated with RPL (53). In a recent study,

investigators looked at the frequency of insulin resistance in

unselected women who presented to a clinic with a diagnosis

of RPL. Insulin resistance was identified significantly more

frequently in women with RPL when compared with age-,

race-, and body mass indexmatched controls (55).

Hypersecretion of LH

Initial reports suggested that a high level (10 IU/L) of LH

on cycle day 8 was associated with an increased risk of

miscarriage (56). Later studies, however, could not confirm

the impact of high follicular LH on the risk of miscarriage

(57), and the suppression of LH with GnRH analogues did

not affect the miscarriage risk (58).

The prevalence of high LH levels in the follicular phase of

women with recurrent miscarriage, defined as plasma con-

centration 10 IU/L, varies enormously, from 0 to 37%

(59). With the use of improved methodology, including daily

blood samples collected in the mid- to late follicular phase,and with the use of newer, more precise immunometric

assays, hypersecretion of LH has been found to occur in only

8% of women (59). Studies have also suggested that a

too-low LH level in the follicular phase could affect miscar-

riage rates negatively in IVF cycles (60); however, there is

no evidence that this is a cause of RPL.

Polycystic Ovaries

In a recent study (61), the prevalence of polycystic ovaries

(PCO) among women with RPL was reported to be 40.7%

(852 of 2,199), but in another study of 102 women with RPLwho underwent transvaginal ultrasonography, only 8 (7.8%)

had the typical features of PCO (59). In the study by Rai et

al. (61), the live birth rate was similar in women with PCO

(60.9%) and in women with normal ovarian morphology

(58.5%). The above evidence leads to the conclusion that

PCO pathology is not predictive of pregnancy loss among

ovulatory women with RPL who spontaneously conceived.

A study on the impact of laparoscopic ovarian diathermy

on reproductive performance in patients with PCO (62)

suggests some benefit of this treatment in infertility, but

randomized studies are needed in RPL patients.

Metformin treatment of infertile women with polycystic

ovary syndrome (PCOS) decreases insulin resistance and

might improve the implantation rate after IVF (63, 64). It is

still uncertain whether metformin decreases the rate of mis-

carriage in patients with PCOS, because no properly de-

signed placebo-controlled studies concerning this treatment

have been conducted.

High Androgen LevelsTwo recent studies have shown that androgen levels in the

follicular phase are higher in women who have RPL than in

normal fertile controls (65, 66). The observation seems to be

independent of the association between PCOS and RPL.

Further studies are required to examine the relationship

between hyperandrogenism and RPL.

Hyperprolactinemia

It is debatable whether hyperprolactinemia is associated with

RPL. In a recent study (59), 3 of 122 subjects (2.5%) with

RPL had marginally elevated PRL levels, and one of themhad significantly high (1,000 IU/L) PRL. Additionally, in

a subgroup of women with RPL (n 23) who had daily

plasma PRL measured in the midfollicular phase to early

luteal phase, there was no evidence of a preovulatory rise of

PRL. The data suggested that there is no firm evidence of an

association between hyperprolactinemia and RPL.

Luteal Phase Defects

Traditionally, low serum P in the midluteal phase or an

endometrial biopsy out of date according to Noyes criteria

(67) have been taken as evidence for a luteal phase defectthat could cause failed implantation and RPL. However,

previous studies in this area have not dated the blood sam-

ples or biopsies according to the LH peak or ultrasonic

demonstration of ovulation. Recent studies have questioned

the clinical relevance of dating of the endometrium by

Noyes criteria.

Primary Endometrial Defect

In many cases, a luteal phase defect is caused by a primary

defect of P responsiveness of the endometrium or a second-

ary defect caused by, for example, adhesions or fibroids, in

which cases the luteal phase P level is normal. A number of

morphologic and immunohistochemical studies suggest that

a primary endometrial defect might be present in more than

20% of subjects with RPL (68).

