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Neonatal and neurodevelopmental outcome of children aged 3–10years born following assisted oocyte activation

Published:August 28, 2013DOI:https://doi.org/10.1016/j.rbmo.2013.07.013

      Abstract

      Assisted oocyte activation (AOA) using a calcium ionophore has been used for more than a decade following intracytoplasmic sperm injection (ICSI) fertilization failure. However, since AOA does not mimic precisely the physiological fertilization process, concerns exist about its use in human assisted reproduction. This study assessed the neonatal and neurodevelopmental outcome of children aged ⩾3 years who had been born following AOA in our centre. Twenty-one children participated in the study (81% response rate; mean age 63.6 ± 21.07 months). Neonatal data were collected via questionnaires. Neurodevelopmental outcome was tested using the Reynell Developmental Language Scales or Clinical Evaluation of Language Fundamentals, Wechsler Preschool and Primary Scale of Intelligence or Wechsler Intelligence Scale for Children, and the Movement Assessment Battery for Children III. Behaviour was scored by the Social Communication Questionnaire, the Child Behaviour Checklist and the Teachers Report Form. For all tests and questionnaires, the mean outcomes lay within the expected ranges. These are first data on the developmental outcome of AOA children. The high response rate and the robustness of the tests support the data, which are reassuring although still considered preliminary. Therefore, AOA should still be performed only in selected couples.
      Intracytoplasmic sperm injection (ICSI) is a very efficient technique used in couples suffering from severe male subfertility. However, in a minority of those couples, ICSI does not lead to normal fertilization rates. In those couples, only very few or often no embryos at all are available for embryo transfer. Assisted oocyte activation (AOA) is a highly specialized fertilization technique which can be added to conventional ICSI to overcome fertilization failure in those couples. Since the late 1990s, many centres have been performing AOA in this rare group of patients. However, until adequate scientific evidence is provided regarding its safety and efficacy, AOA cannot yet be considered an established treatment. We assessed the outcome of children aged ⩾3 years who had been born following AOA in our centre. Twenty-one children participated in our study (81% response rate; mean age 63.6 ± 21.07 months). We used questionnaires as well as age-adjusted tests to assess neonatal data, the cognitive skills, language skills, motor skills and behaviour of those children. For all tests and questionnaires, the outcomes were within the expected ranges. These are first data on the developmental outcome of AOA children. The high response rate and the robustness of the tests support the data, which are reassuring. However, because the total number of children born following AOA is still low, these results should be considered preliminary. Therefore, AOA should still be performed only in selected couples.

