Recurrent pregnancy loss (RPL) occurs in up to 3% of reproductive age couples [1▪▪]. Over the past decade, a consensus definition of RPL consisting of two or more pregnancy losses has emerged [2▪▪,3,4]. A recent meta-analysis confirmed our earlier report that there were no differences in abnormal findings when evaluating women with two or three or more pregnancy losses [1▪▪,5] However, current standards for the evaluation and management of this distressing disorder for couples seeking to have children are insufficient, with over 50% of RPL cases remaining unexplained. This lack of an explanation has resulted in experimental testing and treatment being requested by patients and sometimes offered by physicians .
A newly devised algorithm for the evaluation and management of RPL is presented in this review suggesting an innovative approach to the management of RPL. In addition to screening for the most prevalent, known and potentially ‘treatable’ maternal causes, this new algorithm involves eliminating parental karyotype analysis from the initial RPL work-up [2▪▪] and recommending a systematic 24-chromosome microarray (CMA) analysis of miscarriage tissue after the second or subsequent loss [7▪].
OVERVIEW OF CURRENT STANDARDS OF PRACTICE
The Royal College of Obstetricians and Gynaecologists (RCOG) Green-top Guideline on recurrent miscarriage in 2011 uses quality of evidence to grade their recommendations for evaluation of RPL from A to D. Best practices included referral of patients to a specialist clinic and an ultrasound for evaluation of the uterus. Grade D recommendations included testing for antiphospholipid syndrome (APS) and genetic testing of products of conception (POC) after the third and subsequent loss. The 2012 American Society for Reproductive Medicine (ASRM) assessment of RPL includes analyzing parental karyotypes, evaluating uterine anatomy, and screening for APS, thyroid and prolactin abnormalities . Karyotype analysis of POC should only be used in the setting of ongoing therapy for RPL . The diagnostic results of the ASRM evaluation based on our data from 1398 cases (Fig. 1) reveal that over 50% will be left without an explanation for their loss . In 2018, the European Society of Human Reproduction and Embryology (ESHRE) published extensive guidelines using the ‘GRADE’ system with ‘strong’ recommendations to test for APS, and thyroid abnormalities, as well as for uterine anatomy assessment; whereas conditional recommendations were given against the routine use of parental karyotyping (to be carried out only after individual risk assessment) and POC genetic analysis (to be performed only for explanatory purposes) [2▪▪] (Table 1).
In addition, none of these guidelines support the routine use of hereditary thrombophilia screening for first trimester pregnancy losses (unless for research or for women with additional risk factors for thrombophilia) and male sperm DNA fragmentation (for explanatory purposes only) [2▪▪,3]. Furthermore, ESHRE does not advise routine testing for polycystic ovaries and insulin resistance, ovarian reserve, or luteal insufficiency [2▪▪].
ASRM recommends parental karyotype analysis as a balanced reciprocal or a Robertsonian translocation is present in about 2–5% of RPL couples thus could represent a major prognostic factor [3,8,9]. ESHRE recommends testing only in couples at an increased risk, evidenced by a prior child with congenital abnormalities, offspring with unbalanced chromosomes, or a translocation in POC [2▪▪,3]. RCOG advises parental karyotypes when an unbalanced structural chromosome is found in POC and referral of the couple for genetic counseling . Recent studies have shown that parental karyotyping for all RPL couples is simply not cost-effective [10,11] and that there is no overall difference in live birth rate when comparing Preimplantation Genetic Testing (PGT) to natural conception in those cases [11,12].
Products of conception cytogenetic analysis
The RCOG issued a Grade D recommendation to obtain cytogenetic analysis on POC after the third or subsequent consecutive miscarriage. They state that knowledge of the POC karyotype provides prognostic value to the clinician in counseling the couple on subsequent successful pregnancies .
