Maternal CD4+ microchimerism in HIV-exposed

Early Human Development 98 (2016) 49–55

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Maternal CD4 + microchimerism in HIV-exposed newborns after spontaneous vaginal delivery or caesarean section H. Buxmann a,⁎,1, A. Reitter b,1,2, S. Bapistella a,3, M. Stürmer c, C. Königs d, H. Ackermann e, F. Louwen b, P. Bader d, R.L. Schlößer a, A.M. Willasch d a

Goethe University, Department for Children and Adolescents, Division for Neonatology, University Hospital Frankfurt/Main, Germany Department of Gynecology and Obstetrics, Division of Obstetrics and Prenatal Medicine, University Hospital Frankfurt/Main, Germany Institute for Medical Virology, University Hospital Frankfurt/Main, Germany d Department for Children and Adolescents, Division for Stem Cell Transplantation and Immunology, University Hospital Frankfurt/Main, Germany e Institute of Biostatistics and Mathematical Modeling, University Hospital Frankfurt/Main, Germany b c

a r t i c l e

i n f o

Article history: Received 9 January 2016 Received in revised form 24 May 2016 Accepted 14 June 2016 Available online xxxx Keywords: CD4+ cell microchimerism Mother-to-child transmission HIV Mode of delivery Newborn

a b s t r a c t Background: Maternal CD4+ cell microchimerism may be greater after caesarean section compared to spontaneous vaginal delivery and could cause mother-to-child transmission (MTCT) in HIV-exposed newborns. Aims: To evaluate maternal CD4+ cell microchimerism in HIV-exposed newborns after spontaneous vaginal delivery or caesarean section. Study design and subjects: In this prospective single-centre study, neonates whose mothers were infected with HIV and had normal MTCT risk according to the German Austrian Guidelines were considered for study enrolment. Maternal CD4+ cell microchimerism in the newborns' umbilical cord blood was measured and compared by mode of delivery. Results: Thirty-seven HIV-infected mothers and their 39 newborns were included in the study. None of the 17 (0.0%) newborns delivered vaginally had quantifiable maternal CD4 + cells (95% confidence interval (CI): 0.00–0.00) in their circulation at birth compared with four of 16 (25.0%) newborns delivered via planned caesarean section, who showed 0.01–0.66% maternal cells (95% CI: −0.06–0.16; P = 0.02) in their circulation. The intention to treat analysis, which included six additional newborns delivered by unplanned caesarean section, showed quantifiable maternal CD4+ cells in one (0.05%; 95% CI: −0.02–0.04) of 23 (4.3%) newborn at birth compared to four of 16 (25.0%) born via planned caesarean section (95% CI: −0.06–0.16; P = 0.04). There was no MTCT in any of the newborns. Conclusion: In this small cohort, spontaneous vaginal delivery in HIV-infected women with normal MTCT risk was associated with lower maternal CD4+ cell transfer to newborns compared to planned caesarean section. © 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Abbreviations: CD, cluster of differentiation; Ct, threshold cycle; HIV, human immunodeficiency virus; MTCT, mother-to-child transmission; min., minimum; max., maximum; cART, combination of antiretroviral therapy; DNA, deoxyribonucleic acid; apH, arterial cord blood pH; RNA, ribonucleic acid; PCR, polymerase chain reaction; MACS, magnetic cell sorting; EDTA, ethylenediamine tetraacetic acid; CS, caesarean section; ITT, intention-to-treat. ⁎ Corresponding author at: University Hospital Frankfurt, Clinic for Children and Adolescents, Division for Neonatology, Theodor Stern Kai 7, D-60590 Frankfurt/Main, Germany. E-mail addresses: [email protected], [email protected] (H. Buxmann). 1 H. Buxmann and A. Reitter authors contributed equally to this work. 2 Current affiliation: Sachsenhausen Hospital, Department of Obstetrics and Prenatal Medicine, Frankfurt/Main. 3 Current affiliation: University Hospital Tübingen, Pediatric Clinic, Department of Neonatology.

http://dx.doi.org/10.1016/j.earlhumdev.2016.06.004 0378-3782/© 2016 Elsevier Ireland Ltd. All rights reserved.