AUTOANTIBODIES AND COAGULATION DEFECTS IN RPLAutoantibodies

Numerous autoantibodies have been reported to be associ-

ated with RPL. Most of the literature has dealt with antiphos-

pholipid antibodies (APLs), which are antibodies directed

against negatively charged phospholipid molecules consti-



10/19

tuting parts of the membranes of the cell surfaces and cell

organelles. There is consensus that the APL lupus anticoag-

ulant (LAC) and anticardiolipin antibodies (ACA) should be

looked for in RPL patients because [1] these can be found

with significantly increased prevalence in RPL patients, [2]

the pregnancy prognosis of patients with these antibodies

seems to be poorer than in those without, and [3] the chance

of live birth seems to be improved if treatment with heparin

and low-dose aspirin is given (6971). Unfortunately, there

is no consensus regarding how to define an APL-positiveRPL patient: very different laboratory methods and cut-off

values for positivity are used, and some centers require at

least two positive tests for the diagnosis (72), whereas others

are content with one. A recent comparison of APLs evalu-

ated by 10 different specialty laboratories was an important

step in developing a consensus on APL test results and

interpretation (73).

However, there is considerable disagreement regarding

the mechanisms of action of APLs in women with RPL: are

the antibodies acting on coagulation factors or placental

endothelial cells and thereby promoting thrombosis of pla-cental vessels; are they acting directly on villous or extra-

villous trophoblast cells, inhibiting implantation processes or

cell fusion; or are they only markers of other immunologic

processes harmful to the pregnancy (e.g., a T helper [Th]1

cytokine bias)?

Another important question concerning APLs that remains

unanswered is whether other APLs, such as antiphosphati-

dylserine and antiphosphatidylethanolamine, should be

looked for and whether anticoagulation treatment should be

offered to patients with these antibodies. Results from one

study (74) suggested that APLs other than LAC and ACA

are associated with RPL and display impact on the progno-sis. It also seems that both unfractionated and low-molecu-

lar-weight heparin inhibit binding of ACA and antiphos-

phatidylserine to their respective phospholipid antigens in

vitro (75).

The prevalence of positivity for ACA and lupus anticoag-

ulant varies significantly between studies. This can partly be

explained by different definitions of a positive ACA test, the

timing of the tests, and the screening procedures used. Some

clinics are only investigating IgG ACA and not IgM ACA

because the stability and the prognostic impact of IgG ACA

seems to be higher. However, 20% of APL-positive RPLpatients will only have IgM RPL, and until further studies

have clarified the importance of this antibody isotype, it is

advisable to continue to screen patients for IgM ACA.

Another important factor explaining some of the hetero-

geneity between studies is the fluctuation of ACAs with

timea fluctuation that is particularly large during preg-

nancy. In one study (69), RPL patients were tested 6 weeks

after the last pregnancy loss and again in early pregnancy,

gestational weeks 20 and 30, and after pregnancy. In only

approximately 60% of the women the titers remained con-

stant. There seem to be three subgroups of patients: those

with constantly positive findings, both before and during

pregnancy; those with borderline titers (1020 IgG-phos-

pholipid/IgM-phospholipid [GPL/MPL] units), and those

who are completely negative. There is probably no need to

repeat testing or anticoagulate those who are completely

negative before pregnancy, but in those with borderline titers

before pregnancy new tests should be taken in early preg-

nancy, after which a decision can be made regarding treat-

ment with heparin/aspirin or no treatment.

Screening for the lupus anticoagulant poses a special

problem. In many clinics, a patient with the lupus anticoag-

ulant is a rare finding. This might partly be owing to the use

of activated partial thrombin time (APTT) as a screening test

for the lupus anticoagulant. The use of only standard APTT

as a screening test will miss half of the cases that are positive

for the lupus anticoagulant because it is too insensitive. This

can be overcome by the use of a more sensitive modification

of the APTT test (PTT-LAC) or by the use of two screening

tests: both the dilute Russells viper venom time and the

PTT-LAC.

Several studies have reported that a high frequency of

women with unexplained infertility or secondary infertility

after RPL are positive for APL (76, 77). However, the

chance of achieving pregnancy after a subsequent IVF at-

tempt seems not to be related to the presence of APL (77),

and from the studies that have been undertaken it is not

justified to offer these patients anticoagulation treatment (76,

78).

A last relevant question is the significance of non-APL

autoantibodies, such as antithyroid antibodies and antinu-

clear antibodies, in RPL. Although these antibodies can

probably be found with increased prevalence in RPL pa-

tients, most studies have not been able to document that they

show a negative impact on subsequent pregnancy outcome.