      Keywords

      Introduction

      Intracytoplasmic sperm injection (ICSI) is typically used in cases of severe semen dysfunction or following failure of conventional IVF. In general, fertilization rates of 70–75% are obtained (
      • Palermo G.D.
      • Neri Q.V.
      • Takeuchi T.
      • Rosenwaks Z.
      ICSI: where we have been and where we are going.
      ). In contrast, failed fertilization still occurs in 1–3% of ICSI cycles, even when a sufficient number of mature oocytes and motile spermatozoa are available (
      • Esfandiari N.
      • Javed M.H.
      • Gotlieb L.
      • Casper R.F.
      Complete failed fertilization after intracytoplasmic sperm injection–analysis of 10 years’ data.
      ,
      • Flaherty S.P.
      • Payne D.
      • Swann N.J.
      • Mattews C.D.
      Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection.
      ). The principal reason for fertilization failure following ICSI is defective oocyte activation (
      • Flaherty S.P.
      • Payne D.
      • Matthews C.D.
      Fertilization failures and abnormal fertilization after intracytoplasmic sperm injection.
      ,
      • Liu J.
      • Nagy Z.
      • Joris H.
      • Tournaye H.
      • Smitz J.
      • Camus M.
      • Devroey P.
      • Van Steirteghem A.
      Analysis of 76 total fertilization failure cycles out of 2732 intracytoplasmic sperm injection cycles.
      ,
      • Rawe V.Y.
      • Olmedo S.B.
      • Nodar F.N.
      • Doncel G.D.
      • Acosta A.A.
      • Vitullo A.D.
      Cytoskeletal organization defects and abortive activation in human oocytes after IVF and ICSI failure.
      ). The primary cellular event leading to mammalian oocyte activation is the initiation of intracellular calcium oscillations in the oocyte’s cytosol. Although doubts have risen recently, substantial evidence suggests that the spermatozoon supplies an activating protein, phospholipase C zeta, into the oocyte’s cytosol which triggers calcium oscillations via an inositol-3-phosphate-mediated pathway (
      • Aarabi M.
      • Yu Y.
      • Xu W.
      • Tse M.Y.
      • Pang S.C.
      • Yi Y.J.
      • Sutovsky P.
      • Oko R.
      The testicular and epididymal expression profile of PLCζ in mouse and human does not support its role as a sperm-borne oocyte activating factor.
      ,
      • Saunders C.M.
      • Larman M.G.
      • Parrington J.
      • Cox L.J.
      • Royse J.
      • Blayney L.M.
      • Swann K.
      • Lai F.A.
      PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development.
      ). The oocyte responds to this by the calcium-dependent activation of several down-stream pathways, which are necessary for successful oocyte activation, pre- and post-implantation embryo development (
      • Ducibella T.
      • Huneau D.
      • Angelichio E.
      • Xu Z.
      • Schultz R.M.
      • Kopf G.S.
      • Fissore R.
      • Madoux S.
      • Ozil J.P.
      Egg-to-embryo transition is driven by differential responses to Ca(2+) oscillation number.
      ,
      • Kashir J.
      • Heindryckx B.
      • Jones C.
      • De Sutter P.
      • Parrington J.
      • Coward K.
      Oocyte activation, phospholipase C zeta and human infertility.
      ,
      • Parrington J.
      • Davis L.C.
      • Galione A.
      • Wessel G.
      Flipping the switch: how a sperm activates the egg at fertilization.
      ). Therefore, both the spermatozoon and the oocyte are considered to be responsible for successful oocyte activation and embryo development. In this study centre, the mouse oocyte activation test (MOAT), a heterologous ICSI model, is performed to diagnose the activation capacity of a patient’s spermatozoa (
      • Rybouchkin A.
      • Dozortsev D.
      • Pelinck M.J.
      • De Sutter P.
      • Dhont M.
      Analysis of the oocyte activation capacity and chromosomal complement of roundheaded human spermatozoa by their injection into mouse oocytes.
      ).
      Several reports show that most, but not all, of the couples suffering from ICSI failure benefit from the application of ICSI combined with assisted oocyte activation (AOA) (
      • Heindryckx B.
      • Van der Elst J.
      • De Sutter P.
      • Dhont M.
      Treatment option for sperm- or oocyte-related fertilization failure: assisted oocyte activation following diagnostic heterologous ICSI.
      ,
      • Heindryckx B.
      • De Gheselle S.
      • Gerris J.
      • Dhont M.
      • De Sutter P.
      Efficiency of assisted oocyte activation as a solution for failed intracytoplasmic sperm injection.
      ,
      • Montag M.
      • Köster M.
      • van der Ven K.
      • Bohlen U.
      • van der Ven H.
      The benefit of artificial oocyte activation is dependent on the fertilization rate in a previous treatment cycle.
      ,
      • Vanden Meerschaut F.
      • Nikiforaki D.
      • De Gheselle S.
      • Dullaerts V.
      • Van den Abbeel E.
      • Gerris J.
      • Heindryckx B.
      • De Sutter P.
      Assisted oocyte activation is not beneficial for all patients with a suspected oocyte-related activation deficiency.
      ). Different activating agents have been used successfully in human assisted reproduction, e.g. calcium ionophore, electrical pulses and strontium chloride (
      • Baltaci V.
      • Ayvaz O.U.
      • Unsal E.
      • Aktas Y.
      • Baltaci A.
      • Turhan F.
      • Ozcan S.
      • Sönmezer M.
      The effectiveness of intracytoplasmic sperm injection combined with piezoelectric stimulation in infertile couples with total fertilization failure.
      ,
      • Kyono K.
      • Kumagai S.
      • Nishinaka C.
      • Nakajo Y.
      • Uto H.
      • Toya M.
      • Sugawara J.
      • Araki Y.
      Birth and follow-up of babies born following ICSI using SrCl2 oocyte activation.
      ,
      • Rybouchkin A.
      • Van der Straeten F.
      • Quatacker J.
      • De Sutter P.
      • Dhont M.
      Fertilization and pregnancy after assisted oocyte activation and intra-cytoplasmic sperm injection in a case of round-headed sperm associated with deficient oocyte activation capacity.
      ,
      • Yanagida K.
      • Morozumi K.
      • Katayose H.
      • Hayashi S.
      • Sato A.
      Successful pregnancy after ICSI with strontium oocyte activation in low rates of fertilization.
      ). However, until adequate scientific evidence is provided regarding its safety and efficacy, ICSI combined with AOA cannot yet be considered an established treatment. The current study centre has been performing ICSI combined with AOA during more than a decade, using the injection of calcium chloride (CaCl2) along with the spermatozoon into the oocyte, followed by a 2-fold calcium ionophore exposure of the injected oocyte (
      • Rybouchkin A.
      • Van der Straeten F.
      • Quatacker J.
      • De Sutter P.
      • Dhont M.
      Fertilization and pregnancy after assisted oocyte activation and intra-cytoplasmic sperm injection in a case of round-headed sperm associated with deficient oocyte activation capacity.
      ) and has built up a large experience in the treatment of this rare but challenging group of patients. Rewarding results have been obtained regarding fertilization and pregnancy rates (
      • Heindryckx B.
      • De Gheselle S.
      • Gerris J.
      • Dhont M.
      • De Sutter P.
      Efficiency of assisted oocyte activation as a solution for failed intracytoplasmic sperm injection.
      ). Nevertheless, the artificially induced calcium rises do not mimic precisely the physiologically sperm-induced calcium oscillations and little is known yet about the possible adverse effects of ionophores on post-implantation embryo development. Therefore, concerns exist about the use of a calcium ionophore as oocyte-activating agent in human assisted reproduction. However, the influence of ionomycin exposure during the zygote stage on pre- and post-implantation development has been evaluated in the mouse (
      • Heytens E.
      • Soleimani R.
      • Lierman S.
      • De Meester S.
      • Gerris J.
      • Dhont M.
      • Van der Elst J.
      • De Sutter P.
      Effect of ionomycin on oocyte activation and embryo development in mouse.
      ). Zygotes treated with ionomycin showed normal pre- and post-implantation development, and normally developing and fertile pups were born originating from ionomycin-treated zygotes. Nevertheless, since these results were obtained from highly fertile animals, results should not be extrapolated without caution to a subfertile human population (
      • Harper J.
      • Magli M.C.
      • Lundin K.
      • Barratt C.L.R.
      • Brison D.
      When and how should new technology be introduced into the IVF laboratory?.
      ). Even though the exposure to the calcium ionophore for the purpose of AOA is short and limited to the zygote stage, it is appropriate to examine possible adverse effects of ionomycin in children born following AOA.
      Since the main aim of a IVF-clinic is to ensure the birth of healthy babies, the goal of this study was to report on the neonatal outcome of children born following AOA and on their cognitive, language and motor development and behaviour from the age of 3 years onwards and to compare their outcomes with age-matched norms.

      Materials and methods

      Participants

      All children born following AOA at University Hospital Ghent from October 2001 until September 2008 were eligible for this study. Inclusion criteria for the neurodevelopmental and behavioural assessment were: (i) age ⩾3 years; (ii) the ability to visit the outpatient clinic; and (iii) Dutch as the mother tongue. Written informed consent was given by the parents and the study was approved by the Ethical Committee of the Ghent University Hospital (EC number: 2011/188; Belgian registry number: B670201110978, approved 19 May 2011).

