Informatie aan de pers

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1 Informatie aan de pers Datum : 22 januari 2004 Vrije Universiteit Brussel Partner Universitaire Associatie Brussel Rectoraat Dienst Interne & Externe Communicatie Jeroen De Samblancx T Fax Pleinlaan 2, B-1050 Brussel Betreft : Geassisteerde voortplanting met zaadcellen uit de zaadbal i.p.v. uit zaadstaal blijkt veilig tot zover. Kunstmatige bevruchting met zaadcellen uit de zaadbal i.p.v. uit een zaadstaal is een veilige procedure Onderzoekers van het Centrum voor Reproductieve Geneeskunde van de Vrije Universiteit Brussel, hebben aangetoond dat Intra-Cytoplasmatische Sperma Injectie (kunstmatige bevruchting) met zaadcellen uit de zaadbal i.p.v. uit het zaadstaal zoals gebruikelijk bij deze techniek geen belangrijke aangeboren afwijkingen veroorzaakt bij de kinderen. Deze procedure wordt vooral toegepast bij mannen met obstructieve azoospermie (d.w.z. een verstopping van de afvoerkanalen) of met niet-obstructieve azoospermie (d.w.z. een sterk verminderde zaadcelproductie in de zaadbal zelf). In de studie werden 274 zwangerschappen, bekomen na kunstmatige bevruchting, onderzocht. 70 ervan waren bekomen met zaad van mannen die aan niet-obstructieve azoospermie lijden. Deze groep werd vergeleken met 204 zwangerschappen, waar de man lijdt aan obstructieve azoospermie. De onderzoekers stelden respectievelijk 4% en 3% majeur aangeboren afwijkingen vast bij kinderen van mannen met niet-obstructieve en obstructieve azoospermie, wat vergelijkbaar is met het voorkomen in de algemene bevolking. Uit deze gegevens kan men momenteel besluiten dat Intra-Cytoplasmatische Sperma Injectie gebruik makend van zaadcellen uit de zaadbal, ook bij mannen met niet-obstructieve azoospermie, een veilige procedure is. Men mag echter niet vergeten dat het om een beperkte reeks gaat en dat verder onderzoek zeker noodzakelijk is. Deze toepassing is bovendien veel recenter dan ICSI en er zijn dan ook nog maar weinig gegevens beschikbaar omtrent de zwangerschap en de kinderen geboren na het gebruik van zaadbalzaadcellen. Voor meer informatie kan u terecht bij : Dr. Valerie Vernaeve, Tel: , GSM : , valerie.vernaeve@az.vub.ac.be Het wetenschappelijk artikel Pregnancy outcome and neonatal data of children born after ICSI using testicular sperm in obstructive and non-obstructive zoals verschenen in Human Reproduction vindtu u hier : Een afbeelding van zaadbalcellen onder de microscoop, gezien tussen onrijpe zaadvormende cellen vindt u hier :

2 Human Reproduction Vol.18, No.10 pp. 2093±2097, 2003 DOI: /humrep/deg403 Pregnancy outcome and neonatal data of children born after ICSI using testicular sperm in obstructive and non-obstructive V.Vernaeve 1,3, M.Bonduelle 2, H.Tournaye 1, M.Camus 1, A.Van Steirteghem 1 and P.Devroey 1 1 Centre For Reproductive Medicine and 2 Center for Medical Genetics, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan 101, B-1090 Brussels, Belgium 3 To whom correspondence should be addressed. valerie.vernaeve@az.vub.ac.be BACKGROUND: Registries on outcome of ICSI pregnancies obtained with testicular sperm do not differentiate between obstructive (OA) and non-obstructive (NOA). We evaluated the pregnancy outcome and neonatal data on children born after ICSI using testicular sperm of men with histologically proven OA or NOA. METHODS: Pregnancies obtained after ICSI using testicular sperm of men with de ned NOA (n = 70) were compared with those of men with OA (n = 204). RESULTS: Multiple birth rates in NOA and OA couples, respectively, were 21 versus 27% (P = NS), overall preterm delivery rates were 38 versus 26% (NS), and prematurity rates were 24 versus 13% for singletons (NS) and 86 versus 54% for twins (relative risk 1.59, 95% con dence interval 1.04±2.42). Median gestational age for singletons was 38.3 versus 39.3 weeks, respectively (P < 0.05). The low birth weight rates were 34 versus 31%, respectively (NS). The early perinatal mortality rate was 66 versus 15 per 1000 births, respectively, (NS). Major congenital malformations were observed in 4 versus 3%, respectively, of the live born babies (NS). Prenatal karyotypes showed 7% de-novo abnormalities in the NOA group versus 1% in the OA group (NS). CONCLUSIONS: Our data do not show differences between NOA and OA pregnancies except for a strong tendency towards a lower gestational age in singletons and a higher percentage of premature twins in the NOA group. Although our data are based on a limited sample, the differences observed call for further analysis. Given the low pregnancy rates after ICSI with NOA, a multicentre study, differentiating NOA and OA patients, would be recommended. Key words: /follow-up/icsi/pregnancy outcome/testicular sperm Introduction Since its introduction in 1992, ICSI has become a popular assisted fertilization technique with a high ef ciency in treating severe oligoasthenoteratozoospermia (Palermo et al., 1992). Because ICSI has been considered from the start as a procedure not without risks, several studies have been performed on the follow-up of pregnancies and on