The ASRM Practice Committee recommended against the use of karyotype analysis of the POC except in the setting of ongoing therapy for RPL . According to the committee, ‘if the evaluation identifies a remediable cause, cytogenetic analysis of subsequent losses can be employed to evaluate whether the event was random and not a treatment failure per se.’ ASRM acknowledges the probable psychological value for RPL couples.
The 2017 ESHRE Guidelines issued a conditional recommendation against the routine use of POC genetic analysis but issued a strong recommendation in favor of using microarray CMA whenever genetic analysis was performed on POC for explanatory purposes [2▪▪,13]. Furthermore, ESHRE recognized that the ‘genetic analysis of pregnancy tissue has the benefit of providing the patient with a reason for the pregnancy loss and may help to determine whether further investigations or treatments are required’ [2▪▪].
The ASRM and RCOG positions on POC genetic testing for RPL were based on the then current standard of conventional G-banding karyotype analysis [3,4]. ESHRE recommends CMA as the preferred modality for POC genetic testing because it is not limited by tissue culture failure or false negative results secondary to maternal cell contamination [2▪▪]. Up to 50% of ‘46, XX normal’ reports from POC testing result from maternal cell contamination, so methods to ensure the correct results are required [14▪,15–17]. A recent report on CMA of 26 101 miscarriages had a successful read in over 86% of samples, detected 59% with a chromosomal anomaly that could explain a pregnancy loss, but reported 13% of total results were due to maternal cell contamination [14▪]. Conventional cytogenetic results of 5457 consecutive POC samples yielded only 75% culture successes . Limitations of CMA technology include the inability to detect balanced structural chromosomal rearrangements and low-level mosaicism [16–18].
Benefits of recurrent pregnancy loss evaluation based on products of conception cytogenetic analysis
When the ASRM guidelines were published, we and others proposed a new algorithm for the evaluation and management of RPL based on the results of cytogenetic analysis of POC by G-banding  or CMA  at the time of the second or subsequent first trimester miscarriage. Following this original algorithm, if the cytogenetic analysis revealed aneuploid POC, no further evaluation or treatment was recommended. If an unbalanced chromosomal translocation or inversion was identified, then parental karyotyping and genetic counseling was advised. Finally, if the miscarriage tissue was found to be euploid and maternal cell contamination had been ruled out, a full 2012 ASRM RPL work-up was recommended [19,20].
To clinically validate that this algorithm would be more effective and cost-efficient than the ASRM RPL evaluation alone (Table 1), Popescu et al.[7▪] prospectively evaluated RPL patients using the full ASRM workup (parental chromosomal abnormalities, uterine anomalies, endocrine imbalances, autoimmune factors) as well as CMA analysis of POC [7▪]. Results indicated that in over 90% of cases, at least one potential explanation for the miscarriage could be identified when CMA analysis of POC was added to the standard ASRM RPL evaluation. Moreover, this algorithm was found to be significantly more cost-effective, producing savings of over 50% for the health system based on US numbers [7▪]. We here present additional data on a total of 378 RPL cases in which POC CMA was performed. This large cohort confirms that in 91.8% of cases an explanation for miscarriage can be identified when combining the ASRM work-up with POC CMA (Fig. 2).
Based on our experience, we agree with RCOG, ASRM, and ESHRE statements that there is a psychological benefit to the couples experiencing RPL when they learn that the pregnancy loss was the result of an abnormal chromosome complement. Clinically we have observed a great sense of relief in many couples, alleviating some feelings of guilt once they have an explanation for their loss.
NEW PROPOSED EVALUATION FOR RECURRENT PREGNANCY LOSS
It is appropriate to remember that human reproduction is an extremely inefficient process. Approximately 70% of human conceptions never achieve viability, and nearly 50% spontaneously fade before ever being noticed [21,22]. Spontaneous miscarriage is ultimately the most common complication of pregnancy. Of clinically recognized pregnancies that ultimately miscarry, 60% contain a chromosomal anomaly that can explain the loss [13,14▪]. In spite of this high rate of aneuploidy, it is important to realize that genetic factors alone cannot be the only causative factor for miscarriages in many RPL cases [1▪▪,5].
