General search strategy

A literature search was performed in January 2012 for all papers available at that time in EMBASE and MEDLINE looking specifically for studies of diagnostic tests, systematic reviews, and randomized controlled trials of therapy for RPL. The following search terms were used: RPL, recurrent miscarriage, antiphospholipid antibodies, APS, RPL prognosis, Genetics of miscarriage. Reports were limited to clinical human data including guidelines. All articles considered were investigator initiated trials and published in the scientific literature. In addition, the Cochrane library was searched for systematic reviews of treatment strategies in RPL. If a systematic review was identified, recent updates were sought in the Cochrane Library, MEDLINE, and EMBASE in order to identify randomized controlled trials that became available after publication of the systematic review. However, as positive results have a better chance of being published the selection of studies used for assessment may be biased.

Critical appraisal of the literature

1. In patients with RPLs, what are diagnostic tests which should be performed, and the various risk factors which should be assessed in order to establish a diagnosis of cause?

Search Strategy

• MEDLINE and EMBASE (risk factors): RPL risk factors.
  
• Karyotype and RPL, APS, Thrombophilia and RPL, hormones and RPL, uterine anomalies, natural killer (NK) cells and RPL, infections, and RPL.
  
• AND (risk factors OR risk factors.mp) AND clinical trial AND case – control studies AND cohort studies.

There are various guidelines available with investigation protocols. These include the Royal College of Obstetricians [5], the American College of Obstetricians and Gynecologists (ACOG) [6], the European Society of Human Reproduction and Embryology (ESHRE), [7], and numerous others. However, to date there is no consensus on the optimal evaluation and management strategy. The above protocols differ as to the criteria for investigation. The ACOG protocol recommends investigation after two or more pregnancy losses, whereas the Royal College of Obstetricians and Gynaecologists (RCOG) and ESHRE protocols recommend assessment after three or more losses. The author tends to agree with the conclusions laid out by Farquharson et al. [8], that RPL needs to be much better defined before any relevant investigation or treatment protocols can be determined.

The RCOG protocol [5], was last updated in 2011. Recommendations are made for and against various factors causing miscarriage and methods of treatment are graded according to the level of evidence available. Areas lacking evidence are called “Good practice points.” The guideline recommends parental karyotyping, fetal karyotyping, ultrasound or hydrosonography for uterine anomalies, APS testing, and interpretation according to the “Sapporo” criteria [9]. The guideline claims that there is insufficient evidence to assess progesterone or hCG supplementation, bacterial vaginosis, factor V Leiden (FVL) or the other hereditary thrombophilias. Assessment of Thyroid function, the glucose challenge test, antithyroid antibodies, alloimmune testing and immunotherapy, and assessment of TORCH and other infective agents are not recommended. The guideline states that a significant proportion of cases of RPL remain unexplained, despite detailed investigation, and that the prognosis for a successful future pregnancy with supportive care alone is in the region of 75%. However, the guideline takes no account of specific types of pregnancy loss, and does not distinguish between different types of patient. There are no suggestions regarding patients who subsequently miscarry despite the reassurance of a 75% prognosis for a live birth.

The ACOG guideline [6] has not been revised since 2001, and is now considered out of date. However, the guideline was less dogmatic than the RCOG guideline. Two pregnancy losses are recognized as warranting investigation. The ACOG guideline does not base its recommendations on a strictly evidence based approach, and states clearly that it should not be construed as dictating an exclusive course of treatment or procedure. The guideline states that new and controversial etiologies may be investigated or treated, if they have been discussed between physician and patient. The guideline also states that variations in practice may be justified according to the needs of the individual patient, resources, and limitations in the institution or type of practice. As the RCOG guideline, the ACOG guideline recommends parental karyotyping, and suggests that the couple should be offered prenatal diagnosis if one parent has a chromosomal aberration. The guideline abstains from giving an opinion on karyotyping of the abortus, and reserves judgment on assessment of the uterine cavity. The guideline claims that assessment of the uterine cavity is based on consensus alone, without good evidence. As in the RCOG guideline, there is said to be insufficient evidence to assess luteal phase defect, progesterone or hCG supplements. The ACOG does not recommend assessment of antithyroid antibodies, infections such as Chlamydia, mycoplasma or bacterial vaginosis. Alloimmune testing, paternal leucocyte immunization and IVIg are also not recommended.