children born after ICSI. The different percentages found in the published studies about major and minor congenital malformations cannot be compared, but overall the data in large and reliable surveys do not indicate a higher rate of malformations in ICSI children than in IVF- or naturally conceived children (Wennerholm et al., 2000a; Bonduelle et al., 2002a). However, the study by Ericson and KaÈllen (2001) found an increased risk of the total malformation rate after IVF which could mainly be explained by maternal factors. Furthermore, the limited available data on ICSI fetal karyotypes reveal that, in comparison with a general neonatal population, there is a slight but increased risk of chromosomal anomalies, predominantly affecting the sex chromosomes (Bonduelle et al., 2002b). Today, ICSI is also widely used for patients with even when severe testicular failure is present. Since there is increasing evidence that in these patients spermatozoa have an increased chromosomal aneuploidy rate (Bernardini et al., 2000; Martin et al., 2000; Levron et al., 2001; Mateizel et al., 2002), follow-up of the pregnancies obtained after the use of testicular sperm from these patients is very important. Few studies involving a small number of patients have been published on the outcome of pregnancies and the health of children born after ICSI with testicular sperm (Aytoz et al., 1998; Wennerholm et al., 2000b; Bonduelle et al., 2002a; Ludwig et al., 2002). Furthermore, a distinction between normal spermatogenesis and spermatogenetic failure is lacking. Human Reproduction 18(10) ã European Society of Human Reproduction and Embryology 2003; all rights reserved 2093

3 V.Vernaeve et al. Table I. Outcome of the pregnancies in non-obstructive and obstructive Non-obstructive Obstructive RR 95% CI Pregnancies n Subclinical pregnancies n (%) 9 (13) 24 (12) ±2.24 Clinical miscarriages n (%) 11 (16) 28 (14) ±2.18 Extra-uterine pregnancies n 1 1 Selective reductions n 0 1 a Deliveries n Singletons n (%) 38 (79) 110 (73) ±1.29 Twins n (%) 7 (15) 37 (24) ±1.25 Triplets n (%) 3 (6) 4 (3) ±1.83 a Selective reduction from triplet to twin pregnancy. The aim of this study is to analyse the pregnancy outcome and neonatal outcome of children born after ICSI with testicular sperm in patients with obstructive and non-obstructive. Materials and methods All pregnant patients whose male partner had a testicular sperm recovery for ICSI in the period from January 1994 to December 2000 were included in this retrospective, cohort study. The two study cohorts were de ned according to testicular histology. The nonobstructive (NOA) group included patients with complete or incomplete maturation arrest, complete or incomplete germ cell aplasia (Sertoli cell-only) and tubular sclerosis and atrophy. All these patients had absolute (i.e. no spermatozoa were found in any of the semen analyses after centrifugation at high speed), but had sperm found after testicular biopsy. Klinefelter's syndrome patients were excluded from this study. Pregnancies obtained after ICSI with both fresh and frozen testicular spermatozoa were included in the study. Technical procedures, including testicular sperm recovery, ovarian stimulation protocols, micro-injection procedure, embryo culture and transfer, and luteal phase support, have been described previously (Verheyen et al., 1995; Tournaye et al., 1997; Joris et al., 1998; Van Steirteghem et al., 1998). A rise in serum HCG on two consecutive occasions from 11 days after transfer indicated pregnancy. Each pregnancy with at least one sac revealed by ultrasonography ~5 weeks after transfer was considered as a clinical pregnancy. If there was no sac, the pregnancy was considered subclinical. Gestational age was calculated as the time between the beginning of the last menstrual period and the date of birth of the child. The last menstrual period was calculated by adding 15 days to the date of embryo transfer. We evaluated both early (before 20 weeks of gestation) and late (>20 weeks of gestation) pregnancy outcome in the NOA and obstuctive (OA) cohorts. Abortion was de ned as pregnancy loss before 20 weeks of gestational age, and preterm delivery was de ned as delivery of a live born or stillborn infant before 37 weeks of gestational age. A live born or stillborn infant weighing <2500 g at birth was considered a low birth weight infant. A live born or stillborn infant weighing <1500 g at birth was considered a very low birth weight infant. The death of a fetus of at least 20 weeks' gestation before delivery was de ned as intrauterine death, and the death of an infant during the rst week following delivery was de ned as early neonatal death. The early 2094 perinatal mortality rate was expressed as the sum of stillbirths and early neonatal deaths per 1000 births. Major malformation was de ned as those malformations causing death or functional impairment, or requiring surgical correction. The remaining malformations were considered minor malformations. Since all outcome measures were not available for all patients, calculations were performed