RhD alloimmunization in pregnancy: Management

INTRODUCTION — Despite the development and implementation of anti-D immune globulin prophylaxis, hemolytic disease of the fetus and newborn (HDFN) due to maternal RhD alloimmunization continues to occur worldwide. Ideally, pregnancies complicated by alloimmunization should be managed by a maternal-fetal medicine specialist with appropriate experience and credentialed to perform the invasive diagnostic and therapeutic procedures that may be needed. With appropriate pregnancy monitoring and intervention, this disorder can be treated successfully in almost all cases, with minimal long-term sequelae in offspring.

This topic will provide our approach to management of pregnant people with RhD alloimmunization. Related topics, including a discussion of the Rh system, diagnosis and prevention of RhD alloimmunization, diagnosis and management of pregnant people with alloimmunization from other red cell antigens (eg, Kell), in utero transfusion, and neonatal issues, are reviewed in detail separately:

●(See "RhD alloimmunization in pregnancy: Overview".)

●(See "Management of non-RhD red blood cell alloantibodies during pregnancy".)

●(See "Intrauterine fetal transfusion of red blood cells".)

●(See "RhD alloimmunization: Prevention in pregnant and postpartum patients".)

●(See "Alloimmune hemolytic disease of the newborn: Postnatal diagnosis and management".)

MANAGEMENT OF THE FIRST ALLOIMMUNIZED PREGNANCY — A patient's first pregnancy complicated by RhD alloimmunization is managed differently from subsequent pregnancies because their anti-D titer is usually low at the beginning of the first affected pregnancy; severe fetal anemia may not develop or develops in the late second trimester or the third trimester. In subsequent affected pregnancies, fetal anemia usually is more severe and develops earlier in gestation.

Our approach to managing the first pregnancy complicated by red cell alloimmunization is shown in the algorithm (algorithm 1) and discussed below [1].

Determine whether the fetus is at risk — An RhD-negative fetus is not at risk for complications from maternal anti-D antibodies since it does not carry the target antigen; therefore, one of the initial steps in antenatal management of maternal RhD alloimmunization is to determine the fetal RhD type.

●Father of fetus is RhD-negative – If the biologic father of the fetus is RhD-negative, the fetus must also be RhD-negative. Maternal alloimmunization occurred as a result of a previous pregnancy with an RhD-positive partner or from some other source of RhD-positive red cells (eg, incompatible blood transfusion, needle sharing). An RhD-negative fetus is not at risk for hemolytic disease, so further evaluation, monitoring, and intervention are unnecessary, unless maternal alloimmunization involving non-RhD red cell antibodies has also occurred. Obviously, certainty as to paternity is imperative, and nonpaternity is more common than one might assume [2]. The clinician should consider documenting the discussion regarding assured paternity in the medical record.

●Father of fetus is or may be RhD-positive – If the biologic father of the fetus is RhD-positive, we determine paternal zygosity. (See 'Paternal zygosity testing' below.)

•All offspring of RhD-positive homozygotes will be RhD-positive so further testing for fetal RhD type is unnecessary.

•Offspring of heterozygotes have a 50 percent chance of being RhD-positive, so in these cases, we test for the paternally derived fetal RHD (ie, the gene that encodes the RhD protein) by performing cell-free DNA (cfDNA) testing on maternal plasma. (See 'Cell-free DNA testing' below.)

•If the biologic father of the fetus is unavailable for testing or if paternity is uncertain, then we test for the paternally derived fetal RHD by performing cell-free DNA (cfDNA) testing on maternal plasma. (See 'Cell-free DNA testing' below.)

Paternal zygosity testing — Paternal zygosity is determined using quantitative polymerase chain reaction (PCR) to identify the number of RHD genes [3]. In the past, laboratories used anti-sera to the Rh antigens (D, C/c, E/e) and gene frequency tables based upon race/ethnicity to estimate zygosity at the RHD locus. Although useful, these estimates are less reliable than direct genetic testing, which is commercially available from at least two vendors in the United States (Versiti, Inc; ARUP laboratories).

As discussed above, all offspring of RhD-positive homozygotes will be RhD-positive, so further testing for fetal RHD is unnecessary. Zygosity testing will reveal a heterozygous paternal genotype in approximately 40 percent of RhD-positive White individuals. Heterozygotes have a 50 percent chance of having RhD-negative offspring, so cfDNA testing for fetal RHD is performed.