Two prospective trials have failed to show any benefit from

intervention with low-dose prednisone (79)or any difference

in outcome if patients with normal thyroid function and

thyroid antibodies are followed (80). It is advisable that

patients with antithyroid antibodies are monitored for thy-

roid function during pregnancy (e.g., TSH measurements),

but no special intervention should be offered.

Acquired and Congenital ThrombophiliasInvestigators in one study evaluated 80 women with three

consecutive pregnancy losses, fathered by the same partner,

and whose prior evaluation for RPL had been negative

(Kutteh et al., unpublished data). Evaluation for thrombo-

philias included the following: antiphospholipid antibodies

(APA), LAC, protein C activity, protein S activity, anti-

thrombin III activity, factor V Leiden mutation, activated

protein C resistance, prothrombin (factor II) activity, and

fasting plasma homocysteine. Thirty-two of eighty women

(40%) with unexplained RPL had at least one abnormal test

result from the thrombophilic workup, compared with 9



11/19

(11%) in the control group (P.01). The women with un-

explained RPL differed from controls, especially with regard

to hyperhomocystinemia, elevated APA or LAC, and the

presence of the factor V Leiden mutation. These results are

in concordance with another study that found that 66% of

RPL patients had at least one thrombophilic defect compared

with 28% of controls; however, in this study an accumula-

tion of thrombophilic defects characterized RPL patients

because at least two defects were found in 21% of patients

and in 5.5% of controls (81). These data suggest that hyper-

coagulable states might be an important factor in RPL.

A series of 31 relevant studies judged to be of good quality

has now been subjected to meta-analysis, which concluded

that there is an increased prevalence of several acquired and

inherited thrombophilias in populations of women with un-

explained RPL compared with women without a history of

adverse pregnancy outcome (82). Factor V Leiden mutation

was weakly associated with early RPL (odds ratio [OR], 2.0;

95% confidence interval [CI], 1.13.6) but strongly associ-

ated with late RPL (OR, 7.8; 95% CI, 2.821.7). The pro-thrombin G20210A mutation and protein S deficiency

seemed also to be associated with RPL, but the ORs had very

wide confidence limits owing to small studies, and more

studies are needed to document their association with RPL.

The meta-analysis could not document any association be-

tween methylenetetrahydrofolate mutation (a risk factor for

hyperhomocystinemia), protein C, and antithrombin defi-

ciencies.

A second meta-analysis evaluated the association of factor

V Leiden and prothrombin gene mutation G20210A in

women with RPL (83). The combined ORs for the associa-tion between RPL and factor V Leiden and between RPL and

G20210A were 2.0 (95% CI, 1.52.7; P.00l) and 2.0 (95%

CI, 1.04.0; P.03), respectively.

An unsolved question is whether RPL patients with con-

genital thrombophilic factors, such as the factor V Leiden

mutation should be treated with heparin and aspirin. There

are no placebo-controlled trials documenting the efficacy of

this treatment in these women. So far, there is only evidence

for the use of heparin and aspirin in APL-positive RPL

patients.

IMMUNE CELLS AND CYTOKINES IN RPL

One of the causes of pregnancy loss is thought to be the

immunologic rejection of the fetus due to a breakdown in the

mechanisms that normally prevent the maternal immune

system from becoming activated by the paternal antigens

expressed on the developing fetus. Recent studies have in-

vestigated the role of specific immune cells and molecules in

the cause of repeated miscarriage, by comparing their ex-

pression in peripheral blood, endometrium, and decidua of

women with RPL and control women.

Natural Killer Cells in the Blood and Endometrium

Several studies have shown that the immune cell distribution

within the endometrium and decidua has a profile different

from that in peripheral blood, in the former tissue consisting

mainly of CD56CD16-CD3- natural killer (NK) cells (84).

Endometrial expression of these cells increases during the

secretory phase of the menstrual cycle and in early preg-

nancy. Macrophages and T cells make up the remaining

leucocyte population, but their numbers show little variation

in the menstrual cycle. Studies comparing the expression of

these various cell populations in control and RPL women

have shown very varied and inconsistent results. Numbers of

CD56 NK cells can be either increased or decreased in

women with RPL, depending on the compartment that is

studied (85, 86). Peripheral blood and peri-implantation en-

dometrial CD56 NK cells in early pregnancy are de-

creased, whereas numbers in the nonpregnant endometrium

are increased. A murine strain without NK cells can only

reproduce if NK cells are transferred to the mice (87). This

observation challenges the notion that NK cells are harmful

for pregnancy.