The implementation of preventive interventions for mother-to-child transmission (MTCT) of HIV, namely optimized antiviral therapy of the mother, postnatal prophylaxis of the newborn, avoidance of breastfeeding and appropriate mode of delivery, has resulted in transmission rates of b2% in developed countries in recent decades [1–3]. Regarding mode of delivery, planned caesarean section was the strategy of choice in the era prior to the use of combination antiretroviral therapy (cART) [4–6]. Now that the use of cART is established, the viral load can be suppressed below the limit of detection in the blood of pregnant women. In recent years, large studies have shown no difference in MTCT rates in the setting of successful cART after spontaneous vaginal delivery or planned caesarean section [2,7,8]. Unfortunately, even under optimal conditions, MTCT of HIV still occurs [2], and the reasons are poorly understood [9]. We aimed to study relevant factors other than viral load

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H. Buxmann et al. / Early Human Development 98 (2016) 49–55

that play a crucial role in MTCT. Lin et al. [10] and Kaneda et al. [11] found higher levels of maternal microtransfusions to the newborn after vaginal delivery compared to planned caesarean section in HIVnegative women. Data on the transfer of maternal CD4+ cells to newborns (maternal microchimerism) are not yet available in HIV-positive mothers. Because HIV can persist as proviral DNA in CD4 + cells [12] and reactivate HIV replication even after years of sufficient cART [13], elevated CD4+ materno-foetal cell transfer after spontaneous vaginal delivery could be associated with an elevated risk of MTCT. Therefore, our objective was to evaluate quantitative maternal CD4 + cell microchimerism after spontaneous vaginal delivery compared to planned caesarean section and to determine whether there is an increased risk of MTCT due to proviral DNA transfer in CD4+ cells. 2. Methods 2.1. Overall study design and ethics committee approval This prospective single-centre multidisciplinary study is registered at ClinicalTrials.gov (NCT01450059). The study was conducted in accordance with the latest version of the Declaration of Helsinki. The protocol for the study was reviewed and approved by the Ethics Committee of the Johann Wolfgang Goethe-University Clinic, Frankfurt am Main. Written informed consent was obtained from all parents or single mothers prior to study-related blood draws. According to parental wishes and possible obstetric options, the neonates were born either by planned caesarean section, spontaneous vaginal delivery or unplanned caesarean section after intended spontaneous vaginal delivery. 2.2. Study participants Neonates born during the study period from 31 August 2011 to 30 April 2014 whose mothers were HIV-infected and had normal MTCT risk according to the German Austrian Guidelines [14,15] were considered for enrolment. Women had to have normal MTCT risk because they are the only group of HIV-infected women eligible to give birth by spontaneous vaginal delivery. According to the 2008 guidelines, normal MTCT risk was defined as an HIV viral load ‘below the limit of detection’ at the end of pregnancy [14]; the 2011 guidelines specified that HIV viral load had to be below 50 copies/ml [15]. Exclusion criteria were an elevated or very high risk of MTCT, newborns from underage mothers, infants born at other hospitals, severe perinatal asphyxia, chromosomal defects, severe birth defects, and newborns who were breastfed. All newborns received immediate postnatal support from the attending paediatrician according to the current German Austrian Guidelines [14,15] and daily ward rounds during their hospital stay. Data collection at birth included mode of delivery, maternal CD4 + and CD8+ cells in neonatal blood, weight, length, and head circumference at birth, Apgar score, arterial cord blood pH (a-pH) and sex. HIV-1 RNA was measured by PCR twice in the first year of life, with the second measurement conducted after the third month of life at the earliest. Infants' HIV infection status was classified as ‘HIV uninfected’ after two negative HIV-1 RNA PCR results, a negative HIV antibody test, or a negative Western blot during follow up. Maternal information included age, placenta location at birth, antiretroviral therapy, and CD4+ and CD8+ cell counts, as well as viral load (HIV-1 RNA PCR) within four weeks prior to birth. 2.3. Blood collection Every birth was attended by a paediatrician, who supported the newborns according to the current German Austrian Guidelines [14, 15]. The umbilical cord was clamped twice, near the newborn and the placenta, and was cut between the two clips next to the baby. The