Cell-free DNA testing — Noninvasive assessment of fetal RHD using cfDNA is widely available in the United Kingdom and Europe. It is also available in the United States from at least one commercial laboratory.

The fetal RHD genotype is determined by testing a sample of maternal plasma after 10 weeks of gestation.

●If the fetus is RHD-positive, then maternal indirect Coombs titers (ie, indirect antiglobulin test) are obtained serially until a critical titer is reached. (See 'Follow maternal anti-D titers in at-risk fetuses until the critical titer is reached' below.)

●If the fetus is RHD-negative and the mother has no additional red cell antibodies, it is not at risk for hemolytic disease of the fetus and newborn (HDFN) and further maternal or fetal monitoring for HDFN is unnecessary.

●If the fetus is RHD-negative but the mother has an additional antibody (such as anti-C or anti-E), which is not uncommon, the pregnancy needs to be monitored for HDFN with serial maternal indirect Coombs titers and additional fetal middle cerebral artery (MCA) peak systolic velocity (PSV) Doppler testing once a critical titer is reached. (See "Management of non-RhD red blood cell alloantibodies during pregnancy".)

Test methodology, performance, and interpretation — Fetal cfDNA can be detected in the maternal circulation as early as 38 days of gestation. It comprises 10 to 15 percent of the total cfDNA in the maternal circulation during the late first and early second trimesters, increases with advancing gestation, and disappears soon after giving birth. Fetal RHD status is determined by evaluation of cfDNA sequences in maternal plasma using a reverse transcriptase PCR. A meta-analysis of studies of cfDNA for RHD determination reported sensitivity of 99.3 percent (95% CI 98.2-99.7) and specificity of 98.4 percent (95% CI 96.4-99.3) in the first and second trimesters; real-time quantitative PCR sensitivity was higher than conventional PCR [4].

Assays for the RHD exon 4; exons 5 and 7; exons 4, 5, and 7; or exons 4, 5, 7, and 10 have been recommended and should be performed after approximately 10 weeks of gestation so there will be adequate fetal cfDNA [3,5-9]. Detection of these RHD exons in maternal plasma indicates fetal cfDNA is present and the fetus is RHD-positive. If RHD exons are absent, the fetus is RHD-negative as long as it can be proven that fetal cfDNA and not maternal cfDNA was tested. Identification of Y chromosome gene sequences (SRY) in the plasma sample confirms the presence of fetal cfDNA and validates the test results. If the fetus is female, the maternal white blood cells are analyzed for single nucleotide polymorphisms (SNPs) and these results are compared with the SNPs in the cfDNA sample. If a discordance in SNPs is noted, the SNPs that are different from the mother are assumed to be paternal in origin, thus confirming the presence of fetal DNA [10]. The hypermethylated RASSF1A promoter has also been reported as a universal fetal marker to confirm the presence of fetal DNA [11-13].

Fetal RHD testing via cell-free DNA is available in the United States as part of the UNITY Complete test [14]. The test is performed with multiplex next-generation sequencing technology and includes results from additional fetal screening for:

●Trisomy 21, 18, and 13

●Sex chromosome aneuploidy (X, XXY, XYY, XXX)

Exons 4 (two sites), 5, 7, and 10 of RHD are analyzed, which enables detection of the RHD pseudogene. The test is accurate when the fetal fraction of DNA is above 0.5 percent. An internal control of 64 single nucleotide polymorphisms (SNPs) is used to confirm the presence of fetal DNA in the sample. If at least four paternally-inherited SNPs are detected, the presence of fetal DNA is confirmed. This technology negates the need for drawing a paternal sample. Internal testing has revealed a sensitivity of 99.9 percent with a negative predictive value of 99.8 percent.

False positives — Cell-free DNA derived from a vanishing twin or an organ transplant are potential sources of false-positive results (ie, not reflecting the RHD-type of the potentially affected fetus) [15,16].

False negatives — False-negative results would be more serious as appropriate monitoring and interventions might be withheld. More than one D region (eg, exons 7 and 10, intron 4) should be examined to ensure that negative results reflect true RHD negativity and not the presence of a D variant (see "RhD alloimmunization in pregnancy: Overview", section on 'D variants'). False negatives can also be due to a low level of fetal cfDNA in a maternal sample because it was drawn too early in gestation (<8 weeks of gestation) or to insensitive laboratory techniques [17].