It is indeed unclear whether NK cells are good or bad,

particularly in humans. In mice, NK cells were initially

regarded as a potential threat to the fetus, because studies

showed increased numbers in abortion-prone mice. How-

ever, more recent studies have shown that the placenta in

NK-deficient mice is hypertrophic and is associated with

fetal death, suggesting that NK cells play a positive role. The

situation in humans is even less clear. Furthermore, because

many T lymphocytes also have NK cell markers, evaluating

the importance of NK cells becomes more complicated.

There seem to be no differences in total T cell numbers,

but studies have suggested that expression of peri-implanta-

tion endometrial or peripheral blood T cell subsets, such as

-T cells and the ratio of CD4 to CD8 T cells might be

altered in women with RPL (88, 89).

Cytokines

Cytokines are immune molecules that control cells in both

the immune and nonimmune systems. They are divided into

groups according to their function. Some cytokines produced

by Th1 lymphocytes include interleukin (IL)2, interferon

(IFN)-, and tumor necrosis factor (TNF)- and stimulate

cell-mediated immune responses. Other cytokines producedby T helper 2 (Th2) lymphocytes include IL4 and IL10 and

stimulate antibody-mediated responses. Experiments in ani-

mals suggest that Th1 cytokines are detrimental to preg-

nancy and cause pregnancy loss, whereas Th2 cytokines are

beneficial and prevent pregnancy loss (90, 91). There is

some evidence for an abnormal balance of Th1 and Th2

cytokines in women with RPL (9294). Peripheral blood

mononuclear cells taken either before pregnancy or during

the first trimester of pregnancy from women with RPL

produce IL2 and IFN- (Th1 cytokines) when stimulated

with either trophoblast antigen or the mitogen phorbol my-



12/19

ristate acetate (PMA), whereas those from control women

produce IL4 and IL10 (Th2 cytokines) (92). In addition, T

cell clones produced from CD4 cells isolated from decidua

of RPL women produced less IL4 and IL10 than similarly

prepared cells from control women (93). Another group of

cytokines are proinflammatory cytokines, such as IL1, IL6,

and leukemia inhibitory factor. Studies have shown that

expression of these in peri-implantation endometrium is de-

creased in women with RPL (95, 96). Thus, there is some

evidence for differences in the expression of specific im-mune cells and molecules in the peripheral blood, endome-

trium, and decidua of women with RPL.

Most studies have not been able to find that genetic

polymorphisms resulting in low cytokine production are

associated with RPL. There could still be a problem of

producing adequate levels of the right cytokines in these

patients, but the failure could be at the level of transcription.

The hypothesis of Th1 and Th2 cytokine response as

being important for pregnancy is probably too simple and

rigid. It is important to look at the totality of cytokine

function at the place where things are happening in theuterus. Both Th1 and Th2 cells can secrete what is normally

considered a Th2 cytokine, IL10, in humans but not in mice.

Indeed, human and murine reproductive and immune sys-

tems are very different, and extrapolation of findings from

the murine to the human setting should be done very cau-

tiously. Furthermore, there is much evidence that the neuro-

logic, endocrine, and immune systems interact. It is very

likely that psychological stress in pregnant women with RPL

results in increased levels of stress hormones that can pro-

duce miscarriage through immunologic mechanisms. Stress-

induced fetal loss exists in mice. There is also evidence that

P can interact with proinflammatory cytokines.

To advance this area of research further and to limit the

variation seen in the results of different studies, care should

be taken both with respect to the compartment (peripheral

blood, endometrium, or decidua) in which the cells and

molecules are measured and the timing of the sampling, both

with respect to the menstrual cycle and pregnancy and

whether it is at the time of or just after miscarriage.

THE ROLE OF HLA IN RPL

Prospective studies in inbred populations clearly show an

influence of HLA genes or closely linked non-HLA loci onreproductive processes. Prospective, population-based stud-

ies in the Hutterites, a population isolate of European origin

that has settled in the United States, showed significantly

increased fetal loss rates in couples who shared alleles for

HLA-B or HLA-C. The most significant effects were ob-

served when partners were identical for a complete 16-locus

haplotype, which covered the HLA class I, class II region

and the HLA class III genes (97). However, results obtained

in this population isolate cannot be generalized to outbred

populations in an attempt to understand the pathophysiology

of RPL.