The ESHRE guideline [7] restricts the definition of RPL to three or more consecutive miscarriages. It does take account of different types of patient as the introduction states, “The number of previous miscarriages and maternal age are the most important covariates and they have to be taken into account when planning therapeutic trials. The ideal trial should have stratification for the number of previous miscarriages and maternal age, with randomization between control and experimental treatments within each stratum”. The protocol discusses investigations of cause and treatment interventions separately, and unlike the RCOG or ACOG guidelines does not quote the level of evidence for its recommendations. The protocol does recommend testing blood sugar levels and thyroid function tests, antiphospholipid antibodies (LA and aCL), parental karyotyping and assessment of the uterine cavity by pelvic ultrasound or hysterosalpingography (HSG). Hysteroscopy and laparoscopy are reserved as “advanced investigations” but the protocol does not define which patients warrant “advanced investigations.” There is a category of investigations, known as investigations which should be used in the framework of a clinical trial. These include: fetal karyotyping, testing of NK cells, luteal phase endometrial biopsy, and homocysteine levels. Treatment is classified separately from investigation in this protocol. Both tender loving care and health advice such as diet, abstention, or reduction of coffee intake smoking and alcohol are described as established treatments. However, no evidence, results or references are quoted to justify calling these treatment modalities established treatment.

The author uses an approach which differentiates between patients with a good, medium, or poor prognosis. This approach has been fully described elsewhere [10].

Specific risk factors

An abnormal fetal karyotype is the only definitive cause of miscarriage. Five series have assessed the embryonic karyotype in RPL [11–15]. The mean number of miscarriages was 4.12 ± 0.48. The incidence of chromosomal aberrations varied between 25 and 57%, (mean 41.6% ± 13.9). Ogasawara et al. [12] have also shown that the incidence of chromosomal aberrations decreases as the number of miscarriages increases. Embryonic chromosomal aberrations can be found in the presence of other causes of RPL. A 30% incidence has been reported in two small series of patients with APS [12, 16]. In the present patient, the fifth pregnancy was triploid, and therefore not related to the other pregnancies. The author has reported embryonic chromosomal aberrations in four patients with hereditary thrombophilias [17].

Karyotyping of the abortus allows the patient to be given prognostic information regarding subsequent pregnancy outcomes. Two studies [12, 13] have examined the outcome of the subsequent pregnancy according to the karyotype of the miscarriage. In the series of Ogasawara et al. [12], there was a statistically significant trend for a patient with an aneuploidic abortion to have a better prognosis. The same trend was apparent in Carp et al.’s [13] series. However, repeat aneuploidy may occur and has been assessed in an observational study of the subsequent miscarriage by Sullivan et al. [15]. Of 30 patients with an aneuploid abortion, only three (10%) had a subsequent aneuploid abortion. In the author’s series (unpublished), 43 abortuses were aneuploid, and a subsequent abortion was karyotyped. Only 8 of the 43 abortuses were aneuploid (19%). Hence, approximately 15% of aneuploid abortions may be followed by a subsequent aneuploid abortion. Therefore, 85% of patients with an aneuploid abortion can be assured that the prognosis is good, and that the aneuploid abortion may be a chance occurrence.

Parental karyotyping is usually investigated in the interval between pregnancies. In approximately 3–10% of couples with recurrent miscarriage, one of parents carries a balanced structural chromosomal rearrangement [18–20], most commonly a balanced reciprocal or Robertsonian translocation. Although the risk of miscarriage is often said to be greater with parental chromosomal; rearrangements, four papers which have examined the subsequent live birth rate in RPL and parental chromosomal rearrangements [18, 19, 21, 22], have reported a live birth rate of 53.6% for patients with a mean of 4.19 previous miscarriages. This is the expected rate for patients with 4.19 abortions. Patients are often advised that the presence of a parental karyotypic aberration diagnoses the cause of the miscarriage, as the aberration may be carried to the embryo in an unbalanced form. However, Carp et al. [23], have examined the karyotype of abortuses from parents with karyotypic aberrations. Thirty‐nine abortuses from recurrently miscarrying couples with parental karyotypic aberrations were karyotyped. Of the 39, 17 (26%) were euploid. Another 10 (26%) had the same balanced translocation as the parent. Hence, 69% were chromosomally normal. Only five (13%) abortuses had unbalanced translocations. Seven (18%) of subsequent miscarriages were numerical aberrations unrelated to the parental chromosomal disorder (five trisomies and two embryos with monosomy X). Hence, parental karyotyping is of limited value.

APS. All the guidelines above recommend testing for anticardiphospholipid antibodies (aPL), aCL, and LA. In order to be meaningful, there should be two positive values 12 weeks apart. Most pregnancy losses in APS are in the later stages of pregnancy. Rai et al. [24] found that fetal heart activity was previously present in 86% of recurrently miscarrying women with APS, but in only 43% of recurrently miscarrying women without APS. Lockshin [25] has reported that typically pregnancies start normally, and a fetal heart is detected early in the first trimester. IUGR or second or third trimester fetal death ensues. The author [26] has found an increased prevalence of second trimester miscarriages in APS compared to women with unexplained RPLs. In the above patient aPL were assessed due to three fetal deaths in the second trimester.