Amniocentesis — If cfDNA testing is not available, fetal RHD status can be determined by PCR on uncultured amniocytes obtained by amniocentesis after 15 weeks of gestation [18]. Because this is an invasive procedure, it is reserved for pregnancies in which a critical titer is reached or exceeded (discussed below) and the father is heterozygous for RHD, paternal zygosity is unknown, paternal RhD type is unknown, or noninvasive testing was inconclusive due to a RHD pseudogene or a low amount of fetal DNA. However, if a patient with titers below the critical level is undergoing amniocentesis for another indication (eg, diagnosis of aneuploidy), it is reasonable to determine fetal RHD status at that time as well, instead of cfDNA testing.

Whenever amniotic fluid is obtained for fetal testing and the fetus is RHD-negative, a false-negative result due to maternal cell contamination should be excluded by performing SNP testing on maternal DNA and DNA derived from amniocytes.

Transplacental amniocentesis should be avoided, if possible, as it may worsen alloimmunization (chorionic villus biopsy is avoided for the same reason [19]). The magnitude of this risk is unknown.

Follow maternal anti-D titers in at-risk fetuses until the critical titer is reached — General principles include:

●In the first alloimmunized pregnancy with a fetus at risk of HDFN, the indirect Coombs titer (ie, indirect antiglobulin test) is repeated monthly as long as it remains stable; rising titers should be repeated every two weeks until the titer reaches the "critical" level. (See 'Critical titer' below.)

●Serial titers should be determined by the same laboratory since variation in titers among laboratories is common. Furthermore, as a quality control measure, the laboratory should consider repeating the titer from the prior sample each time it performs a titer on a newly submitted sample.

●Intralaboratory variation occurs, but a truly stable titer should not vary by more than one dilution when repeated in a given laboratory. As an example, a titer of 2 that increases to 4 may not represent a true increase in the amount of antibody in the maternal circulation, but a rise to 8 is likely real.

•Gel microcolumn assay (GMA) – The GMA card is gaining widespread acceptance by blood banks to replace the traditional tube agglutination tests for determining anti-D antibody titers. Advantages of GMA over tube tests include the following: it is less susceptible to interlaboratory and intralaboratory sources of variability, yields clear objective results that are stable, takes less time, and is compatible with automation [20]. However, GMA may yield higher titer values than tube tests. In several studies, the increase was usually only one or two dilutions, which is not clinically significant (a fourfold difference is clinically significant) [20,21], but some studies reported greater discordance [22,23]. More data are needed to establish the correlation between GMA titer and severity of fetal anemia (ie, the threshold for a "critical titer") before this assay can be used to manage alloimmunization in pregnancy. For this reason, standard tube titers should still be used for clinical management of the alloimmunized pregnancy.

●Maternal administration of anti-D immune globulin after the primary immune response to the D antigen has occurred will not prevent a rise in titer, and anti-D immune globulin should not be administered to sensitized patients [24,25].

Critical titer — A critical titer has historically been defined as the titer associated with a risk for development of severe anemia and hydrops fetalis at a specific institution. Below the critical titer, the fetus is at risk for developing mild to moderate, but not severe, anemia. However, given the decreased incidence of RhD alloimmunization in pregnancy, most institutions lack a sufficient number of alloimmunized patients to establish a critical titer and therefore consider an anti-D titer of 16 to 32 as a critical value. In Europe and the United Kingdom, a threshold value of 15 international units/mL is the critical value, based upon comparison with an international standard [26].

If the critical titer is reached or exceeded, further assessment is required to determine whether the RhD-positive fetus is severely anemic. Maternal titers are screening tests, not diagnostic of severe anemia, and should be discontinued once a critical titer is reached. In one series of 590 patients alloimmunized to anti-RhD, 70 percent developed a critical titer of ≥16 in their second pregnancy (first alloimmunized pregnancy) [27]. The average gestational age for the development of a critical titer in those patients whose fetuses/neonates developed HDFN was 26+3 weeks of gestation.