The contribution of HLA genes to the etiology of this

pregnancy complication is still under discussion. Immuno-

logically, the fetus is a semi-allograft that has inherited one

set of maternal and one set of foreign paternal HLA

antigens. It was initially suggested that maternal recognition

of paternally derived HLA is required or might at least be

beneficial in normal pregnancy (98). As a result, early stud-

ies on HLA in RPL were based on the hypothesis that

increased HLA similarity between partners would lead to

inadequate maternal protective immune responses and tofetal loss. Although a considerable number of studies of

HLA-sharing in couples with RPL have been performed,

convincing evidence for this hypothesis is still lacking.

Additionally, recent research on the expression of HLA

genes in early embryonic and trophoblast tissues revealed

that, with the exception of HLA-C, classic HLA class I and

II antigens are not expressed on human preimplantation

embryos or at the maternalfetal interface. Thus, maternal

immune recognition of classic HLA antigens expressed by

the fetus is unlikely to be the key mechanism that leads to

maternalfetal tolerance in pregnancy.Alternatively, the existence of maternal high-risk alleles

for RPL has been proposed under the assumption of allele-

specific differences in immune responsiveness or genetic

linkage of high-risk HLA alleles to non-HLA loci, which

predispose to hypersecretion of certain cytokines (e.g., TNF-

). A meta-analysis of 18 studies on HLA-DR1 and -DR3

frequencies in women with RPL documented a slightly in-

creased risk for RPL in women carrying the HLA-DR1 allele

(OR, 1.29; 95% CI, 1.051.58) (99), and a large case

control study found that HLA-DR3 was associated with RPL

in Danish women (OR, 1.40; 95% CI, 1.11.9) (7). Thus, the

maternal genetic constitution with regard to the HLA-DR1and -DR3 alleles or closely linked non-HLA genes might be

one of the contributing factors in RPL.

Considerable progress has been made in the elucidation of

the immunologic composition of the fetalmaternal inter-

face. After the nonclassic HLA-G had been identified as the

dominant HLA antigen on trophoblast cells (100), coexpres-

sion of HLA-C and HLA-E, albeit at lower concentrations,

was demonstrated more recently (101). Although the exact

functions of these molecules in the placenta have not been

fully elucidated, they seem to participate in an intrinsic

network that guarantees the survival of the fetal allograft.However, studies on polymorphisms of HLA-C and -E or

sharing of HLA-C or -E alleles in patients with RPL could

not provide evidence for a key role of those antigens in the

pathogenesis of RPL (102106).

Human leukocyte antigen G is the dominant HLA antigen

at the maternalfetal interface, and different HLA-G alleles,

including a functional null-allele, G*0105N, has been iden-

tified (107). Clinical data on HLA-G*0105N homozygotes

provided the important information that the major mem-

brane-bound isoform of HLA-G protein, HLA-G1, is not

essential for fetal survival (108, 109). However, the serum



13/19

level of soluble HLA-G seems to be a predictor of pregnancy

success; decreased HLA-G expression in trophoblasts has

been described in spontaneous abortions (110112). Further-

more, embryos expressing HLA-G seem to have a better

chance of implanting (113, 114). Those data provide some

evidence that HLA-G or some of its soluble isoforms, al-

though not essential for fetal survival, might still influence

pregnancy development.

Several studies have investigated a potential role forHLA-G in the pathogenesis of RPL. Recent studies based on

molecular determination of HLA-G alleles and HLA-G nu-

cleotide polymorphisms suggest that women with RPL who

carry specific HLA-G alleles might have lower chances for a

successful pregnancy (106, 115), but the results were not

confirmed in a third study (116), and the pathophysiologic

basis of those allele associations has yet to be clarified.

However, HLA-G polymorphisms are associated with dif-

ferences in the levels of expression of HLA-G messenger

RNA and protein and in the pattern of alternatively spliced

HLA-G messenger RNA isoforms, which might be involved

in the observed link between detection of HLA-G and preg-nancy success, as described above (117, 118).