Based on our data summarized in Fig. 2, we now propose an updated algorithm for the evaluation and management of RPL. This proposal includes CMA analysis of POC after the second or subsequent loss combined with a modified ASRM evaluation that omits parental karyotypes.
MATERIALS AND METHODS
The retrospective cohort and the prospective studies were approved by the Institutional Review Boards at the University of Tennessee Health Sciences Center and Rhodes College, respectively, for exempt status because the research included the collection and study of existing data recorded by the investigators in such a manner that the subjects could not be identified directly or indirectly through identifiers linked to the subjects [5,7▪]. The original prospective study has been expanded for this report.
Patients included women with two or more consecutive, clinically documented pregnancy losses. Pregnancy loss was defined as any natural miscarriage occurring at or before 20 weeks of gestation or with a fetal weight of 500 g or less . For this study, patients who had losses after 20 weeks, ectopic pregnancies, molar pregnancies, and pregnancy terminations were excluded. All women were included regardless of socioeconomic status or race. Laboratory and uterine cavity evaluation as well as interpretation of normal and abnormal results has been described in detail elsewhere .
The average age of patients at the time of the loss for inclusion in this report was 33.4 ± 4.6. The estimated gestational age at the time of loss was 8.2 ± 3.5 weeks with 96% occurring before 13 weeks of gestation. Primary RPL occurred in 68% of women (951/1398) whereas secondary RPL was diagnosed in the remaining women (447/1398; 32%). The majority of women had two losses (747/1398; 53.4%) or three losses (393/1398; 28.1%), while fewer had four or more losses (258/1398; 18.5%). It has previously been reported that the frequency of abnormal findings does not differ in women who had two, three, or four or more losses [1▪▪,5].
EVALUATION AND MANAGEMENT
Couples who have experienced RPL want their medical team to provide compassionate care and clear information on the causes and treatments of RPL [23,24]. Healthcare providers should support, understand, and acknowledge the pregnancy losses are significant life events . Furthermore, in keeping with ASRM, RCOG, and ESHRE guidelines, couples should be informed of the negative impacts of smoking, excessive alcohol intake and weight extremes on general health as well as on their reproductive potential, and referred to qualified providers if necessary [2▪▪,3,4] (Fig. 3, Box 1, item 1).
Modified evaluation of patients with recurrent pregnancy loss
In this new algorithm, all known, prevalent, and potentially treatable abnormalities in first trimester loss RPL cases, whether anatomic, immunologic, and/or endocrine, are to be addressed regardless of POC cytogenetic analysis results (Fig. 3, Box. 1). This recommendation differs from the original proposals of Bernardi et al. who suggested that the results of G-banding karyotypes on POC or Brezina and Kutteh  who suggested that the results of 24-CMA on POC be used to direct testing. Because it has been shown that approximately 25% of women with an aneuploid result on POC CMA analysis will also have a treatable cause of RPL a modified ASRM workup is necessary [7▪] (Fig. 3, Box 1, items 2–4).
The modified evaluation omits parental karyotype analysis from the initial work-up, replacing that with systematic CMA evaluation of miscarriage tissue. The frequency of abnormal parental karyotypes is 2–5% and the self-pay cost for a karyotype analysis is $800. Thus, cost for each couple to obtain a karyotype is $1600. Conversely, the self-pay cost for a 24-CMA is $249, which will provide an explanation for the miscarriage in almost 60% of cases [7▪,14▪,15,17]. Potentially treatable maternal causes that will be found in about 40% of cases recognized by RCOG, ASRM, or ESHRE include uterine abnormalities, APS, and thyroid and prolactin endocrine disorders [1▪▪,2▪▪,3–5].