If fetal RHD has not yet been determined by cfDNA testing, examination of amniocytes, or certain RHD homozygous paternity, then we recommend fetal RHD evaluation at this point to avoid potentially unnecessary serial Doppler ultrasound monitoring and fetal blood sampling. Maternal indirect Coombs titers can rise even though the fetus is RhD-negative; the reason is unclear.

Assess for severe anemia using MCA-PSV in fetuses at risk — When the critical titer is reached or exceeded and the fetus is RHD-positive, Doppler velocimetry of the MCA-PSV is performed to identify fetuses that are likely to be severely anemic. Doppler assessment of the fetal MCA-PSV is based on the principle that the fetal hemoglobin level determines blood flow in the MCA: MCA-PSV increases as fetal hemoglobin level falls [28].

●A 2009 meta-analysis including nine observational studies (675 fetuses) provided compelling evidence that Doppler interrogation of the MCA-PSV performs well as a screening tool for severe fetal anemia of any etiology [29]. When severe anemia was defined as fetal hemoglobin <0.55 multiples of the median (MoMs) for gestational age, sensitivity and specificity were 75.5 and 90.8 percent, respectively.

In the seminal study included in this review, both hemoglobin level in blood obtained by cordocentesis and MCA-PSV were measured in 111 fetuses at risk for anemia due to maternal red cell alloimmunization and compared with values in 265 normal fetuses [30]. The sensitivity of increased MCA-PSV (above 1.5 MoMs) for the prediction of moderate or severe anemia was 100 percent (95% CI 86-100), either in the presence or absence of hydrops, with a false-positive rate of 12 percent.

The same authors conducted a follow-up prospective study of 125 fetuses at risk for alloimmune anemia and reported the overall performance of MCA-PSV for moderate to severe anemia (hemoglobin level below 0.65 MoMs) was sensitivity 88 percent, specificity 87 percent, positive predictive value 53 percent, and negative predictive value 98 percent [31]. One of nine fetuses with severe anemia was missed, possibly due to a screening interval longer than two weeks.

●A subsequent meta-analysis of 12 studies (696 fetuses) published from 2008 to 2018 found that MCA-PSV ≥1.5 MoMs had sensitivity and specificity of 86 percent (95% CI 75-93) and 71 percent (95% CI 49-87), respectively, for prediction of moderate to severe anemia in nontransfused fetuses [32]. However, sensitivity progressively fell with an increasing numbers of transfusions. (See "Intrauterine fetal transfusion of red blood cells", section on 'Scheduling subsequent transfusions'.)

Timing, frequency, and performance — MCA-PSV is performed, when clinically indicated, after 20 weeks of gestation in the first affected pregnancy because severe anemia is unlikely in the first half of pregnancy and fetal blood sampling and transfusion are difficult at this gestational age. Proper technique for measuring MCA-PSV is important and is described elsewhere [33,34]. Ideally, the measurement is obtained when the fetus is in a quiet behavioral state as results are inaccurate when the fetus is active [35,36].

Because MCA-PSV increases across gestation (figure 1), results should be adjusted for gestational age. Conversion calculators, such as the one found at perinatology.com, can be used to convert the actual MCA-PSV in cm/second to MoMs to correct for gestational age.

The optimal interval between examinations has not been determined. Experts suggest one to two week intervals based on clinical experience and what is known about progression of fetal anemia in this setting [30]. The frequency is increased if indicated by MoMs approaching 1.5.

MCA-PSV ≤1.5 MoMs for gestational age — An MCA-PSV ≤1.5 MoMs for gestational age is consistent with absence of moderate to severe anemia.

If MCA-PSV remains at this level, we schedule delivery at 37+0 to 38+6 weeks of gestation, consistent with Society for Maternal-Fetal Medicine and American College of Obstetricians and Gynecologists guidelines [33,37]. In addition, we begin weekly antenatal testing at 32 weeks of gestation. Historically, alloimmunization has been considered an indication for antepartum fetal surveillance, although no well-designed studies have evaluated the utility, type, or frequency of testing [38]. (See "Overview of antepartum fetal assessment".)