      Assisted oocyte activation

      ICSI combined with AOA was performed as previously described (
      • Heindryckx B.
      • De Gheselle S.
      • Gerris J.
      • Dhont M.
      • De Sutter P.
      Efficiency of assisted oocyte activation as a solution for failed intracytoplasmic sperm injection.
      ). Briefly, a spermatozoon was injected into the oocyte together with a small amount of 0.1 mol/l CaCl2 (corresponding to the diameter of the oocyte). Thereafter, the oocytes were incubated for 30 min at 37°C in a 6% CO2 air atmosphere. Next, the oocytes were exposed to a calcium ionophore (10 μmol/l ionomycin, I9657; Sigma-Aldrich, Bornem, Belgium) for 10 min. Following ionophore exposure, the oocytes were washed with Cook Cleavage medium (Cook Ireland, Limerick, Ireland) and were incubated again. After another 30 min, the calcium ionophore treatment was repeated for 10 min. Finally, the oocytes were washed and incubated overnight under 5% O2.

      Neonatal data

      A questionnaire concerning obstetric and neonatal parameters was filled in by the parents shortly after the birth of their child. This questionnaire was retrieved from the patients’ file at the moment of this study and informed consent was given by the parents to use this questionnaire for data analysis. Additionally, some parents were consulted on the day of the neurodevelopmental assessment if data were missing or unclear.

      Follow up by assessment

      All children aged ⩾3 years were invited to the study centre for neurodevelopmental follow up using a set of three standardized tests. For each child, all tests took place on one single day and were performed with a period of rest in between. The order of testing could be different due to planning. The cognitive, language and motor development tests were performed by two clinical psychologists, one speech therapist and one physiotherapist, respectively.

      Cognitive development

      To assess cognitive development, the Wechsler Preschool and Primary Scale of Intelligence (WPPSI-III-NL, ⩽7 years) or the Wechsler Intelligence Scale for Children (WISC-III-NL) was used (
      • Hendriksen J.
      • Hurks P.
      WPSSI-III-NL Wechsler Preschool and Primary Scale of Intelligence.
      ,
      • Kort W.
      • Schittekatte M.
      • Bosmans M.
      • Compaan E.L.
      • Dekker P.H.
      • Vermeir G.
      • Verhaeghe P.
      WISC-III-NL, Wechsler Intelligence Scale for Children. Handleiding en verantwoording.
      ). Both tests measure verbal IQ (VIQ), performance IQ (PIQ) and full-scale IQ (FSIQ) and can be completed without reading or writing. Dutch/Flemish norms were used for both tests.

      Language development

      Language skills were assessed by the Reynell Developmental Language Scales (Reynell Taalontwikkelingsschalen, RTOS) or by the Clinical Evaluation of Language Fundamentals (CELF-IV-NL) (
      • Kort W.
      • Schittekatte M.
      • Compaan E.
      Clinical Evaluation of Language Fundamentals.
      ,
      • Schaerlaekens A.
      • Zink I.
      • Van Ommeslaeghe K.
      Reynell Taalontwikkelingsschalen RTOS: Handleiding.
      ). The RTOS is used for children <5 years and is a Dutch/Flemish norm-based practical tool for everyday clinical use. The CELF-IV-NL is used for children aged ⩾5 years. For both tests, the outcomes are percentile scores and include two scales next to a total percentile score: the comprehension and the expressive scale.

      Motor development

      The Movement Assessment Battery for Children (Movement ABC-II-NL) was used to identify and evaluate movement problems (
      • Henderson S.E.
      • Sugden D.A.
      • Barnet A.L.
      Movement Assessment Battery for Children-II-NL.
      ). It provides a comprehensive assessment of movement competence on age-appropriate tasks within three subtests: manual dexterity, ball skills and static and dynamic balance. Outcome score is a percentile score based on Flemish/Dutch norms. Additionally, a questionnaire regarding motor skills in everyday life was given to the parents (Vragenlijst voor Motorische Vaardigheden van Kleuters (VMVK, <5 years) or Coördinatievragenlijst Voor Ouders (CVO, ⩾5 years)). The CVO was officially translated from Developmental Coordination Disorder Questionnaire (
      • Wilson B.N.
      • Kaplan B.J.
      • Crawford S.G.
      • Campbell A.
      • Dewey D.
      Reliability and validity of a parent questionnaire on childhood motor skills.
      ).

      Follow up by questionnaires

      The behaviour of the AOA children aged ⩾3 years was assessed by three standardized questionnaires. The Social Communication Questionnaire (SCQ) is a worldwide-used screening tool for autism spectrum disorders (ASD) (
      • Rutter M.
      • Bailey A.
      • Lord C.
      Social Communication Questionnaire (SCQ).
      ,
      • Warreyn P.
      • Raymaekers R.
      • Roeyers H.
      Handleiding vragenlijst sociale communicatie.
      ). Furthermore, the Child Behaviour Checklist (CBCL) and the Teacher’s Report Form (TRF) were used. These questionnaires are identical, filled in by the parent and teacher, respectively, on which the child is rated on various behavioural and emotional aspects. The CBCL is one of the most widely used standardized measures in child psychology for evaluating maladaptive behavioural and emotional problems in children (
      • Achenbach T.M.
      • Rescorla L.A.
      Manual for the ASEBA School-age Forms and Profiles. Research Centre for Children, Youth and Families.
      ). For the CBCL, the usefulness of existing US norms within a Flemish community sample were explored before and the reliability was excellent (
      • Braet C.
      • Callens J.
      • Schittekatte M.
      • Soyez V.
      • Druart C.
      • Roeyers H.
      Assessing emotional and behavioural problems with the child behaviour checklist: exploring the relevance of adjusting the norms for the Flemisch community.
      ). In addition to the total problem score, the CBCL and TRF give two broad syndrome scores: internalizing and externalizing behaviour. All the above mentioned questionnaires were in Dutch and filled in by one or both parents, except for the TRF, which was filled in by the teacher of the child. The TRF was a non-mandatory questionnaire.