umbilical vein was punctured, and at least 0.5 ml of blood was collected in an ethylenediamine tetraacetic acid (EDTA) tube. From the mothers, 5 ml of blood was collected into an EDTA tube by venous puncture. All probes were cooled immediately to 2–8 °C and processed either in real time during regular working hours or within 72 h at the latest on weekends. 2.4. Microchimerism analysis 2.4.1. Isolation of CD4+ cells As previously published by Willasch et al. [16], CD4+ cell isolation from umbilical cord blood was conducted using the MACS (magnetic cell sorting) technique. Here “Whole Blood MicroBeads” conjugated to a monoclonal anti-human CD4 antibody were used for magnetic cell separation conducted by the AutoMACS separator device (Miltenyi Biotec, Bergisch Gladbach, Germany). The positive fraction was used for DNA isolation as described elsewhere [17]. 2.4.2. Quantification of maternal CD4+ cell microchimerism Maternal microchimerism was quantified by real-time PCR (qPCR, TaqMan chemistry), as published by Willasch et al. [17]. Briefly, 29 sequence polymorphism markers were available for the screening of a maternal specific marker. After primer selection, amplification curves generated from artificial chimeric dilution series, consisting of maternal and foetal DNA, were used for quantification of maternal microchimerism (limit of detection of 0.01%). All probes were measured a minimum of two times. If the results of the two measurements of the same probe differed by less than one threshold cycle (Ct), they led to positive quantifiable or negative results. When the findings of a probe differed repeatedly by one Ct or more, the result was assessed as not quantifiable. 2.5. Extraction and amplification of HIV-specific nucleic acids To determine whether HIV-specific nucleic acids were present in any of the tested compartments, whole DNA from CD4 + cells was used as a source of amplification with HIV-specific primers located in the env region (C2V5). The protocol described here has been used for routine genotypic tropism testing with either viral RNA or proviral DNA as the PCR template. Whole DNA from isolated CD4 + cells was extracted using the QIAamp Viral RNA Kit (Qiagen) as described elsewhere [18]. Amplification of the C2V5 region was performed using the SuperScript™ One Step RT-PCR kit (Invitrogen) according to the manufacturer's recommendations. The reverse primers were 7791 m and 8001 m, and 6501 and 6631 were used as the forward primers. Cycling conditions were as follows: reverse transcription at 50 °C for 30 min, denaturation at 94 °C for 2 min followed by 30 cycles (94 °C 30 s, 50 °C 90 s, 72 °C 180 s), and a final step at 72 °C for 10 min. Afterwards, a nested PCR was run using the Platinum® Taq DNA Polymerase (Invitrogen) according to the manufacturer's recommendations with 7401 as the reverse primer and 7001 as the forward primer [19]. Amplification conditions were denaturation at 95 °C for 3 min, 40 cycles (95 °C 30 s, 55 °C 45 s, 72 °C 180 s), and a final step at 72 °C for 10 min. 2.6. Statistical analyses The required sample size was calculated using preliminary data [20]. With respect to the measured maternal microchimerism of CD4+ cells in the Caesarean Section and the Spontaneous Group, a range of 0.26 was expected. From that, the calculated standard deviation was σ = 0.065 (R ~ 4 * SD). The two random check t-test (α = 0.05, β = 0.20, power = 0.80) required 30 cases (n1 = n2 = 15). Micochimerism results were compared categorical with the Mantel–Haenszel test. Differences in dichotomous variable were calculated with Fisher's exact test, while ordinal and quantitative data were compared with the Wilcoxon


Mann–Whitney U test. P-values b 0.05 were considered statistically significant. Statistical analysis was performed with BIAS 8.4 (Dr. Hanns Ackermann, Biomathematics, Johann Wolfgang Goethe University, Frankfurt/Main, Germany). Data were managed and analysed using Microsoft Excel 2011 (Microsoft Corp., Redmond, Washington, USA), Adobe Creative Suit 5 (Adobe Systems, San José, California, USA), GraphPad Prism 6 (GraphPad® Software, Inc., San Diego, California, USA) and Microsoft Word 2011 (Microsoft Corp., Redmond, Washington, USA). 3. Results During the study period, 74 neonates from 71 HIV-positive mothers were born in our perinatal centre. Seven newborns from six mothers could not be included in the study for one of the following reasons:

