In order to evaluate the safety of the intracytoplasmic sperm injection (ICSI) procedure, a prospective follow-up study of 423 children born after ICSI was carried out. The aim of this study was to compile data on karyotypes, congenital malformations, growth parameters and developmental milestones. Before starting the infertility treatment, couples were asked to participate in a follow-up study including genetic counselling and prenatal diagnosis. The follow-up study of the child was based on a visit to the paediatrician-geneticist at birth or at 2 months of age, at 1 year and at 2 years of age when a physical examination for major and minor malformations and a psychomotoric evaluation were done. Between April 1991 and September 1994, 320 pregnancies obtained after ICSI led to the birth of 423 children (222 singletons, 186 twins and 15 triplets). Prenatal diagnosis determined a total of 293 karyotypes, one of which was abnormal (0.3%), and four were benign familial structural aberrations, all inherited from the paternal side. A total of 14 (3.3%) major malformations were observed, defined as those causing functional impairment or requiring surgical correlation. Neurological or developmental problems at the age of 2 months were found in 14 children, four of whom were multiples. Compared to most registers of children born after assisted reproduction and to registers of malformations in the general population, the figure of 3.3% major malformations is within the expected range. Before drawing any firm conclusion, further careful evaluations of the available data are necessary.

A prospective follow-up study of 877 children born after ICSI was carried out. The aim of this study was to compile data on karyotypes, congenital malformations, growth parameters and developmental milestones so as to evaluate the safety of this new technique. The follow-up study included agreement to genetic counselling and prenatal diagnosis and was based on a physical examination at the Centre for Medical Genetics (Dutch-speaking Brussels Free University, Brussels, Belgium) at 2 months, 1 year and 2 years, when major and minor malformations and a psychomotor evolution were recorded. Between April 1991 and July 1995, 904 pregnancies obtained after intracytoplasmic sperm injection (ICSI) led to the birth of 877 children (465 singletons, 379 twins and 33 triplets). Prenatal diagnosis determined a total of 486 karyotypes, of which six were abnormal (1.2%) and six (1.2%) were familial structural aberrations, all transmitted from the father. This slight increase in de-novo chromosomal aberrations and the higher frequency of transmitted chromosomal aberrations are probably linked directly to the characteristics of the infertile men treated rather than to the ICSI procedure itself. In all, 23 (2.6%) major malformations were observed in the children born, defined as those causing functional impairment or requiring surgical correction. No particular malformation was disproportionately frequent. Compared with most registers of children born after assisted reproduction and with registers of malformation in the general population, the figure of 2.6% was within the expected range. These observations should be further completed by others and by collaborative efforts. In the meantime, patiens should be counselled about the available data before any treatment: the risk of transmitted chromosomal aberrations, the risk of de-novo, mainly sex chromosomal, aberrations and the risk of transmitting fertility problems to the offspring. Patients should also be reassured that there seems to be no higher incidence of congenital malformations in children born after ICSI.

The aim of this prospective follow-up study of children born after intracytoplasmic sperm injection (ICSI) was to compile data on karyotypes, congenital malformations, growth parameters and developmental milestones in order to evaluate the safety of this new technique. The study design included karyotyping of the parents and their agreement to genetic counselling and prenatal diagnosis and it was based on a physical examination of the child at the Centre for Medical Genetics at the ages of 2 months, 1 year and at 2 years, where major and minor malformations and psychomotor evolution are recorded. Here we describe the first 57 children born from 40 ICSI pregnancies with epididymal spermatozoa (group 1), the first 50 children born from 34 ICSI pregnancies with testicular spermatozoa (group 2) and the first 58 children born from 48 pregnancies after replacement of cryopreserved ICSI embryos (group 3). Parental karyotypes were obtained from only 72/246 (29%) parents and were all normal. Prenatal karyotypes were determined for a total of 70 samples (40%): 21 in group 1, 15 in group 2 and 34 in group 3. In this last group 2 abnormal 47,XXY karyotypes (5.8%) and no structural aberrations were found. This increase in de-novo sex-chromosomal aberrations has already been described with regard to the first 877 children born after ICSI carried out at our Centre and is probably linked directly to the characteristics of the infertile men treated rather than to the ICSI procedure itself. Major malformations, defined as those causing functional impairment or requiring surgical correction, were observed in four children: two born after ICSI with epididymal spermatozoa, one after ICSI with testicular spermatozoa and one after ICSI and cryopreservation. No particular malformation was disproportionally frequent. In the follow-up examinations at 2 months (107/161 or 66.5%) and at 1 year (37/161 or 22.9%), no additional anomalies were observed. Lost for follow-up rate at 2 months was 33.5%. These observations on a limited number of children do not suggest a higher incidence of diseases linked to imprinting, nor do they suggest a higher incidence of congenital malformations. These observations are still limited in number and should be further completed by others and by collaborative efforts. In the meanwhile patients should be told about the available data before any treatment: there appears to be some risk of transmitted chromosomal aberrations, of de-novo, mainly sex-chromosomal aberrations and of transmitting fertility problems to the offspring. Patients should also be reassured that until now there seems to be no higher incidence of congenital malformations in children born after ICSI with epididymal or testicular spermatozoa or after replacement of ICSI embryos.