      Statistical analysis

      To compare birthweight and gestational age between the singletons and twins, the Mann–Whitney U-test was used. The Fisher’s Exact test was used to compare the amount of birth defects in the study population to the amount of birth defects present in a large ICSI population (
      • Davies M.J.
      • Moore V.M.
      • Willson K.J.
      • Van Essen P.
      • Priest K.
      • Scott H.
      • Haan E.A.
      • Chan A.
      Reproductive technologies and the risk of birth defects.
      ). The Mann–Whitney U-test was used to compare mean results on the developmental tests between study groups. The Wilson’s confidence interval for a single proportion method was used to calculate the 95% confidence intervals around the proportion of children with test scores within pathological thresholds (
      • Altman D.S.
      • Machin D.
      • Bryant T.
      • Gardner S.
      Statistics with Confidence: Confidence Intervals and Statistical Guidelines.
      ). The Statistical Package for the Social Sciences version 20 (SPSS Statistics, IBM Corp, NY, USA) and R version 2.15.1 (The R Foundation for Statistical Computing) were used for statistical analysis. A P-value < 0.05 was considered statistically significant.

      Results

      Study population

      From 2001 onwards, 53 pregnancies were achieved following AOA in this study centre, of which 41 ended in childbirth (77.4% ongoing pregnancy rate). Six out of those 41 pregnancies were twin pregnancies (14.6%). Eight spontaneous miscarriages (15.1%) and three biochemical pregnancies (5.7%) occurred. One pregnancy was interrupted because of the fetus carrying trisomy 21. From those 47 children born following AOA, 26 were aged 3 years or older at the time of this study and therefore eligible for this study. Twenty-one out of those 26 children aged 3–11 years could be included in the study (81% response rate). The mean ± standard deviation and median (range) age of the included children was 63.6 ± 21.07 and 62.0 (38–124) months, respectively. The mean maternal and paternal age at childbirth was 32 ± 3.0 years and 34 ± 3.6 years, respectively. Two singleton sisters had a mother with Asian descent. All other parents were Caucasians. Two-thirds of the parents had a higher level of education (beyond 18 years of age). All children lived together with both their biological parents.
      The fertility history and indications for AOA are shown in Table 1. In 11 couples, the indication for AOA was low or zero fertilization in previous conventional ICSI cycles, whereas for the remaining three couples, AOA had been advised because of total globozoospermia, a condition in which the sperm acrosome is lacking and typically leading to very low or zero fertilization rates using conventional ICSI. Without AOA the mean fertilization rate in this group of 14 couples was 19.9%, whereas following AOA treatment the fertilization rate was restored to 74.1%. A mean of 2.1 ± 1.75 conventional ICSI cycles were performed in these couples before they were referred to this study centre. Pregnancies following AOA treatment were achieved after a mean of 2.5 ± 1.16 fresh AOA cycles. For 12 out of 14 couples, a diagnostic MOAT was performed before proceeding to AOA treatment. Nine of the couples were classified into MOAT group 1 or 2, which strongly suggests a sperm deficiency (
      • Vanden Meerschaut F.
      • Leybaert L.
      • Nikiforaki D.
      • Qian C.
      • Heindryckx B.
      • De Sutter P.
      Diagnostic and prognostic value of calcium oscillatory pattern analysis for patients with ICSI fertilization failure.
      ).
      Table 1Fertility history and indications for AOA.
      CharacteristicStudy population (n =14 patients)
      Fertility history
      Data for all 14 parent couples.
       Conventional ICSI without pregnancy
        Cycles2.1 ± 1.75
        Fertilization rate (%)19.9 ± 19.30
       ICSI + AOA
        Cycles2.5 ± 1.16
        Fertilization rate (%)74.1 ± 20.81
      Indication to perform AOA
      Data for all 14 parent couples.
       Zero fertilization following conventional ICSI3 (21.4)
       Low fertilization (<33.3%) following conventional ICSI8 (57.1)
       Globozoospermia3 (21.4)
      MOAT results
      Data for 12 couples.
       0–20% activation: certain sperm deficiency5 (41.7)
       21–84% activation: probable sperm deficiency4 (33.3)
       85–100% activation: no sperm deficiency3 (25.0)
      Values are mean ± standard deviation or n (%).
      AOA = assisted oocyte activation; ICSI = intracytoplasmic sperm injection; MOAT = mouse oocyte activation test.
      a Data for all 14 parent couples.
      b Data for 12 couples.

      Neonatal outcome and birth defects

      Eleven singletons and 10 twins born following ICSI combined with AOA were included in this study. Neonatal data were retrieved for all of the included children (Table 2). The birthweight of the singletons was significantly higher compared with the twins (P < 0.01). Seven out of 21 children (33.3%) were born following a Caesarean section, four of whom were twins. Mean gestational age at birth was 39.4 ± 0.92 weeks and 36.8 ± 2.53 weeks for the singletons and twins, respectively (P < 0.001). Five children (solely twins) were admitted to the neonatal intensive care unit (NICU) (mean duration of stay 35 ± 18.8 days, range 1–44 days). Reasons for admission to the NICU were: prematurity (n = 2), poor condition of the mother (n = 2) and low temperature (n = 1). Birth defects were diagnosed in three children (14.3%, 95% CI 5.0–34.6%) at toddler age. In one child, a small arachnoid cyst was found by chance at the age of three. The condition is considered to be benign and did not require any intervention. In another child, a pectus excavatum and genu varum were found. In a third child, vesico-ureteral reflux was diagnosed at the age of two.
      Table 2Neonatal data.
      OutcomeSingletons (n =11)Twins (n =10)Total (n =21)
      Obstetric
       Delivery <37 weeks0 (0)2 (20.0)2 (9.5)
       Vaginal delivery8 (72.7)6 (60.0)14 (66.7)
       Caesarean section3 (27.3)4 (40.0)7 (33.3)
      Neonatal
       Birthweight (g)3623 ± 324.5
      P<0.01 (Student’s t-test).
      (3085–4140)
      2696 ± 676.7
      P<0.01 (Student’s t-test).
      (1640–3450)
      3181 ± 695.5 (1640–4140)
       Birthweight <2500 g0 (0)3 (30)3 (14.3)
       APGAR <7 at 5 min0 (0)0 (0)0 (0)
       Neonatal intensive care0 (0)5 (50.0)5 (23.8)
      Duration (days)
       ⩽70 (0)1 (10.0)1 (4.8)
       >70 (0)4 (40.0)
      Two of these children were admitted to NICU because of severe illness of their mother (aorta dissection).
      4 (19.0)
       Perinatal mortality0 (0)0 (0)0 (0)
      Gender
       Female8 (72.7)6 (60.0)14 (66.7)
       Male3 (27.3)4 (40.0)7 (33.3)
      Values are n (%) or mean ± standard deviation (range).
      a P < 0.01 (Student’s t-test).
      b Two of these children were admitted to NICU because of severe illness of their mother (aorta dissection).