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four neonates from three mothers had elevated risk of MTCT prior to birth, and three babies from three mothers had an intended spontaneous vaginal delivery after the recruitment period had closed. Further, 13 parents did not consent to participate in the study. The remaining 54 neonates from 52 mothers were included in our survey. Fifteen neonates from 15 mothers were removed from the study for one of the following reasons: elevated perinatal risk of MTCT according to the current German Austrian Guidelines [14,15] was present in five cases, three cases were lost to follow up, three neonates had improper sampling operations and breastfeeding occurred in one case. In three mother–childpairs, no sequence polymorphism markers could be found, and thus the chimerism analysis could not differentiate between mother and child. As a result, data from 39 newborns from 37 mothers were analysed. Sixteen neonates from 15 mothers were born via planned caesarean section (‘Caesarean Section Group’, CS group). The indications were

Fig. 1. Flow chart of the recruitment process for the maternal microchimerism study.

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request from the HIV-positive expectant mother in six cases, planned repeat caesarean section in four cases, placenta praevia totalis and tubal sterilization in two women each and a viral load approximately 50 copies at the end of pregnancy in one case. Locations of the placentas in this group were as follows: anterior uterine wall in eight cases, posterior uterine wall in five cases and lateral uterine walls in three cases. Seventeen babies from 16 mothers were spontaneously delivered vaginally and allocated to the ‘Spontaneous Group’. In six additional neonates from six mothers, a vaginal delivery was planned, but they underwent unplanned caesarean section (Intention-to-treat [ITT]Spontaneous Group; Fig. 1) due to failure to progress in the first or second stage of labour. Neonates' characteristics are shown and the groups are compared in Table 1. Thirty-five of the 37 (96.6%) mothers in the study population had been seen by an HIV specialist prior to their pregnancy, and HIV was initially diagnosed during pregnancy in two cases (3.4%). The various antiretroviral therapies of the pregnant women are shown in Table 2. 3.1. Maternal CD4+ cell microchimerism in the newborns' umbilical cord blood In 15/17 (88.2%) samples of the Spontaneous Group, no positively quantifiable maternal CD4 + cells (95% CI: 0.00–0.00) were detected in the infants' circulation, including in a pair of diamniotic–dichorionic twins. Two samples provided no quantifiable results. Within the Caesarean Section Group, nine (9/16; 56.3%) newborns were negative for maternal CD4 + microchimerism, including one infant from a set of diamniotic–dichorionic twins. Three samples yielded no quantifiable results (See Table 3). In four (4/16; 25.0%) cases of the Caesarean Section Group, including the second infant from the set of twins in this group, quantifiably positive maternal CD4+ microchimerism was found (range: 0.01–0.66%; 95% CI: − 0.06–0.16; one twin: 0.02%). In three (3/4; 75%) infants, the placenta was located on the anterior uterine wall, two of them were located somewhat to the right of the middle of the anterior uterine wall. The maternal CD4+ microchimerism of the Caesarean Section Group significantly differed from the Spontaneous Group (P = 0.02; Fig. 2). In the ITT-Spontaneous Group, 19/23 (82.6%) samples were negative, one (1/23; 4.3%) was positive (0.05% maternal cells; 95% CI: − 0.02–0.04) and three showed no quantifiable results (Table 2). This result differed significantly from the Caesarean Section Group (P = 0.04; Fig. 3). 3.2. Mothers' CD4+ cell count at the time of delivery in relation to the cases with positively quantifiable maternal CD4+ microchimerism The maternal CD4+ cell counts at the time of delivery were as follows: 30 mothers had CD4+ cells N 350/μl; in two cases (2/30; 6.7%) their offspring showed positively quantifiable maternal CD4 + microchimerism. Six mothers in our study had 200–350/μl CD4+ cells