MCA-PSV >1.5 MoMs for gestational age — For pregnancies with MCA-PSV >1.5 MoMs for gestational age, we obtain fetal blood by cordocentesis for hemoglobin determination and have blood readily available for intrauterine fetal transfusion, but only perform the transfusion if fetal hemoglobin is more than two standard deviations below the mean value for gestational age; reference values have been established (table 1). A hematocrit less than 30 percent can also be used as the threshold for fetal transfusion [39]. If the hemoglobin is above this threshold, we obtain another fetal blood sample in one to two weeks, depending on the value. Fetal hemoglobin level should be checked before transfusion because a high MCA-PSV is not definitive proof of clinically significant fetal anemia; false positives occur [30,31].

Transfusion at a moderately reduced hemoglobin level results in a better fetal outcome than waiting until development of severe anemia (hemoglobin deficit >7 g/dL below the normal mean for gestational age [40]) or hydrops (typically hemoglobin is less than 5 g/dL) [30].

Intravascular intrauterine transfusion is generally limited to pregnancies between 18 and 35 weeks of gestation because before 18 weeks, the small size of the relevant anatomic structures poses technical challenges, and after 35 weeks, intrauterine transfusion is considered riskier than delivery followed by postnatal transfusion therapy [41]. Thus, at ≥35 weeks of gestation, we would deliver a fetus with MCA-PSV >1.5 MoMs for gestational age without fetal blood sampling to check the fetal hemoglobin. (See "Intrauterine fetal transfusion of red blood cells".)

Other findings and methods for assessing fetal anemia

●Sinusoidal heart rate – In severely anemic fetuses, the fetal heart rate may show a sinusoidal pattern. (See "Intrapartum fetal heart rate monitoring: Overview", section on 'Sinusoidal pattern'.)

●Fetal blood sampling – Fetal blood can be sampled to precisely determine the severity of fetal anemia, but this procedure carries a 1 to 2 percent risk of fetal loss, with the highest risk at lower gestational ages and in hydropic fetuses (see "Fetal blood sampling"). We reserve fetal blood sampling for pregnancies in which MCA-PSV suggests moderate to severe anemia.

●Amniotic fluid bilirubin level – Amniocentesis to determine amniotic fluid bilirubin levels (delta OD₄₅₀) had been the traditional method for indirectly estimating the severity of fetal anemia. Bilirubin in amniotic fluid derives from fetal pulmonary and tracheal effluents and correlates with the degree of fetal hemolysis [42,43]. However, Doppler velocimetry is as, or more, sensitive and specific for detection of severe fetal anemia and has the advantage of being noninvasive [44]. For these reasons, delta OD₄₅₀ assay is no longer readily available at most commercial laboratories.

DELIVERY

Timing

●In pregnancies complicated by alloimmunization in which no intrauterine transfusion was performed and the MCA-PSV remains <1.5 MoMs for gestational age, we would proceed with delivery between 37 and 38 weeks of gestation. This is a prudent approach because the sensitivity of MCA-PSV for detecting severe fetal anemia decreases after 35 weeks, thus the benefits of delivery, newborn evaluation, and treatment (if needed) in this setting likely exceed the small risk of mild neonatal immaturity from an early term birth.

●In pregnancies complicated by alloimmunization in which no intrauterine transfusion was performed and the MCA-PSV reaches >1.5 MoMs for gestational age at ≥35 weeks of gestation, we would proceed with delivery and evaluate/treat the newborn for HDFN rather than initiate these interventions in utero.

●In pregnancies complicated by alloimmunization in which intrauterine transfusion was performed, labor is induced approximately three weeks after the last transfusion, typically at 37+0 or 38+0 weeks of gestation, or a cesarean birth is scheduled if indicated for standard obstetric indications. (See "Intrauterine fetal transfusion of red blood cells", section on 'Timing delivery'.)

Delayed cord clamping — We do not consider alloimmunization a contraindication to delayed cord clamping, even in severe hemolytic disease of the fetus and newborn. Limited data on delayed cord clamping in alloimmunized pregnancies suggest a short-term benefit and lack of harm.

In the only randomized trial evaluating the outcome of delayed versus early cord clamping in Rh-alloimmunized neonates (70 neonates at 28 to 41 weeks of gestation), delayed clamping improved packed cell volume at two hours of life without significantly increasing risks for double volume exchange transfusion, partial exchange transfusion, and duration of phototherapy during the hospital stay, or blood transfusion through 14 weeks of life [45]. Approximately 50 percent of the neonates in each group had received intrauterine transfusions (mean three transfusions, range two to five).