In 163 couples referred for assisted fertilization, pregnancy was established by subzonal insemination (SUZI), intracytoplasmic sperm injection (ICSI) or a combination of both techniques. These couples agreed to participate in a prospective study that included a prenatal diagnosis and clinical follow-up of the children. No cytogenetic aberrations were observed in 43 children tested. In 23 pregnancies occurring after SUZI, 15 women gave birth to 21 children. After replacement of combined SUZI and ICSI embryos, 10 pregnancies resulted in 8 deliveries with 10 children. Transfer of ICSI embryos led to 130 pregnancies ending in 20 deliveries with 24 children, with many others still ongoing successfully. In total, 55 children have been examined: 29 boys and 26 girls. One child from a singleton pregnancy presented multiple congenital malformations; one twin child presented a quadriparesis. In this observational study on a limited number of children, the incidence of major malformations was not different from the incidence in the general population.

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STUDY QUESTION Is there a relationship between karyotype abnormalities in fetuses and children conceived by ICSI and their father’s semen parameters? SUMMARY ANSWER The de novo chromosomal abnormality rate in pre- and postnatal karyotypes of ICSI offspring was higher than in the general population and related to fathers’ sperm parameters. WHAT IS KNOWN ALREADY Several studies have reported a higher rate of de novo chromosomal anomalies in ICSI fetuses but recent data from large cohorts are limited. Overall, reported prevalences of non-inherited karyotype aberrations are increased in fetuses conceived after ICSI and vary between 1.6% and 4.2%. Only a few studies focus on the relation between karyotype anomalies in ICSI offspring and semen parameters of their fathers. Furthermore, an increased incidence of abnormal karyotypes in ICSI neonates has been described, but the rates vary widely across studies. STUDY DESIGN, SIZE, DURATION We report on karyotype results from prenatal testing by means of chorionic villus sampling and amniocentesis and results from postnatal blood sampling in offspring conceived by ICSI in a single center. Ongoing pregnancies resulting from an oocyte retrieval between January 2004 and December 2012 and after transfer of fresh ICSI embryos obtained using ejaculated or non-ejaculated sperm (fresh or frozen-thawed) were considered. Pregnancies following frozen embryo transfer, oocyte or sperm donation, IVF, preimplantation genetic testing and IVM were excluded. All abnormal prenatal results after sampling are reported irrespective of the outcome of the pregnancy. PARTICIPANTS/MATERIALS, SETTING, METHODS From the 4816 ongoing ICSI pregnancies, information on pregnancy outcome was available for 4267 pregnancies. Prenatal testing was performed in 22.3% of the pregnancies, resulting in a diagnosis in 1114 fetuses. A postnatal karyotype was obtained in 29.4% of the pregnancies in which no invasive prenatal diagnosis was performed, resulting in a total of 1391 neonates sampled. The prevalence of chromosomal anomalies according to maternal age and semen quality was analyzed with logistic regression. For definitions of normal semen quality, the World Health Organization reference values for human semen characteristics were adopted. MAIN RESULTS AND THE ROLE OF CHANCE An abnormal fetal karyotype was found in 29 singletons and 12 multiples (41/1114; 3.7%; 95% CI 2.7–4.9%): 36 anomalies were de novo (3.2%; 95% CI 2.3–4.4), either numerical (n = 25), sex (n = 6) or structural (n = 5), and five were inherited. Logistic regression analysis did not show a significant association between maternal age and a de novo chromosomal fetal abnormality (odds ratio (OR) 1.05; 95% CI 0.96–1.15; P = 0.24). In all but one case, fetuses with an abnormal karyotype were conceived by ICSI using ejaculated sperm. Abnormal karyotypes were found in 14 (1.0%; 95% CI 0.6–1.7) out of 1391 postnatal samples of children born after ICSI who were not tested prenatally: 12 were de novo anomalies and two were inherited balanced karyotypes. The 14 abnormal karyotypes were all found in children born after ICSI using ejaculated sperm. The odds of a de novo karyotype aberration increased with maternal age when combining pre- and postnatal data (OR 1.11; 95% CI 1.04–1.19). A higher rate of de novo chromosomal abnormalities was found in fetuses and children of couples with men having a sperm concentration <15 million/ml (adjusted OR (AOR) 2.10; 95% CI 1.14–3.78), sperm concentration <5 million/ml (AOR 1.9; 95% CI 1.05–3.45) and total sperm count <10 million (AOR 1.97; 95% CI 1.04–3.74). LIMITATIONS, REASONS FOR CAUTION We cannot exclude that the observation of a higher prevalence of karyotype anomalies in ICSI offspring compared to literature data in the general population is due to enhanced surveillance after ART given the lack of a control group. Although we did not find more chromosomal anomalies after ICSI with non-ejaculated sperm, the small numbers do not allow firm conclusions. WIDER IMPLICATIONS OF THE FINDINGS The observed increased risk of a de novo karyotype anomaly after ICSI conception in couples with poor sperm warrants continued counseling toward prenatal testing. The current and widespread use of innovative non-invasive prenatal testing will result in larger datasets, adding to a balanced estimation of the prevalence of karyotype anomalies in ICSI offspring. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Methusalem grants issued by the Vrije Universiteit Brussel. All authors declared no conflict of interest related to this study. TRIAL REGISTRATION NUMBER N/A