In summary, although HLA-mediated effects on reproduc-

tion were clearly shown in prospective studies in inbred

populations (97), the involvement of the human HLA com-

plex in the pathophysiology of RPL has not yet been clearly

defined. In addition to maternalfetal immunologic interac-

tions, which involve classic and nonclassic HLA loci, effects

of HLA-linked non-HLA genes on fetal development might

play an important role. Future research will have to delineate

which of the many hypotheses of HLA-mediated effects on

pregnancy outcome are valid for this special group of pa-

tients.

INDUCTION OF IMMUNOLOGIC TOLERANCE AS ANAPPROACH FOR TREATING RPL

When the immune system encounters an antigen, such as a

pathogen, vaccine, or allogeneic cells, the result is either

sensitization or tolerance (119). During pregnancy, the

mother encounters paternal antigens borne by the semi-

allogeneic fetus, and the potential arises for a damaging

immune response being mounted against the fetoplacental

unit. On the basis of hypotheses that RPL in humans could

be caused by an immunologic attack, treatments have beendeveloped aimed at resetting the immune system so that

the mother becomes tolerant of her fetus. The first substantial

trial with an immunologic approach was published by Mow-

bray and colleagues in 1985 (120). In that trial, immuniza-

tion with paternal peripheral blood mononuclear cells

(PBMC) given intravenously, intradermally, and subcutane-

ously, led to successful pregnancy in 71% of patients (n

35) given paternal cells, compared with 47% of patients (n

30) given their own (i.e., maternal) cells. The successful

outcomes reported by this group led to considerable interest

in the idea that RPL can be treated by prior immunization of

the mother with paternal PBMC. Immunologic changes that

have been observed after such procedures include a decrease

in the mixed lymphocyte reaction, decreased soluble IL2

receptor levels, decreased NK cell activity, an increase in

blocking factors, and a decreased Th1/Th2 ratio (121

124).

A number of clinical trials have now been carried out,

with some studies showing a beneficial effect and others no

beneficial effect; in some instances such procedures seem tohave worsened the prognosis. Unfortunately, heterogeneity

in cell numbers, number of injections, type of placebo used,

and patients reproductive histories make comparisons

among the trials somewhat difficult. A recent review for the

Cochrane library of 11 trials of paternal PBMC immuniza-

tion found that, overall, paternal PBMC treatment led to a

live birth rate of 62.2%, compared with 60.0% in controls

(21). This analysis concluded that paternal cell immuniza-

tion, along with other immunologic treatments, such as third-

party donor leukocytes, trophoblast membranes, and intra-

venous Ig, provide no significant beneficial effect over

placebo in preventing further miscarriages. However, theresults of another meta-analysis suggest that such treatment

has value in a subgroup of women with primary RPL who

have suffered a large number of miscarriages (125).

It has been argued that the current immunologic therapies

for the treatment of RPL do not have a sound scientific basis.

Are there other immunologically based options that could be

explored? Despite much research, the key mechanisms al-

lowing a mother to tolerate her semi-allogeneic fetus re-

main to be established (126, 127). Recently, evidence for a

role of CD4 CD25 T regulatory cells has been put

forward (128). With respect to immune responses in general,

although some of the criteria that determine whether expo-

sure to an antigen leads to sensitization or to immunologic

tolerance have been defined, the details again remain

sketchy. Polymeric antigen, given at a moderate dose either

SC or IM will usually lead to sensitization in the presence of

costimulatory signals, such as those provided by adjuvants.

On the other hand, monomeric antigens or antigens given at

very low or very high doses, particularly if given IV, muco-

sally, or intrathymically will, in the absence of additional

costimulatory signals, preferentially lead to tolerance induc-

tion. Indeed, an encounter with antigen in the absence of

costimulatory signals, such as cytokines and the cell-surface

B7 molecules, leads to either apoptosis or anergy (functional

inactivation) of the relevant lymphocytes. The encounter of

T lymphocytes with allogeneic HLA in the absence of co-

stimulation has also been shown to result in tolerance (129).

Several strategies aimed at preventing rejection of allo-

grafts are being developed by transplantation biologists

(130). Here there are some parallels but also some funda-

mental differences from the situation regarding a semi-allo-

geneic fetus. Nonetheless, if the immune system has the

potential to destroy the fetus, any way of preventing immu-

nologic destruction of a fully allogeneic tissue or organ graft



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might provide some signposts for developing immunologic

treatments of RPL. Current experimental approaches that are

being investigated in transplantation biology involve expos-

ing the recipient immune system to donor antigen in the

presence of T cell modulators, such as monoclonal antibod-

ies, directed toward important T cell surface molecules,

including CD3, CD4, or CD154 (CD40L). Another approach

is to create mixed allogeneic chimeras by infusion of allo-

geneic bone marrow in combination with T cell modulators.