PROGNOSIS IN THE FIRST ALLOIMMUNIZED PREGNANCY — In one retrospective series of 106 patients in their first alloimmunized pregnancy, 60 patients (57 percent) did not develop a critical anti-RhD titer >16 [27]. Among those who developed a critical titer, 54 percent of fetuses/neonates developed hemolytic disease of the fetus and newborn: 26 percent severe disease (hydrops fetalis, fetal demise, or need for intrauterine transfusion), 4 percent moderate disease (need for neonatal exchange transfusions), and 24 percent mild disease (neonatal phototherapy/simple blood transfusion).

PROGNOSIS AND MANAGEMENT IN SUBSEQUENT PREGNANCIES — Pregnancies after the first alloimmunized pregnancy are characterized by increasingly severe fetal hemolytic disease due to the entry of fetal red cells into the maternal circulation at each birth, which causes an anamnestic maternal antibody response. A patient whose prior pregnancy was complicated by the need for intrauterine fetal transfusion, fetal hydrops, preterm birth because of severe fetal anemia, or neonatal exchange transfusion can expect development of severe fetal anemia in subsequent pregnancies with an RhD-positive fetus. The severe anemia typically occurs earlier in gestation than in the prior pregnancy; one case report described severe anemia as early as 15 weeks of gestation [46]. Management of these pregnancies is illustrated in the algorithm (algorithm 2). We determine fetal RHD type early in gestation using cell-free DNA and begin middle cerebral artery (MCA) peak systolic velocity (PSV) monitoring of RHD-positive fetuses at 16 to 18 weeks of gestation. MCA-PSV-based management is similar to that described above for first alloimmunized pregnancies, except measurement of MCA-PSV is usually performed weekly [33]. (See 'Assess for severe anemia using MCA-PSV in fetuses at risk' above.)

A patient whose prior pregnancy was complicated by the need for only neonatal phototherapy should also be managed with initiation of serial MCA-PSV Dopplers in the next pregnancy if the fetus is RhD-positive, beginning at 18 weeks gestation. The principle of initiating fetal Doppler surveillance based on a critical titer should be limited to the management of the first alloimmunized gestation. Serial maternal titers in the next pregnancy are less informative than in the first affected pregnancy as they are not predictive of the onset of fetal anemia. However, we obtain a baseline maternal titer early in gestation as an extremely high titer (≥1028) suggests the need for immunomodulation. (See 'Management of pregnancies with severe fetal anemia before 24 weeks of gestation' below.)

SPECIAL ISSUES

Management of pregnancies with severe fetal anemia before 24 weeks of gestation — For the rare patient with a history of previous very early and severe alloimmunization, aggressive monitoring with weekly MCA-PSV determinations as early as 16 weeks of gestation is indicated. Ultrasound assessment showing absence of ascites/hydrops should not be considered reassuring. In one series of 30 fetuses with severe anemia at less than 22 weeks of gestation, 71 percent failed to exhibit signs of hydrops [47].

Administration of intravenous immunoglobulin G (IVIG) with or without plasma exchange may maintain the fetal hematocrit above life-threatening levels long enough to achieve a gestational age when intrauterine transfusion is technically feasible and less likely to be associated with fetal death. Fetal death occurred in 6 of the 30 fetuses described above who underwent intrauterine transfusion before 24 weeks [47]. In a multicenter retrospective review, initiation of weekly infusion of IVIG before 13 weeks of gestation was associated with a delay in the development of severe anemia by 25 days compared with the previous pregnancy [48]. A variety of therapeutic regimens have been described in case reports and small cases series [48,49]. The American Society for Apheresis guidelines on use of therapeutic apheresis describe intrauterine transfusion as the mainstay of treatment, but state IVIG and/or therapeutic plasma exchange may be indicated if there is a high risk of fetal demise or signs of hydrops prior to 20 weeks [50].

Intraperitoneal transfusion is technically feasible in early gestations, even prior to 20 weeks, and has a lower risk of procedure-related complications than intravascular transfusion. (See "Intrauterine fetal transfusion of red blood cells", section on 'Choosing a fetal access site'.)

A monoclonal antibody to block the FcRn receptors of the placenta is in a phase II clinical trial in patients with severe hemolytic disease of the fetus and newborn (HDFN) [51].