Hereditary thrombophilias are genetic tendencies to thrombosis. They have been reported to predispose to thrombosis in decidual vessels, leading to fetal anoxia and possibly pregnancy loss. The hereditary thrombophilias include: antithrombin deficiency, protein C deficiency, protein S deficiency, activated protein C resistance and FVL, homozygosity for the methylenetetrahydrofolate reductase mutation (MTHFR, C677T), and the prothrombin gene (FII) mutation G20210. A meta‐analysis [3] of 68 retrospective studies has reported a strong association between second trimester miscarriage and hereditary thrombophilias: FVL, FII gene mutation, and protein S deficiency. Hereditary thrombophilias have also been reported to be associated with an increased risk of early fetal loss (less than 25 weeks) in women with protein C, protein S, or antithrombin deficiencies [27] and in FVL [28]. There are five studies examining the incidence of pregnancy complications in the presence of thrombophilias. Neither Ogasawara [29], nor Carp et al. [30] found an increased subsequent miscarriage for patients. However, both Jivraj et al. [31] and Lund et al. [32] found a lower live birth rate in women with FVL and the prothrombin gene (G20210A) mutation in Lund et al.’s [32] series.

Hormone testing has not been shown to be valuable in the interval between pregnancies in RPL. There is no question that adequate hormone support is essential in early pregnancy. Progesterone enhances implantation, affects cytokine balance, inhibits NK cell activity at the feto‐maternal interface, inhibits the release of arachidonic acid, prevents myometrial contractility and prevents cervical dilatation. Lutectomy prior to seven weeks causes miscarriage, and mifepristone blocks the progesterone receptor, leading to fetal death and placental separation. However, assessment of mid luteal progesterone levels have been unreliable as predictors of the progesterone status in pregnancy, as progesterone secretion is pulsatile. Blood may be drawn at a pulse peak or nadir. These may vary 10‐fold. There is also a lack of correlation between plasma progesterone levels and endometrial histology.

Polycystic ovary syndrome (PCOS) has been linked to an increased risk of miscarriage, but elevated serum luteinizing hormone levels or testosterone levels do not predict an increased risk of future pregnancy loss. An elevated free androgen index may be a prognostic factor for a subsequent miscarriage in women with recurrent miscarriage [33].

A recent systematic review and meta‐analysis [34] reported a strong association between (sub) clinical hypothyroidism and recurrent miscarriage (OR 2.3, 95% confidence interval (CI) 1.5–3.5). There may therefore be value in assessing thyroid function. Anti‐thyroid antibodies have been linked to recurrent miscarriage. However, in the authors series [35] there was no increased prevalence of antithyroid antibodies in women with RPL. One prospective study [36] has reported that the presence of thyroid antibodies in RPL does not affect future pregnancy outcome if the patient is euthyroid.

Uterine cavity assessment can be performed by HSG, two or three dimensional ultrasound, hydrosonography or hysteroscopy. HSG, probably the most painful of the procedures, cannot differentiate between a septate uterus and a bicornuate uterus, nor determine the myometrial extension or the size of intra‐uterine lesions. However, HSG has the advantage of assessing tubal patency if there is concurrent infertility. Two or three dimensional ultrasound can diagnose congenital anomalies such as a septum, fibroids, polyps, etc. and has the ability to visualize both the uterine cavity and the myometrium. A three dimensional scan facilitates the diagnosis of uterine anomalies and enables easy differentiation between subseptate and bicornuate uteri. However, ultrasound is not so accurate regarding intrauterine adhesions.

Valenzano et al. [37], have assessed transvaginal sonohysterography (SHG) in the detection of uterine anomalies. SHG was able to detect all uterine anomalies found in a study of 54 patients with primary or secondary infertility or RPL and a sonographically suspected abnormal uterus. The sensitivity and specificity of SHG were the same as for hysteroscopy.

Hysteroscopy can directly visualize intracavitary structures and directed biopsies can be obtained when indicated. A retrospective study [38] has found an association, between the hysteroscopic findings in 344 women with recurrent spontaneous abortion and major, and even minor uterine anomalies. The anomalies were shown to correlate with an increased risk of recurrent miscarriage.

Uterine anomalies have for long been known to be associated with pre‐term labor, but have only recently been definitely associated with RPL. Sugiura‐Ogasawara et al. [4] have performed a case controlled study on 676 patients with two or more pregnancy losses. Twenty‐five (59.5%) of the 42 patients with a bicornuate or septate uterus had a successful first pregnancy after examination, compared to 1096 (71.7%) of the 1528 with normal uteri. However, the incidence of embryonic chromosome aberrations in women with and without uterine anomalies were 15.4% (2 of 13) and 57.5% (134 of 233), respectively. The author’s concluded that congenital uterine anomalies have a negative impact on reproductive outcome in couples with recurrent miscarriage and are associated with further miscarriage with a normal embryonic karyotype. Chan et al. [39] carried out a systematic review to evaluate the prevalence of uterine anomalies in an unselected population, infertility, a history of miscarriage, infertility and recurrent miscarriage combined and preterm delivery. The authors identified 94 observational studies on 89 861 women. The prevalence of uterine anomalies was 5.5% in the unselected population, 13.3% (CI, 8.9–20.0) in women with a history of miscarriage and 24.5% (95% CI, 18.3–32.8) in women with miscarriage and infertility.