      Non-respondent analysis

      The five children who did not participate were excluded because of: (i) a mother tongue different from Dutch (n = 3); and (ii) parents were not able to visit the outpatient clinic (n = 2).
      All five excluded children were singletons (three girls and two boys), and two of them were sisters. Indications for AOA were globozoospermia (n = 4) and failed fertilization following conventional ICSI (n = 1). Their mean ± standard deviation and median (range) age at the moment of recruitment for the study was 73.8 ± 33.66 and 77.0 (40–125) months, respectively. The maternal age at childbirth was 28 ± 4.0 years. Mean gestational age of these children was 39.0 ± 1.32 weeks, with none of them born before week 37 of pregnancy. Mean birthweight was 3107 ± 217.8 g.

      Neurodevelopmental outcome

      The majority of the children were younger than 7 years at the moment of testing. Seven of the 11 singletons were single child. Two singletons were sisters; another pair of singletons comprised a brother and a sister. The results are presented in Table 3.
      Table 3Scores of the follow up by assessment.
      Test and age groupNo. of childrenNormScore
      Cognition21Verbal IQPerformal IQFull scale IQ
       WPPSI-III-NL (⩽7 years)20100102.8 (96.4–109.2) (77–129)105.0 (97.8–112.2) (74–142)104.2 (97.5–110.8) (85–144)
       WISC-III-NL (>7 years)1100123130131
      Language20Receptive scaleExpressive scaleTotal percentile score
       RTOS (<5 years)85082.0 (63.4–100.7)a (35.5–99)73.3 (49.4–97.2)b (30–99)78.2 (57.2–99.2)c (33.3–99.0)
       CELF-IV-NL (⩾5 years)125032.5 (7.3–57.7)a (3.6–94.5)40.9 (16.6–65.2)b (7.1–88.5)40.1 (16.8–63.4)c (9.1–89.7)
      Motor skills19Manual dexterityAiming and catchingBalanceTotal percentile score
       Movement ABC-II-NL (3–6 years)175042.0 (24.4–58.7) (1–95)43.8 (29.5–58.0) (2–95)41.5 (25.1–57.9) (5–91)37.1 (22.3–52.0) (2–84)
       Movement ABC-II-NL (7–10 years)25056.5 (NA) (50–63)21.0 (NA) (5–37)77.0 (NA) (63–91)50.0 (NA) (37–63)
      Autism screening16Score
       SCQ (⩾2 years)16<156.4 (4.5–8.3) (0–13)
      Maladaptive behaviour screening19Internalizing broadbandExternalizing broadbandTotal score
       CBCL (1.5–5 years)11<7050.5 (42.7–58.2) (33–74)47.6 (42.1–53.0) (35–60)48.6 (42.5–54.8) (30–63)
       CBCL (6–18 years)8<7038.4 (33.6–43.2) (33–50)39.1 (35.0–43.2) (34–48)37.3 (32.0–42.5) (29–45)
       TRF (1.5–5 years)4<7047.5 (41.3–53.7) (44–53)44.8 (36.8–52.7) (38–49)45.5 (41.7–49.3) (44–49)
       TRF (6–18 years)4<7043.5 (31.7–55.7) (37–52)42.7 (40.9–44.1) (41–43)42.5 (32.1–52.9) (33–48)
      Values are mean (95% CI) (min–max) or n. Norm: (i) the 50th percentile of the general population (for cognition, language and motor skill assessment); or (ii) the cut-off below which the behaviour is considered normal (for autism and maladaptive behaviour screening).
      CBCL = Child Behaviour Checklist; CELF = Clinical Evaluation of Language Fundamentals; Movement ABC-II-NL = Movement Assessment Battery for Children III; NA = not applicable (n = 2); RTOS = Reynell Developmental Language Scales; SCQ = Social Communication Questionnaire; TRF = Teachers Report Form; WISC = Wechsler Intelligence Scale for Children; WPPSI = Wechsler Preschool and Primary Scale of Intelligence.
      aP < 0.01. b,cP < 0.05 comparing RTOS and CELF scores (Mann–Whitney U-test), respectively.

      Cognitive development

      Cognitive development was assessed for all included children. Mean FSIQ, PIQ and VIQ scores on the WPPSI-III-NL were 104.2, 105.0 and 102.8, respectively (20 children: 10 singletons, 10 twins). One girl and one boy (not genetically related to each other) scored 77 and 74 for VIQ and PIQ, respectively. However, both their FSIQ scores were considered normal: 93 and 85, respectively. The oldest child was tested using the WISC-III-NL: FSIQ, PIQ and VIQ scores were 131, 130 and 123, respectively. Considering the pathological threshold for FSIQ scoring being <70 (cognitive disability), the proportion of children in this cohort falling below this threshold is 0% (95% CI 0–15.5%).