Management of patients with multiple antibodies — Some patients develop antibodies to more than one red cell antigen. There are no specific guidelines for management of these pregnancies; they are typically managed as described above.

The presence of other red cell antibodies (especially anti-C) with anti-D antibody appears to be associated with a more aggressive maternal immune response, thus increasing the risk of severe anemia and need for intrauterine fetal transfusion [52-54]. These pregnancies warrant close observation.

Prevention of an affected fetus in future pregnancies — Each subsequent pregnancy after the first affected pregnancy is likely to manifest more severe HDFN, and at an earlier gestational age. HDFN can be prevented by avoiding conception of an RhD-positive fetus. However, prevention is rarely attempted because of the costs and complexities involved and because HDFN can be treated successfully in most cases.

An RhD-positive fetus can be avoided in the following ways:

●In vitro fertilization (IVF) with preimplantation genetic testing – If the potential biologic father is heterozygous for RHD, IVF with preimplantation genetic testing can be used to identify RHD-negative embryos and only these embryos are considered for embryo transfer [55]. An accuracy of 95 percent is widely quoted by these laboratories, therefore we recommend fetal confirmatory testing with cell-free DNA after 10 weeks of gestation. (See "Preimplantation genetic testing".)

●Use of a gestational carrier – If the potential biologic father is homozygous for RHD, the intended parents can conceive by IVF and the embryo can be carried by a gestational carrier who is not alloimmunized. (See "Gestational carrier pregnancy".)

●Use of donor sperm – Sperm from an RhD-negative donor can be used for intrauterine insemination of the alloimmunized mother. (See "Donor insemination".)

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Rh disease in pregnancy".)

INFORMATION FOR PATIENTS — UpToDate offers two types of patient education materials, "The Basics" and "Beyond the Basics." The Basics patient education pieces are written in plain language, at the 5^th to 6^th grade reading level, and they answer the four or five key questions a patient might have about a given condition. These articles are best for patients who want a general overview and who prefer short, easy-to-read materials. Beyond the Basics patient education pieces are longer, more sophisticated, and more detailed. These articles are written at the 10^th to 12^th grade reading level and are best for patients who want in-depth information and are comfortable with some medical jargon.

Here are the patient education articles that are relevant to this topic. We encourage you to print or e-mail these topics to your patients. (You can also locate patient education articles on a variety of subjects by searching on "patient info" and the keyword(s) of interest.)

●Basics topic (see "Patient education: Pregnancy in Rh-negative people (The Basics)")

SUMMARY AND RECOMMENDATIONS

●First affected pregnancy — The evaluation and management of the first affected pregnancy is described in the algorithm (algorithm 1) and below:

•Risk for severe anemia – In a patient's first pregnancy complicated by RhD alloimmunization, severe fetal anemia develops in only 16 percent of cases, generally in the late second trimester or the third trimester. (See 'Management of the first alloimmunized pregnancy' above.)

•Determine paternal zygosity – If the biologic father of the fetus is RhD-positive, paternal zygosity is determined. All offspring of RHD-positive homozygotes will be RhD-positive so further testing for fetal RhD type is unnecessary. Heterozygotes have a 50 percent chance of having RhD-negative offspring, so in these cases we determine fetal RHD type noninvasively using cell-free DNA (cfDNA) in maternal plasma (algorithm 3). If the biologic father of the fetus is unavailable for testing or paternity is uncertain, we determine fetal RHD type by cfDNA testing. (See 'Cell-free DNA testing' above and 'Paternal zygosity testing' above.)

•Determine fetal RHD type – An RhD-negative fetus is not at risk for complications from maternal anti-D antibodies; therefore, one of the initial steps in antenatal management of maternal RhD alloimmunization is to determine the fetal RHD type. (See 'Determine whether the fetus is at risk' above.)

If the biologic father of the fetus is RhD-negative, the fetus must also be RhD-negative. An RhD-negative fetus is not at risk for hemolytic disease, and further evaluation, monitoring, and intervention are unnecessary. Documentation of the discussion regarding paternity should be included in the medical record.