On the basis of such strategies, what potential therapeutic

options might open up for the treatment of RPL? Paternal

cells could be administered in the presence of immunomodu-

lators, such as IL10, or soluble versions of cell-surface

molecules, such as CD178 (Fas-ligand). Paternal bone mar-

row plus T cellmodulating agents could be used to establish

chimerism. However, such therapies might carry a substan-

tial risk to the recipient, and these types of approaches are

unlikely to be investigated in RPL until much more infor-

mation is available from transplantation studies.

In conclusion, although a substantial review of the trials of

paternal lymphocyte immunization has not shown an overallbenefit (21), there is some evidence that allogenic leukocyte

immunotherapy with freshly prepared cells is beneficial for a

subset of women with primary RPL with no evidence of

antipaternal cytotoxic immunity and in whom there is no

autoimmune disease (125, 131). The current incomplete un-

derstanding of the immune mechanisms involved in the

maintenance of pregnancy makes it hard to devise rational

novel immunotherapies. Potential future directions can be

gleaned from the experience of transplantation biologists and

the experimental therapies being developed in that field. The

problem might not, in fact, be so great in the case of RPL.

After all, an allogenic kidney graft should be for life,

whereas a pregnancy is for only 9 months. Thus, for RPL, it

is not essential to permanently switch off any allo-reactive

response but rather to curtail the response for a relatively

short period.

LIFESTYLE FACTORS AND RPL

Lifestyle factors rarely, if ever, cause RPL; however, epide-

miologic studies have given evidence that a series of lifestyle

factors can increase the risk of miscarriage. There is good

evidence that obesity (132, 133), daily caffeine intake 300mg (134137), alcohol consumption (136, 138), and use of

nonsteroidal anti-inflammatory drugs (139, 140) increase the

risk of miscarriage significantly. It is more controversial

whether smoking increases the risk of miscarriage (136, 138,

141, 142). Social class and occupation also impact the rate of

miscarriage, with the greatest risk among women exposed to

high physical or psychic stress during work (143145). Sev-

eral studies now also indicate that a previous infertility

diagnosis or infertility treatment might increase the risk of

miscarriage (146, 147). It is important to be aware of these

risk factors because, in an optimal casecontrol or random-

ized controlled trial, an equal distribution of the factors

between the groups should be documented.

DISCUSSION

Many of these controversies in RPL research are due to the

apparent lack of appreciation of the many methodologic

pitfalls highlighted in the first part of this article. It is our

hope that the design of future RPL studies will improve so as

not to fall into the methodologic pitfalls inherent in this areaof research. When more properly designed and conducted

studies are published, we believe that consensus about the

investigations and treatments needed for RPL patients can be

reached.

There is no doubt that research in RPL during recent years

has identified a series of new factors that exhibit an associ-

ation with RPL in casecontrol studies and that also seem to

affect the pregnancy prognosis and thus might be character-

ized as causative factors (Table 2). So many risk factors have

now been identified that it is very common to find more than

one of them in the same patient. Both thrombophilic riskfactors (81)and immunogenetic risk factors, such as specific

HLA alleles and genes for low mannan-binding lectin serum

levels (Christiansen et al., unpublished data), seem to aggre-

gate significantly more frequently than expected in RPL

patients, indicating that they might exhibit an additive or

multiplicative effect on RPL risk. So far, it has been com-

mon to divide the causes of RPL into single sufficient factors

like slices of a pieuterine malformations 10%, endocrine

factors 10%, antiphospholipid antibodies 15%, and so on

which together with the unexplained group make up 100%

(Fig. 1). This model is probably not adequate, owing to the

frequent finding of several risk factors in the same patientsand the general belief that disorders with a high population

prevalence (such as RPL) almost always have a multifacto-

rial background. In internal medicine and other disciplines,

the development of many common diseases (e.g., arterial

hypertension, diabetes mellitus, schizophrenia) are thought

to be determined by a threshold model. A number of risk

factors are in themselves not sufficient to cause disease, but

when several intrinsic and extrinsic factors come together in

the same individual, the risk exceeds a threshold level, and

disease develops. Previous family studies (41, 148) found

that the RPL prevalence in siblings of RPL probands indeed

was in accordance with a multifactorial threshold model forinheritance (43). We therefore encourage scientists and cli-

nicians working in the area of RPL to think within the

threshold rather than the pie model.