      Language development

      One child was excluded because of an incomplete test result due to a lack of co-operation. Eight children aged <5 years were tested using the RTOS. Their total percentile score for language development was 78.2, while their percentile scores on receptive and expressive language skills were 82.0 and 73.3. The remaining 12 children aged ⩾5 years were tested using the CELF-IV-NL. Their total, receptive and expressive language percentile scores were 40.1, 32.5 and 40.9, respectively. If both groups were compared, the younger group scored significantly better than the older group of children (P < 0.05). Importantly, the older group consisted of eight twins (8/12, 67%), whereas in the younger group only two twins were present (2/8, 25%). Considering the pathological thresholds for language percentile scoring being <5th percentile (language disability), the proportion of children in this cohort falling below this threshold for the expressive and total language score was 0% (95% CI 0–16.1%), whereas this was 5% (95% CI 0.01–23.6%) for the receptive language score.

      Motor development

      In the two youngest children, the test was cancelled because of fatigue. Seventeen children were tested using the Movement ABC-II-NL for children aged 3–6 years. The mean total percentile score was 37.1. The remaining two older children (both singletons) were tested using the Movement ABC-II-NL test for children aged 7–10 years. Their mean percentile score was 50.0. Subtest percentile scores are shown in Table 3. Six children (31.6%) scored below the 15th percentile for motor skill development. Among them, 5 (83.3%) were twins, of which two were born before week 37 of gestation. No correlation was found between the birthweight of the included children and the total or subtest scores on motor skill development. Considering the pathological thresholds for motor skill percentile scoring being <5th percentile (Developmental Coordination Disorder), the proportion of children in this cohort falling below this threshold is 10.5% (95% CI 2.9–31.4%) for manual dexterity, 5.3% (95% CI 0.01–24.6%) for aiming and catching and the total motor score, and 0% (95% CI 0–16.8%) for balance. Additionally, the parents were asked to fill out a questionnaire regarding motor skills in everyday life (VMVK or CVO questionnaire). All children (n = 21) tested within the normal ranges on these questionnaires (data not shown), meaning that according to the questionnaires no notable co-ordination disorders with influence on everyday life activities are present in these children.

      Behaviour

      The behaviour of all 21 AOA children was assessed using three validated questionnaires. The SCQ questionnaire was filled out and returned valid for 16 children. The mean score was 6.4 (range 0–13). No correlation was found between paternal and maternal age at birth, APGAR score at 5 min, birthweight and the SCQ score. The CBCL and TRF (non-mandatory) were returned for 19 and eight children, respectively. The mean scores for the CBCL and TRF were 43.8 and 44.0, respectively. Again, these scores fell within the normal range for children of similar age, being <70. Also, for both questionnaires, the mean scores for internalizing and externalizing behaviour lied within the normal range. One girl scored above the norm for internalizing behaviour (CBCL score: 74). Nevertheless, this was not confirmed by her teacher (TRF score: 53).