at delivery, whereas three cases (3/6; 50%) had a positive quantifiable microchimerism. There were no statistically significant differences in the maternal CD4+ cell counts between the study groups (Table 2). 3.3. Amplification of HIV-specific nucleic acid in CD4+ cells No HIV-specific nucleic acid was detected in any of the CD4+ cell probes from the newborns' umbilical cord blood in the Caesarean Section Group, the Spontaneous Group or the ITT-Spontaneous Group. 3.4. Maternal CD8 + cell microchimerism in newborns' umbilical cord blood In the Spontaneous Group, 15/17 (88.2%) newborns showed no maternal CD8+ cells in their circulation, whereas one sample (1/17; 5.9%) yielded no quantifiable results, and one yielded a positive quantifiable result (0.09% maternal cells). Nine (9/16; 56.3%) of the specimens in the Caesarean Section Group were negative for maternal CD8 + microchimerism, three showed no quantifiable results and four (4/16; 25.0%) showed maternal CD8+ microchimerism (0.01–0.17% maternal cells). The maternal CD8 + microchimerism differed significantly between the two groups (P = 0.04). In the ITT-Spontaneous Group, 19/23 (82.6%) samples were negative, three (3/23; 13.0%) were positive (0.09–0.24% maternal cells) and one showed no quantifiable result. The difference in the maternal CD8+ microchimerism between these groups did not reach statistical significance (P = 0.14). 3.5. Follow up Thirty-eight of 39 (97.4%) newborns received postnatal antiviral prophylaxis with Zidovudine for four weeks, which follows the current guidelines [14,15]. Parents of one newborn declined this medication. All neonates had follow-up visits in the outpatient clinic and were not infected with HIV during the follow up. 4. Discussion Among women using antiviral therapy and who are not breastfeeding, MTCT occurs mostly at the very end of pregnancy during labour and birth [21]. In addition to ascending HIV infection via the amniotic structures [21] and direct contact with maternal blood or vaginal fluid during birth, MTCT may occur via the transfer of minimal amounts of maternal CD4+ cells. The placental transmission of HIV-1 is a complex and incompletely understood process [9]. Independent of the context of MTCT, it has been described that maternal micro transfusions to the newborns are higher after spontaneous vaginal delivery compared

Table 1 Neonatal characteristics and postnatal antiretroviral therapy in comparison between the study groups. Parameter

Spontaneous Group (n = 17)

Planned Sectio Caesarean Group (n = 16)

P*

Unplanned Sectio Caesarean Group (n = 6)

ITT Spontaneous Group (n = 23)

P**

Gestational age [Med.] Birth weight (g) Length at birth (cm) Head girth at birth (cm) 1 min APGAR-Score [Med.] 5 min APGAR-Score [Med.] 10 min APGAR-Score [Med.] Arterial cord blood-pH (a-pH) Sex (♀:♂)

40 + 3 (37 + 0–42 + 0) 3370 (2670–4430) 51 (49–54) 35 (31–37) 9 (5–10) 10 (8–10) 10 (9–10) 7.25 (7.08–7.42) 4:13

39 + 4 (36 + 4–41 + 5) 3169 (2320–3970) 51 (47–56) 35 (32.5–38) 9 (7–9) 10 (8–10) 10 (9–10) 7.31 (7.17–7.47) 5:11

0.29 0.37 0.52 0.88 0.38 0.63 0.27 0.053 0.46

40 + 4 (39 + 0–41 + 4) 3732 (3000–4130) 54 (50–58) 35 (33–37) 9 (9–9) 10 (10−10) 10 (10–10) 7.25 (7.19–7.33) 4:2

40 + 3 (37 + 0–42 + 0) 3464 (2670–4430) 52 (49–58) 35 (31–37) 9 (5–10) 10 (8–10) 10 (9–10) 7.25 (7.08–7.42) 8:15

0.19 0.13 0.22 0.88 0.20 0.36 0.15 0.02 0.55

6

22

Antiretroviral regimen of the newborns Zidovudin for four weeks 16

16

Mean values are shown, except where median [Med.] is indicated. In parentheses range is indicated; P* = Spontaneous Group versus Planned Sectio Caesarean Group; P** = ITT Spontaneous Group versus Planned Sectio Caesarean Group; ITT = intention-to-treat.