The updated algorithm recommends that the uterine cavity be evaluated for all RPL women (Table 1), given such anomalies are common but also simply treatable. ESHRE prefers three dimensional transvaginal ultrasound to assess the uterine cavity in RPL given its high sensitivity and specificity and its cost-effectiveness compared with hysteroscopy [2▪▪]. ASRM accepts sonohysterography, hysteroscopy, and/or hysterosalpingography as suitable methods . RCOG suggests a pelvic ultrasound on all women with RPL followed by hysteroscopy or three dimensional ultrasonography if a uterine anomaly is suspected . An ESHRE committee concluded that three dimensional ultrasound technology has an accuracy of 97.6% (confidence interval 94.3–100) for the diagnosis of female genital tract anomalies when compared with hysteroscopy ± laparoscopy . The prevalence of congenital and acquired uterine tract anomalies has been estimated to be ∼16.7% in RPL as compared with ∼6.7% in the general population .
The updated proposal recommends screening for APS as a potentially treatable and prevalent (15–20%) diagnosis in RPL women [2▪▪,3,4,27,28]. All three societies recommend testing for APS using lupus anticoagulant, IgG and IgM anticardiolipin in all patients with RPL (Table 1) [2▪▪,3,4]. The International Congress on APS recommends adding antiphosphatidyl serine antibody testing to identify an additional 5% of women with APS who would otherwise not be detected . The clinical value of such an approach goes beyond miscarriage prevention, given that treatment has been shown to reduce late pregnancy complications . ASRM adds testing for IgG and IgM antiβ2 glycoprotein I antibodies, however, RCOG and ESHRE guidelines consider the association weaker [2▪▪,3,4]. A general consensus recommends low-dose aspirin (75–100 mg/day) beginning before conception and a prophylactic dose of heparin starting with a positive pregnancy test and continuing to delivery [2▪▪,3,4,27,30,31].
In the newly proposed algorithm, all cases are to be evaluated for thyroid and prolactin abnormalities along with elevation of HgbA1c. In our current protocol, when thyroid stimulating hormone (TSH) levels are above 2.5 mIU/l, especially in combination with positive thyroid peroxidase (TPO) antibodies, we initiate low-dose levothyroxine. There have been various published recommendations from ESHRE, ASRM, and RCOG for hormonal testing including TSH, TPO antibodies, prolactin, HgbA1c, diabetes, insulin resistance, and ovarian reserve (refer to Table 1 for comparisons) [2▪▪,3,4].
We do not routinely recommend ovarian reserve testing; however, a recent meta-analysis correlated low-ovarian reserve in some women with unexplained RPL . Vitamin D deficiency has been shown to be associated with several obstetric complications including miscarriage  and ESHRE issued a recommendation to consider vitamin D supplementation for all cases as part of preconception counseling [2▪▪]. We believe it judicious to consider Vitamin D supplementation for all women with RPL.
Management based on initial evaluation results including products of conception chromosome microarray
Typically at the initial referral for RPL evaluation, the chromosomal status of the previous pregnancy miscarriage is not available. Studies based on G-banding karyotype analysis, suggest that POC results from the first miscarriage (euploid or aneuploid) are associated with a similar genetic outcome in 65% of second miscarriages . Using CMA analysis of POC revealed similar aneuploidy rates in pregnancy losses from natural conceptions (56.8%), conceptions from IVF (53.6%), women with one prior loss (54.4%), and women with three or more losses (52.1%) . POC aneuploidy is extremely common, and its presence does not rule out the presence of any of the known treatable RPL causes, which could have caused the loss of the previous pregnancy. Indeed, we believe all RPL cases deserve to be assessed for all potentially treatable causes (Fig. 3, Box. 1). One key feature of this new algorithm makes use of POC cytogenetic analysis to direct management toward what would potentially be the most appropriate approach, without making assumptions regarding the chromosomal status of previous miscarriages (Fig. 3, Box 1, item 5 and Box 2).