•Determine indirect Coombs/critical titer – If the fetus is RHD-positive, the indirect Coombs titer (ie, indirect antiglobulin test) is repeated monthly as long as it remains stable; rising titers should be repeated every two weeks. The critical titer (the titer associated with a risk for development of severe anemia and hydrops fetalis) varies among laboratories and by methodologies; however, in most centers, an anti-D titer between 16 and 32 is considered critical. In Europe and the United Kingdom, a threshold value of 15 international units/mL is the critical value, based upon comparison with an international standard. (See 'Follow maternal anti-D titers in at-risk fetuses until the critical titer is reached' above.)

•Follow Doppler MCA-PSV

-When the critical titer is reached or exceeded and the fetus is RHD-positive, further assessment by Doppler velocimetry is performed to determine whether the fetus is severely anemic. Doppler interrogation of the fetal middle cerebral artery (MCA) peak systolic velocity (PSV) is the best tool for predicting moderate to severe fetal anemia in at-risk pregnancies. (See 'Assess for severe anemia using MCA-PSV in fetuses at risk' above.)

-We measure MCA-PSV at one- to two-week intervals. The frequency is increased if indicated by multiples of the median (MoMs) approaching 1.5. An MCA-PSV ≤1.5 MoMs for gestational age is consistent with absence of moderate to severe anemia. If MCA-PSV remains at this level, we schedule delivery at 37 to 38 weeks of gestation. In addition, we begin weekly antenatal testing at 32 weeks of gestation. (See 'MCA-PSV ≤1.5 MoMs for gestational age' above.)

•Criteria for intrauterine transfusion and delivery – For pregnancies with MCA-PSV >1.5 MoMs for gestational age and <35 weeks of gestation, we obtain fetal blood by cordocentesis for hemoglobin determination and perform an intrauterine fetal transfusion if fetal hemoglobin is two standard deviations below the mean value for gestational age (table 1). Intrauterine transfusion is generally limited to pregnancies <35 weeks of gestation because after 35 weeks, intrauterine transfusion is considered riskier than delivery followed by postnatal transfusion therapy. Labor is induced approximately three weeks after the last transfusion, typically at 37+0 or 38+0 weeks of gestation, or a cesarean birth is scheduled if indicated for standard obstetric indications.

For pregnancies with MCA-PSV >1.5 MoMs for gestational age and ≥35 weeks of gestation, we deliver the fetus rather than initiate invasive evaluation and transfusion.

If MCA-PSV remains <1.5 MoMs for gestational age, we proceed with delivery between 37+0 and 38+0 weeks of gestation. (See 'Assess for severe anemia using MCA-PSV in fetuses at risk' above and 'Timing' above.)

●Previously affected fetus/infant – A patient whose prior pregnancy was complicated by fetal hydrops, intrauterine fetal transfusion, preterm birth because of severe fetal anemia, or neonatal exchange transfusion can expect development of severe fetal anemia in subsequent pregnancies with an RhD-positive fetus and the severe anemia typically occurs earlier in gestation than in the prior pregnancy. (See 'Prognosis and management in subsequent pregnancies' above.)

•Evaluation and management – Evaluation and management are described in the algorithm (algorithm 2)

-We determine fetal RHD type using cfDNA and a baseline maternal titer early in gestation (algorithm 3). Subsequent maternal titers are less informative as they may not be predictive of onset of fetal anemia. (See 'Prognosis and management in subsequent pregnancies' above.)

-We measure MCA-PSV weekly, beginning at 16 to 18 weeks gestation. (See 'Prognosis and management in subsequent pregnancies' above.)

-For pregnancies with MCA-PSV >1.5 MoMs for gestational age, we obtain fetal blood by cordocentesis for hemoglobin determination and perform an intrauterine fetal transfusion if fetal hemoglobin is two standard deviations below the mean value for gestational age (table 1). In gestations of <20 weeks, intraperitoneal transfusions should be considered as a bridging therapy until cordocentesis is technically possible. Intrauterine transfusion is generally limited to pregnancies <35 weeks of gestation because after 35 weeks intrauterine transfusion is considered riskier than delivery followed by postnatal transfusion therapy. At ≥35 weeks of gestation, we would deliver a fetus with MCA-PSV >1.5 MoMs for gestational age. (See 'MCA-PSV >1.5 MoMs for gestational age' above and 'Prognosis and management in subsequent pregnancies' above.)