      Discussion

      This is the first report on the neurodevelopmental and behavioural outcome of children born following AOA, which is a rarely required, nonetheless highly specialized fertilization technique. The high response rate and the robustness of the performed tests and questionnaires support the validity of the results found in this study.
      Others described a group of 22 babies born following AOA (
      • Takisawa T.
      • Doshida M.
      • Hattori H.
      • Nakamura Y.
      • Kyoya T.
      • Shibuya Y.
      • Nakajo Y.
      • Tasaka A.
      • Toya M.
      • Kyono K.
      Effect of oocyte activation by calcium ionophore A23187 or strontium chloride in patients with low fertilization rates and follow-up of babies.
      ,
      • Takisawa T.
      • Sato Y.
      • Tasaka A.
      • Ito Y.
      • Nakamura Y.
      • Hattori H.
      Effect of oocyte activation by calcium ionophore A23187 or strontium chloride in patients with low fertilization rates and follow-up of babies.
      ). Of these babies, 10 were born following AOA by the calcium ionophore A23187 and 12 following SrCl2 treatment. Growth and health of these babies at birth were not different between both AOA protocols. In a similar communication, weight and height of 21 AOA children was assessed up to the age of six (
      • Sato Y.
      • Nakamura Y.
      • Sakamoto E.
      • Tasaka A.
      • Usui K.
      • Hattori H.
      • Ito Y.
      • Nakajo Y.
      • Doshida M.
      • Kyono K.
      Follow up of children following new technology: TESE, IVM, and oocyte activation.
      ). For the singletons, the physical growth was within the 10–90% percentile. In the latter report, it was not mentioned whether the children were born following calcium ionophore or strontium treatment.
      The neonatal data of this group of children were reassuring. No birth defects were reported at birth, whereas at toddler age birth defects were reported in 14.3% of the children followed up by assessment. However, the occurrence of birth defects in this cohort was not different from a recent reported birth defect rate of 9.7% in 939 ICSI children (
      • Davies M.J.
      • Moore V.M.
      • Willson K.J.
      • Van Essen P.
      • Priest K.
      • Scott H.
      • Haan E.A.
      • Chan A.
      Reproductive technologies and the risk of birth defects.
      ). Nonetheless, the sample size of the current study group was not adequate to allow for a meaningful conclusion, as it led to a wide confidence interval for the observed birth defect rate, which indicated that the observed birth defect rate is statistically compatible not only with the rate of the reference cohort (9.7%), but also with a rate that is 3-times higher, which should be interpreted with caution. One should also bear in mind that children born following assisted reproduction technology stay more often in neonatal care units compared with naturally conceived children and are checked very carefully, which might lead to overestimation of anomalies that create an observational bias.
      For the assessment of neurodevelopmental health, only children aged 3 years or older were included. It is known that tests of cognitive ability performed on very young children (⩽2 years) are not necessarily reliable predictors of long-term intelligence (
      • Bowen J.
      • Gibson F.
      • Leslie G.
      • Arnold J.
      • Ma P.
      • Starte D.
      Medical and developmental outcome at 1 year for children conceived by intracytoplasmic sperm injection.
      ,
      • Leslie G.I.
      • Gibson F.L.
      • McMahon C.
      • Cohen J.
      • Saunders D.M.
      • Tennant C.
      Children conceived using ICSI do not have an increased risk of delayed mental development at 5 years of age.
      ). In several studies, no delay of cognitive development was found in ICSI children compared with IVF or naturally conceived controls (
      • Cederblad M.
      • Friberg B.
      • Ploman F.
      • Sjöberg N.O.
      • Stjernqvist K.
      • Zackrisson E.
      Intelligence and behaviour in children born after in-vitro fertilization treatment.
      ,
      • Leunens L.
      • Celestin-Westreich S.
      • Bonduelle M.
      • Liebaers I.
      • Ponjaert-Kristoffersen I.
      Follow-up of cognitive and motor development of 10-year-old singleton children born after ICSI compared with spontaneously conceived children.
      ,
      • Place I.
      • Englert Y.
      A prospective longitudinal study of the physical, psychomotor, and intellectual development of singleton children up to 5 years who were conceived by intracytoplasmic sperm injection compared with children conceived spontaneously and by in vitro fertilization.
      ,
      • Ponjaert-Kristoffersen I.
      • Bonduelle M.
      • Barnes J.
      • Nekkebroeck J.
      • Loft A.
      • Wennerholm U.B.
      • Tarlatzis B.C.
      • Peters C.
      • Hagberg B.S.
      • Berner A.
      • Sutcliffe A.G.
      International collaborative study of intracytoplasmic sperm injection-conceived, in vitro fertilization-conceived, and naturally conceived 5-year-old child outcomes: cognitive and motor assessments.
      ). However, one study suggested that IQ scores were significantly lower in ICSI children (
      • Knoester M.
      • Helmerhorst F.M.
      • Vandenbroucke J.P.
      • van der Westerlaken L.A.
      • Walther F.J.
      • Veen S.
      Cognitive development of singletons born after intracytoplasmic sperm injection compared with in vitro fertilization and natural conception.
      ). The latter could not be confirmed by the present study with normal IQ scores. One girl, who scored rather low for VIQ (<85), was known with distinct shyness, especially in new situations, which could have led to a false low score on VIQ. No gross problems were known for this girl at school or in relationship with classroom peers. One boy also scored relatively low for PIQ (<85). This boy was at the lower age limit to perform this test and since this test was performed at the end of the day, tiredness could have been the cause for the lower PIQ score (
      • Murphy K.R.
      • Davidshofer C.O.
      Psychological Testing.
      ). Both children had normal FSIQ scores.
      At the group level, the mean scores for language development fell within the normal ranges. The younger children tested by the RTOS scored significantly higher on language development compared with the older group tested by the CELF-IV-NL. This difference might be attributed to the fact that the majority of the children in the older group were twins and several reports claim that twins on average score lower on language development tests compared with singletons (
      • Rutter M.
      • Thorpe K.
      • Greenwood R.
      • Northstone K.
      • Golding J.
      Twins as a natural experiment to study the causes of mild language delay: I: design; twin-singleton differences in language, and obstetric risks.
      ,
      • Thorpe K.
      Twin children’s language development.
      ).
      It has been shown that motor skill development in ICSI children can lag behind (
      • Knoester M.
      • Vandenbroucke J.P.
      • Helmerhorst F.M.
      • van der Westerlaken L.A.
      • Walther F.J.
      • Veen S.
      Matched follow-up study of 5–8 year old ICSI-singletons: comparison of their neuromotor development to IVF and naturally conceived singletons.
      ). Although the overall results of the present cohort was reassuring, the twins tended to lag behind compared with the included singletons, although the difference was not significant. This might be explained by the significantly lower gestational age at birth for the twins compared with the singletons.
      Extensive research regarding the influence of IVF and ICSI on children behaviour and the incidence of attention-deficit hyperactivity disorder and disorders in the autism spectrum (ASD) has been performed. It was shown that the prevalence of ASD was higher among ICSI children (3.4%) compared with the general population (0.6–0.7%) (
      • Knoester M.
      • Helmerhorst F.M.
      • van der Westerlaken L.A.
      • Walther F.J.
      • Veen S.
      Matched follow-up study of 5–8 year old ICSI singletons: child behaviour, parenting stress and child (health-related) quality of life.
      ). Older paternal as well as maternal age are considered to be risk factors for ASD (
      • Hultman C.M.
      • Sandin S.
      • Levine S.Z.
      • Lichtenstein P.
      • Reichenberg A.
      Advancing paternal age and risk of autism: new evidence from a population-based study and a meta-analysis of epidemiological studies.
      ,
      • Sandin S.
      • Hultman C.M.
      • Kolevzon A.
      • Gross R.
      • Maccabe J.H.
      • Reichenberg A.
      Advancing maternal age is associated with increasing risk for autism: a review and meta-analysis.
      ). On the contrary, another group could not confirm a relationship between IVF/ICSI and ASD when results were adjusted according to maternal age and educational level, parity, smoking, birthweight and multiplicity (
      • Hvidtjørn D.
      • Grove J.
      • Schendel D.
      • Schieve L.A.
      • Sværke C.
      • Ernst E.
      • Thorsen P.
      Risk of autism spectrum disorders in children born after assisted conception: a population-based follow-up study.
      ). In this study, the SCQ questionnaire was used to assess the risk for ASD. The mean score for the AOA children fell within the normal range. Nevertheless, taking into account the research of
      • Snow A.V.
      • Lecavalier L.
      Sensitivity and specificity of the Modified Checklist for Autism in Toddlers and the Social Communication Questionnaire in preschoolers suspected of having pervasive developmental disorders.
      , one younger child (aged 37 months) scored just above the cut-off value. The latter researchers decreased the cut-off score from 15 to 13 for younger children. Importantly, one should keep in mind that a single questionnaire is not sufficient to diagnose ASD. No significant correlations were found in this study between the SCQ score and paternal and maternal age at birth, APGAR score at 5 min or birthweight. According to the CBCL questionnaire, this group of AOA children did not show any maladaptive behaviour. However, one girl scored above the norm for internalizing behaviour. This could be explained by the fact that ICSI girls tend to score higher on the externalizing and internalizing broadband as well as on the total CBCL score in comparison with IVF girls (
      • Knoester M.
      • Helmerhorst F.M.
      • van der Westerlaken L.A.
      • Walther F.J.
      • Veen S.
      Matched follow-up study of 5–8 year old ICSI singletons: child behaviour, parenting stress and child (health-related) quality of life.
      ). Nevertheless, this girl’s CBCL score was not confirmed by her teacher, since her TRF score was normal.
      This cohort consists of almost 50% twins, which is essentially higher compared with the overall twinning rate following assisted reproduction technology, which was 11.2% in Belgium in 2010 (