If the POC CMA reveals an unbalanced translocation or an inversion, then a karyotype analysis of both parents should be performed. In addition to genetic counseling, expectant management versus IVF with PGT should be considered and discussed [4,8].
The POC CMA will reveal aneuploidy in about 55% of cases[7▪,35] (Fig. 2). In a patient who has been evaluated and treated for all of the known causes (according to Fig. 3, Box 1), no further testing is generally recommended. Approximately 25% of women with POC aneuploidy by CMA will have at least one concomitant abnormal finding on the ASRM evaluation [7▪] and should benefit from treatment in any subsequent pregnancy. For women with a normal ASRM work-up with aneuploid POC as the sole identified cause of at least the last miscarriage, we optimistically counsel the couple about the chances of a future live born child based on the maternal age and number of prior losses .
When POC are euploid on cytogenetic analysis (42.3%) (Fig. 2) over 80% of these women will have an abnormal ASRM test result [7▪]. Overall a euploid POC and an abnormal ASRM work-up occur in 28% of all cases [7▪]. Given that at least one of the known and treatable causes would have been addressed, no further testing is recommended at this point for those specific cases.
RPL cases with a normal ASRM evaluation and euploid POC account for 8.2% of 378 cases (Fig. 2). Thus, a new definition for ‘unexplained RPL’ arises when incorporating systematic POC cytogenetic analysis as a routine part of the RPL evaluation. For these specific cases, additional testing can be considered if indicated and appropriate (Fig. 3, Box. 2) by the managing medical team, availability of given test and/or for research purposes. The patient should be counseled that expectant management is also appropriate and that successful outcomes are frequent [36,37].
Management of subsequent miscarriage
Management of a subsequent pregnancy loss should be based on the prior investigation (Fig. 3, Box 3). If no testing has been performed, evaluation should begin according to the new algorithm including POC cytogenetic analysis and modified ASRM workup (starting with Fig. 3, Box 1). POC CMA testing should be performed on the present miscarriage tissue as it could provide an explanation for the loss and be relevant to any prior treatment. Additional testing, which is available and appropriate, should be considered potentially beneficial in specific cases such as when repeated losses are euploid (Fig. 3, Box 2). In cases of repetitive POC aneuploidy, when all possible causes have previously been addressed, IVF with PGT of embryos for aneuploidy remains an unproven option with potential benefit but may offer no benefit over expectant management [38,39] (Fig. 3, Box 3).
We have designed an updated strategy for a more effective and cost-efficient diagnosis and treatment of RPL. The combination of a genetic evaluation on miscarriage tissue with an evidence-based evaluation of RPL will provide a probable or definitive explanation for the loss in 90% of couples and less than 10% of couples with RPL will remain unexplained. In addition to doubling the numbers of couples that will be given an explanation for their pregnancy loss, this new strategy for the management of RPL results in an estimated 50% cost savings to the healthcare system [7▪] (Fig. 3).
These results compare very favorably to the use of RCOG, ASRM, or ESHRE guidelines where over 50% of couples will be categorized as unexplained RPL. This should decrease the tendency for clinicians and patients to move toward unproven therapies out of desperation. This small remaining group of patients with unexplained RPL would be ideal candidates for new research studies and therapies. In our opinion, future studies on novel treatments for RPL should include mandatory testing of POC using CMA.
Couples with RPL want to know the reason for their pregnancy losses. The evaluation and treatment of RPL should be based on the cause(s) of the loss. Explanation of test results and emotional support are very important components of therapy. Realistic expectations for future success should be based on maternal age, numbers of prior losses, and the results of the evaluation. Finally, knowing the cause(s) of miscarriage may provide great psychological and emotional relief, as patients often put the responsibility for the loss on themselves when this is rarely the case.
Author contribution: R.S.P. – writing and review of article. W.H.K. – data collection, analysis, revision and review of article.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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