STUDY QUESTION Does double vitrification and warming of human blastocysts having undergone biopsy once or twice have an impact on the clinical outcome? SUMMARY ANSWER The clinical pregnancy rate obtained with double vitrification single biopsy blastocysts was comparable to that obtained with single vitrification single biopsy blastocysts in our center in the same time period (46%; 2016–2018), whereas that obtained with double-vitrified double-biopsied blastocysts seemed lower and will need further study. WHAT IS KNOWN ALREADY Genetic testing on cryopreserved unbiopsied embryos involves two cryopreservation procedures. Retesting of failed/inconclusive-diagnosed blastocysts inevitably involves a second round of biopsy and a second round of vitrification as well. To what extent this practice impacts on the developmental potential of blastocysts has been studied to a limited extent so far and holds controversy. Additionally, the obstetrical/perinatal outcome after the transfer of double-vitrified/single or double-biopsied blastocysts is poorly documented. STUDY DESIGN, SIZE, DURATION This retrospective observational study included 97 cycles of trophectoderm biopsy and preimplantation genetic testing (PGT) on vitrified-warmed embryos followed by a second round of vitrification between March 2015 and December 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS In 36 warming cycles, no biopsy was performed on the embryos before the first vitrification (single biopsy group). In 61 warming cycles, the embryos had been biopsied on Day 3 (n = 4) or on Day 5/6 (n = 57) before the first vitrification (double biopsy group). A second biopsy was mostly indicated in cycles of failed or inconclusive diagnosis at the first biopsy. Two cycles involved a more specific mutation test for X-linked diseases on male embryos and one cycle involved testing for a second monogenic indication supplementary to a previously tested reciprocal translocation. Post-warming suitability for biopsy, availability of genetically transferable embryos and clinical outcome of subsequent frozen-thawed embryo transfer (FET) cycles were reported. Neonatal follow-up of the children was included. MAIN RESULTS AND THE ROLE OF CHANCE In total, 91 cleavage-stage embryos and 154 blastocysts were warmed, of which 34 (37.4%) and 126 (81.8%), respectively, were of sufficient quality to undergo trophectoderm biopsy and were subsequently vitrified for a second time. Out of these, 92 underwent biopsy for the first time (single biopsy), whereas 68 underwent a second biopsy (double biopsy). After diagnosis, 77 blastocysts (48.1%) were revealed to be genetically transferable (44 in the single biopsy group and 33 in the double biopsy group). In 46 warming cycles, 51 blastocysts were warmed and 49 survived this second warming procedure (96.0%). Subsequently, there were 45 FET cycles resulting in 27 biochemical pregnancies and 18 clinical pregnancies with fetal heartbeat (40.0% per FET cycle: 44.0% in the single biopsy group and 35.0% in the double biopsy group, P = 0.54). Thirteen singletons were born (eight in the single biopsy group and five in the double biopsy group), while three pregnancies were ongoing. A total of 26 embryos (13 in each group) remain vitrified and have the potential to increase the final clinical pregnancy rate. The neonatal follow-up of the children born so far is reassuring. LIMITATIONS, REASONS FOR CAUTION This is a small retrospective cohort, thus, the implantation potential of double vitrification double biopsy blastocysts, as compared to double vitrification single biopsy blastocysts and standard PGT (single vitrification, single biopsy), certainly needs further investigation. Although one could speculate on birthweight being affected by the number of biopsies performed, the numbers in this study are too small to compare birthweight standard deviation scores in singletons born after single or double biopsy. WIDER IMPLICATIONS OF THE FINDINGS PGT on vitrified-warmed embryos, including a second vitrification-warming step, results in healthy live birth deliveries, for both single- and double-biopsied embryos. The neonatal follow-up of the 13 children born so far did not indicate any adverse effect. The present study is important in order to provide proper counseling to couples on their chance of a live birth per initial warming cycle planned and concerning the safety issue of rebiopsy and double vitrification. STUDY FUNDING/COMPETING INTEREST(S) None. TRIAL REGISTRATION NUMBER N/A.

(Abstracted from Hum Reprod 2020;35(11):2488–2496) Despite advances in preimplantation genetic testing (PGT), inconclusive diagnoses following aneuploidy screening, chromosomal rearrangement, or monogenic testing do occur regularly. With PGT these days shifting toward more and more trophectoderm (TE) biopsy, test failures inevitably involve a second round of biopsy and blastocyst vitrification.