The clinical implication is that, in principle, an RPL

patient should be screened for all potential risk factors, and

the investigation should not for economic or other reasons

stop as soon as the first risk factor has been identified. A

problem is that there is no consensus as to the importance of

all risk factors, and there is very little agreement regarding

the level of positivity of the tests. The threshold of detecting

abnormality varies greatly from study to study. This holds



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true for both laboratory tests, such as anticardiolipin anti-

bodies (149), and uterine abnormalities. Indeed, only studies

of the impact of various potential risk factors on the outcome

of future, untreated pregnancies in RPL patients can estab-

lish what degree of abnormality of a factor should be con-

sidered as important for RPL.

It is recommended that examinations in RPL should as a

minimum be carried out according to Table 3 (documenta-

tion A) and, in addition, factors with association and prog-nostic impact documented in studies of adequate size and

quality could be investigated (Table 3,documentation B). A

specific prognosis for the patient can only be given if all risk

factors are investigated. When clinicians encounter a patient

with RPL and several risk factors, it is important to inform

her about all the risk factors and then discuss with her

whether an attempt should be made to treat all risk factors at

the same time or only one of them before or during the next

pregnancy attempt. In many cases, the patient will wish to

have all factors corrected or treated before attempting a

subsequent pregnancy.

As mentioned previously, a number of lifestyle factors,including obesity, occupation, history of alcohol use, and

caffeine consumption, are important for the risk of miscar-

riage. Recurrent pregnancy loss is a complex disorder for

which lifestyle factors are expected to modify the effect of

the non-lifestyle (intrinsic) factors previously discussed. The

prevalence of the most important lifestyle factors should be

reported in publications to establish whether groups studied

for the occurrence of non-lifestyle risk factors or pregnancy

outcome are comparable. Some RPL clinics are already

using a structured information sheet for each patient, which

includes a series of lifestyle factors. It is recommended that

investigators doing research in RPL should use a standard-ized sheet for recovering lifestyle information from patients.

It might be a task for international societies in the field of

fertility and reproduction to design such a standardized

sheet, which can be referred to in publications. However, it

is important to bear in mind that lifestyle factors such as

smoking and alcohol use are very difficult to evaluate from

a sheet. Patients might not provide the correct information

about these matters in a fertility clinic. In a recent study, no

correlation between the patients initial history regarding

tobacco use and subsequent IVF outcome was found; how-

ever, when cotinine levels (a serum marker for tobacco use)

were measured in the blood from Day 3 samples, an excel-

lent correlation between high cotinine concentration and badembryo quality was found in the same patients (150).

A major problem in RPL research is the scarcity of high-

quality randomized controlled trials of treatments. Some

useful trials testing hormonal, anticoagulation, and immuno-

logic therapies have been conducted. However, except for

cervical cerclage, almost none of the surgical interven-

tionsremoval of uterine fibromas and septae, ovarian dia-

thermia, and abdominal cerclagehave been evaluated in

randomized trials. Unfortunately, surgeons are not disposed

to allocate patients to a non-treatment group. Many surgeons

are convinced that there is a narrow window of opportunityto do a randomized study of surgical interventionsbeyond

a certain stage it would be too late to start because the

treatment becomes too established and the patients expect it

to be offered.

Fortunately, there are examples of large randomized trials

of surgery to improve fertility. In Scandinavia, a multicenter

trial was organized to evaluate the effect on implantation rate

and miscarriage rate of removal of hydrosalpinges in IVF

patients (151). Two hundred twenty patients were random-

ized to surgical treatment or no treatment. A similar trial of

removal of fibroids in women with RPL could be done.

However, to facilitate surgical trials we need a change in thewhole concept of being a good scientist. By participating in

a multicenter randomized trial, an investigator will be one of

perhaps 100 collaborators, but only a minority of these will

TABLE 3

Recommended investigations of recurrent pregnancy loss pat

investigations and Treatments of Recurrent Pregnancy Loss

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