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Table 2 Maternal CD4+ cell count and antiretroviral therapy regimen in comparison between the study groups. Parameter

Spontaneous Group (n = 16)

Planned Sectio Caesarean Group (n = 15)

P*

CD4+ cells prior to birth b200 cells/μl 200–350 cells/μl N350 cells/μl Absolute cell count (cells/μl)

1 2 13 576 (167–1212)

0 3 12 487 (242–767)

0 1 5 0.53 421 (277–549)

1 3 18 541 (167–1212)

0.82

Viral load prior to birth b20 copies/ml 20–50 copies/ml 51–65 copies/ml CD8+ cells prior to birth (cells/μl)

13 2 1 1090 (492–2103)

10 4 1 840 (500–1390)

4 1 1 0.20 1094 (678–2098)

17 3 2 1091 (492–2103)

0.23

Antiretroviral regimen antepartum Tenovofir/Emtricitabin + Lopinavir Tenovofir/Emtricitabin + Zidovudin Tenovofir/Emtricitabin + Atazanvir Tenovofir/Emtricitabin + Nevirapin Lopinavir + Raltegravir Tenovofir/Emtricitabin + Ritonavir + Atazanvir Tenovofir/Emtricitabin + Ritonavir + Darunavir Raltegravir + Lopinavir + Saquinavir Raltegravir + Atazanvir + Ritonavir Zidovudin + Ritonavir + Maraviroc Ritonavir + Tenovofir/Emtricitabin + Lamivudin +

3 0 1 0 1 7 1 0 0 1 1

4 1 0 1 1 3 0 2 1 0 0

1 2 1 0 1 1 0 0 0 0 0

4 2 2 0 2 8 1 0 0 1 1

Zidovudin Emtricitabin + Atazanvir + Raltegravir Ritonavir + Atazanvir + Raltegravir +

1 0

1 1

0 0

1 0

3

2

1

4

Unplanned Sectio Caesarean Group (n = 6)

ITT Spontaneous Group (n = 22)

P**

Lamivudin/Zidovudin Antiretroviral regimen intrapartum Zidovudin

P* = Spontaneous Group versus Planned Sectio Caesarean Group; P** = ITT Spontaneous Group versus Planned Sectio Caesarean Group; ITT = intention-to-treat.

to primary caesarean section [10,11]; additionally, transplacental leakage is highest around birth [22]. To evaluate a possible transfer of maternal CD4 + cells that could contain proviral DNA, we evaluated maternal CD4 + cell microchimerism after spontaneous vaginal delivery compared to planned caesarean section. For the microchimerism analysis, we used a combination of realtime PCR and sequence polymorphism markers [17], which is a highly sensitive (detection limit: 0.01% maternal cells) and informative (informative markers for 95% of our primarily included patients were available) method. 4.1. Maternal microchimerism In our study population, there were no positively quantifiable maternal CD4 + cells in the blood of newborns in the Spontaneous Group, which differed significantly (P = 0.02) from the four newborns with positive quantifiable maternal CD4+ microchimerism in the Caesarean Section Group. There was also significantly lower maternal CD4+ microchimerism in the ITT-Spontaneous Group compared to the Caesarean Section Group (P = 0.04). Therefore, in our study population, neither spontaneous vaginal delivery nor intended vaginal delivery with

unplanned caesarean section was associated with a higher risk of maternal CD4+ microchimerism. The reason for the higher rate of maternal CD4+ microchimerism in the Caesarean Section Group compared to the Spontaneous Group remains unclear and is in contrast with the results of Lin et al. [10] and Kaneda et al. [11]. We found that in three (3/4; 75%) of the cases with positively quantifiable maternal CD4+ microchimerism in our Caesarean Section Group, the placenta was located on the anterior wall of the uterus. Therefore, we hypothesize that potential manipulation or lesion of these anterior wall placentas during the section procedure may have led to the transfer of maternal blood to the offspring. Regarding the mothers' CD4+ cell count at the time of delivery, we found a negative correlation with maternal CD4+ microchimerism in the newborns. Specifically, in three (3/6; 50%) mothers with 200–350/μl CD4+ cells at birth, positively quantifiable CD4 + microchimerism was found in their infants, whereas only two (2/30; 6.7%) cases with maternal CD4 + cells N 350/μl around birth had positively quantifiable microchimerism in the newborns. These results are in agreement with the publication of Kwiek et al. [23], who found a negative correlation between CD4 + cell counts in HIV-infected mothers and the amount of maternal DNA in foetal cord blood. Our findings show higher permeability of the blood–placental barrier in HIVinfected mothers with lower CD4 + cell counts.