      Belrap, 2012. Report 2010 of the College of Physicians in Reproductive Medicine. Available from: <http://www.belrap.be/Documents/Reports/Global/Report_IVF10_14DEC12.pdf>.

      ). Since 1 July 2003, assisted reproduction centres in Belgium are legally obliged to follow a strict regulation regarding embryo transfer. In women aged <36 years, only one embryo can be transferred in a first treatment cycle. From the second cycle on, two embryos might be transferred if only moderate embryos are available for transfer. From the third cycle onwards, a maximum of two embryos may be transferred. Since most of the couples undergoing AOA carry a history of several treatment cycles, most of these couples legally applied for the transfer of two embryos, which led to the high twinning rate in the current cohort of children. Since the high success rates of both the study centre’s AOA procedure and the current cryopreservation techniques, the centre now encourages the transfer of only one embryo in AOA couples. Consequently, the twinning rate in this AOA population has gone down significantly in the last 5 years.
      Four singletons of this cohort are siblings, which is one-third of the singleton cohort.
      • Kramer L.
      • Conger K.J.
      What we learn from our sisters and brothers: for better or for worse.
      pointed out the important role of brothers and sisters in a child’s development. Sibling relations provide an important context for the development of children’s understanding of their social, emotional, moral and cognitive worlds. This should be kept in mind when interpreting the results of the current study.
      The strengths of this case series are both the high response rate and the use of a range of different validated age-adjusted tests and questionnaires. Although AOA is rarely necessary in modern assisted reproduction, its possible impact on offspring development should not be neglected. However, this follow-up study is also characterized by certain limitations that must be taken into consideration. First of all, since AOA is a rarely used technique, the absolute number of included children is very low which severely limits conclusion making. Also, the fact that all tests were pooled on one day could have influenced the results, since some children showed fatigue and a lack of interest during the course of the day. Unfortunately, due to practical considerations (mostly related to the parents’ professional activities), all tests had to be performed on one day. Another concern which needs to be addressed is the fact that two-thirds of the parents of the children that were followed up by assessment had a higher level of education (beyond 18 years of age). Parental education, together with family income, occupational status and place of residence describes the socioeconomic status (SES) of an individual. The influence of SES on the development of children born at term is well established (
      • Ardila A.
      • Rosselli M.
      • Matute E.
      • Guajardo S.
      The influence of the parents’ educational level on the development of executive functions 1.
      ). Research shows that SES is associated with a wide range of health, cognitive and socioemotional outcomes in children, with effects beginning prior to birth and continuing into adulthood (
      • Bradley R.H.
      • Corwyn R.F.
      Socioeconomic status and child development.
      ,
      • Turkheimer E.
      • Haley A.
      • Waldron M.
      • D’Onofrio B.
      • Gottesman I.I.
      Socioeconomic status modifies heritability of IQ in young children.
      ). As a matter of fact, parental education is a major component of the SES variables. It is generally accepted that IVF/ICSI children comprises a selected group of children, born to parents with on average higher educational and socioeconomic levels. Therefore, appropriate reference values for IQ and percentile scores may be higher, so that the apparent lack of departure from the norm, or advantage over it, may be less reassuring than it looks. Finally, this analysis did not include a group of control children, since for all used tests and questionnaires, validated norm values were available. Individual performance was compared with established norms for Dutch-speaking children. Also, since the current cohort of AOA children is very small due to the fact that AOA is a rarely required technique, the prior aim was to describe the neurodevelopmental performance of those children specifically. In future, the optimal design would be a multicentre-based child age-matched control study.
      The overall results of this case series of children born following ICSI combined with AOA showed that neonatal, developmental as well as behavioural outcome is reassuring, although the results are still to be considered preliminary. No serious adverse effects were observed; however, the size of this study is too small to reach a definite conclusion. Therefore, AOA should still not be considered routine practice yet. The sample size of this cohort was too small to detect chromosomal aberrations and malformations following AOA.
      Couples undergoing AOA (or any other assisted reproduction procedure) should be counselled in advance regarding increased risks of pregnancy complications and higher rates of obstetric interventions. Although conflicting data exist, studies of children born following IVF and ICSI suggest increased rates of congenital malformations, imprinting disorders and a marginally increased risk of cancer (
      • Basatemur E.
      • Sutcliffe A.
      Follow-up of children born after ART.
      ). However, the current study and evidence is inadequate to draw definite conclusions about long-term outcomes after assisted reproduction technology and AOA specifically. Prospective multicentre long-term studies are needed to eliminate the effect of confounders. The long-term outcomes after AOA are difficult to evaluate because of the variability in AOA methods and data reporting. Therefore, clinicians should aim for standardization of the AOA procedure as well as the methodology for follow up of children born following AOA. ICSI combined with AOA should still be performed only in cases where a clear indication is present.

      Acknowledgements

      The authors are grateful to the children and parents who came to our centre voluntarily to participate in this study and to the team of the Centre for Developmental Disturbances (Centrum voor Ontwikkelingsstoornissen, COS) of the University Hospital Ghent, whose collaboration made this study possible.

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