NK cells are large granular lymphocytes bearing the CD56+ antigen, and are part of the innate immune system. These cells have been scrutinized as to their role in the immune response to pregnancy, as they are the only lymphocytes to be found in the uterine mucosa. Uterine NK cells seem to be involved with immunosurveillance of the pregnancy, but their exact role is unclear. It has been suggested that NK cells may be responsible for remodeling of spiral arteries into utero‐placental arteries, or that NK cells may be responsible for immune attack on the placenta, if lymphokine activated. Since Aoki et al.’s original report [40] showing that increased numbers of NK cells in the peripheral blood of women with RSA predict the likelihood of another miscarriage, there have been two trends, both to identify subgroups of patient with RPL and to include all patients with RPL in megatrials. Shakhar et al. [41] have found increased numbers of peripheral NK cells in primary (patients who lose all their pregnancies), but not secondary aborters (live birth or births, followed by a string of miscarriages). Perricone et al. [42] have reported that patients with APS and RPL have higher levels of NK cells than patients with APS and no RPL. However, when all patients with RPL are assessed as a homogeneous group, it is difficult to see the association between NK cells and RPL. Tang et al. [43] carried out a literature search for relevant publications from 1950 to 2010. The study included peripheral blood and uterine NK cell numbers or activity in women with RPL, or infertility. The search identified 12 publications which fulfilled the inclusion criteria. However, there were too few women entered into the observational studies to assess whether high peripheral blood NK cell numbers or activity predicted subsequent miscarriage in women with idiopathic RPL. Similarly, the studies of uterine NK cells were not large enough to assess whether abnormal uterine NK cell density predicted subsequent miscarriage in women with idiopathic RPL. At present, more studies are needed to confirm or refute the role of NK cell assessment as a predictive test for subsequent miscarriages.

Infections such as toxoplasmosis, Listeria monocytogenes, mycoplasma, Chlamydia, and Parvovirus B19 have been implicated in RPL. However, the role of infection in RPL is unclear. For an infective agent to be causative, it must be asymptomatic in the interval between pregnancies, and capable of persisting in the genital tract. There is some evidence that bacterial vaginosis may predispose to second‐trimester miscarriage and preterm delivery [44], but there is little evidence of an association with first trimester miscarriage. It is conceivable that infections may cause miscarriage of live embryos when the uterus contracts and expels a live embryo or fetus, or that a retroplacental hematoma may become infected. However, there are no reports of research in these subgroups of patients. At present no screening for infections has been shown to be helpful.

1. 2. In pregnant patients with previous pregnancy losses, what is the diagnostic value of single and serial measurements of serum β‐hCG or serial ultrasound examinations to determine the likelihood of the pregnancy developing?

Search strategy

• MEDLINE and EMBASE: hCG and prognosis, β‐hCG and prognosis.
  
• AND clinical trial AND case–control studies AND cohort studies.

At the beginning of pregnancy, repeated or serial hCG measurements are the only practical test to provide information about fetal viability. hCG can be used clinically to diagnose pregnancy from nine days after the luteinizing hormone (LH) surge. In the first trimester, hCG values approximately double every two days in women with developing pregnancies [45]. If the rise of hCG is slower, pregnancy development may be abnormal, or may indicate an ectopic pregnancy. Osmanağaoğlu et al. [46] carried out a study to determine the value of β‐hCG, progesterone, CA125 and their combined use in the prediction of first trimester abortions. A total of 140 singleton pregnant women between 5 and 13 weeks of gestational age whose pregnancies resulted in missed, incomplete, complete, or inevitable abortion were compared to 129 normal pregnancies. When using the free β‐hCG level of 520 ng ml⁻¹ as a cut‐off point, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 91%, 82%, 46%, and 98%. When a progesterone level of 515 ng ml⁻¹ was used as a cut‐off point, they were 91%, 89%, 59%, and 98% respectively. The authors concluded that a single measurement of free β‐hCG or progesterone levels can be useful in the prediction of first trimester spontaneous abortions. CA125 levels are not found to be an effective marker.

Others have also assessed the predictive value of progesterone levels. Al‐Sebai et al. [47] assessed 358 threatened abortions before 18 weeks of pregnancy, and found a single progesterone level ≤ 45 nmol l⁻¹ (14 ng ml⁻¹

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