Table 3 Quantifiable and non-quantifiable maternal CD4+ microchimerism results. Maternal CD4+ microchimerism

Spontaneous Group (spontaneous delivery) (n = 17)

Caesarean Section Group (planned sectio caesarean) (n = 16)

ITT Spontaneous Group (spontaneous delivery + unplanned sectio caesarean) (n = 23)

Negative Not quantifiable Positive

15 2 0

9 3 4

19 3 1

ITT = intention to treat; Not quantifiable: if the measurements of one probe yielded different results of one threshold cycle (Ct) or more, the analysis was assessed as not quantifiable.

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Fig. 2. Maternal CD4+ microchimerism in the Spontaneous and the Caesarean Section Group.

Similar to our results of CD4+ microchimerism, we found statistically significantly more maternal CD8+ cells in the newborns' circulation in the Caesarean Section Group compared to the Spontaneous Group (P = 0.04). In relation to the ITT-Spontaneous Group, there were more cases of maternal CD8+ microchimerism in the Caesarean Section Group, but this difference did not reach statistical significance (P = 0.14). In summary, the CD8+ microchimerism results affirm our CD4 + microchimerism results, with the lowest amount of maternal

cells in the newborns' circulation following spontaneous vaginal delivery. 4.2. Limitations of the study Limitations of this study include the relatively few patients with positively quantifiable maternal CD4 + microchimerism. Therefore, it would be reasonable to verify these results in further studies with larger

Fig. 3. Maternal CD4+ microchimerism in the ITT-Spontaneous and the Caesarean Section Group.


cohorts and, if possible, with an even more sensitive method of microchimerism analysis [24]. 5. Conclusion This is the first study to investigate maternal CD4 + cell microchimerism in newborns of HIV-infected mothers with normal MTCT risk after spontaneous vaginal delivery and planned caesarean section. Our results show lower rates of maternal CD4 + cells in the blood of the newborns after spontaneous vaginal delivery and intended spontaneous vaginal delivery with unplanned caesarean section. Therefore, the data presented suggests that intrapartum materno-fetal CD4 +-cell transfer does not contribute to an increased rate of MTCT in HIV-positive women with low viral load who intend to deliver vaginally. Conflicts of interest and source of funding This work was supported by an unrestricted grant of GILEAD Förderprogramm Infektiologie; GILEAD Sciences GmbH, 82152 Martinsried, Germany. We, the authors of this article, certify that there is neither a conflict of interest with any financial organization nor a non-financial interest in the subject matter discussed in this manuscript. Acknowledgements Our sincere thanks go to all participating families for their consent and support, which made this study possible. We gratefully acknowledge all staff members of our multidisciplinary team who provided clinical and laboratory support for this study. References [1] A. Dorenbaum, C.K. Cunningham, R.D. Gelber, M. Culnane, L. Mofenson, P. Britto, et al., Two-dose intrapartum/newborn nevirapine and standard antiretroviral therapy to reduce perinatal HIV transmission: a randomized trial, JAMA 288 (2002) 189–198. [2] C.L. Townsend, M. Cortina-Borja, C.S. Peckham, A. de Ruiter, H. Lyall, P.A. Tookey, Low rates of mother-to-child transmission of HIV following effective pregnancy interventions in the United Kingdom and Ireland, 2000–2006, AIDS 22 (2008) 973–981. [3] M. Pammi, J.E. Garley, E.M. Carlin, Pregnancy outcomes of HIV-positive women in a tertiary centre in the UK, J. Obstet. Gynaecol. 35 (2) (2015) 136–138. [4] Collaboration TEMOD, Elective caesarean-section versus vaginal delivery in prevention of vertical HIV-1 transmission: a randomised clinical trial, Lancet 353 (1999) 1035–1039. [5] K.L. Dominguez, M.L. Lindegren, P.J. D'Almada, V.B. Peters, T. Frederick, T.A. Rakusan, et al., Increasing trend of cesarean deliveries in HIV-infected women in the United States from 1994 to 2000, J. Acquir. Immune Defic. Syndr. 33 (2003) 232–238.

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