on appropriate data subsets. Comparisons of the NOA and OA groups for qualitative variables were performed using a Fisher exact test. A Mann±Whitney test was used when data were not normally distributed. A P-value of <0.05 was considered to be statistically signi cant. Additional relative risk (RR) with corresponding 95% con dence interval (CI) was calculated whenever relevant. This study was approved by our institutional review board. Results In all, 299 pregnancies obtained with testicular sperm were evaluated. Eighty-three pregnancies were obtained with testicular sperm from NOA patients (72 pregnancies were obtained after the use of fresh sperm and 11 pregnancies with frozen-thawed sperm). Two hundred and sixteen pregnancies were obtained with testicular sperm from OA patients (189 pregnancies were obtained after the use of fresh sperm and 27 pregnancies with frozen-thawed sperm). In all, 25 patients were lost for follow-up (8%): 13 (16%) in the NOA and 12 (6%) in the OA group (P = 0.006). Data for analysis were thus available for a total of 274 pregnancies (70 in the NOA group and 204 in the OA group). Six patients were pregnant more than once in the NOA group (9%) compared with 28 in the OA group (14%) (P = 0.3). The mean age of the women was 31.4 years (95% CI, 29.7± 33.0) versus 32.7 years (95% CI, 31.8±33.6) in the NOA and OA group, respectively. Primigravidae were more common in the NOA group (51 out of 67 with known gestational status versus 127 out of 203 with known status, i.e. 76 versus 63%), but statistical signi cance was not reached (P = 0.053). The parity was not statistically different between the two cohorts. There were no statistical signi cant differences with respect to the outcome of the pregnancies in the two groups studied (Table I). Of the 61 children in the NOA group, 58 were live born, three were stillborn and one died in the immediate post-partum period. Thirty-eight children were from singleton pregnancies,

4 Pregnancy outcome after ICSI with testicular sperm Table II. Gestational age at birth, preterm deliveries and birth weight of the children in non-obstructive and obstructive Non-obstructive Obstructive RR 95% CI P-value a Gestational age b Singletons 38.3 (37.0±38.3) 39.3 (37.8±39.3) Twins 36.0 (34.7±36.0) 36.6 (35.4±36.6) 0.15 Triplets 36.2 (32.1±34.2) 32.5 (31.2±32.5) 0.86 Preterm deliveries c Overall n (%) 18/47 (38) 38/148 (26) ±2.35 Singletons n (%) 9/37 (24) 14/107 (13) ±3.93 Twins n (%) 6/7 (86) 20/37 (54) ±2.42 Triplets n (%) 3/3 (100) 4/4 (100) Birth weight d Singletons 3200 (2555±3485) 3184 (2975±3580) 0.7 Twins 2450(1890±2770) 2517 (2215±2840) 0.5 Triplets 2055 (1270±2312) 1772 (1479±2264) 0.7 Low birth weight (<2500 g) e Overall n (%) 20/59 (34) 59/192 (31) ±1.67 Singletons n (%) 5/36 (14) 11/106 (10) Twins n (%) 8/14 (57) 36/74 (49) Triplets n (%) 7/9 (78) 12/12 (100) Very low birth weight (<1500 g) e 6/59 (10) 7/192 (4) ±7.98 Singletons + twins n (%) 3/50 (6) 4/180 (2) ±11.67 a Mann±Whitney test. b Median gestational age in weeks, with interquartile range in parentheses. c Number/number of deliveries with known outcome, with percentage in parentheses. d Median birth weight in grams, with interquartile range in parentheses. e Number/number of children with known outcome, with percentage in parentheses. Table III. Major and minor malformations in live born children after ICSI with testicular sperm of men with non-obstructive and obstructive Non-obstructive Obstructive Children with major malformations Polydactyly pre-axial ngers Unilateral inguinal hernia with unilateral cryptorchidism requiring orchidopexy at 37 weeks Bilateral cleft lip Unilateral mega-ureter with an important vesico-uretheral re ux and uretero hydronefrosis and large congenital nevus Atrioventricular septal defect with cleft mitral valve Vesico-uretheral re ux requiring surgery Bilateral ureteral stenosis Children with minor malformations Bilateral inguinal hernia in Hypomelanosis in both children of a twin pregnancy premature child Inguinal hernia Single umbilical artery Two children with open foramen ovale Two children with a hip luxation 14 were from twin pregnancies and nine were from triplet pregnancies. Of the 196 children in the OA group, 193 were live born and three were stillborn. One-hundred and ten children were from singleton pregnancies, 74 were from twin pregnancies and 12 were from triplet pregnancies. There was a strong tendency towards a lower gestational age among the singletons and a higher percentage of preterm twins in the NOA group. The gestational age at birth and percentages of preterm deliveries are summarized in Table II No differences were observed between both groups regarding the birth weight of the children. The results of birth weights of the non-obstructive and obstructive groups are summarized in Table II. Three fetuses died in utero after 20 weeks in both groups, and one infant died in the early neonatal period in the NOA group. The early perinatal mortality rate was thus 66 per 1000 births (n = 4) and 15 per 1000 (n = 3) in the NOA and OA group, respectively (RR: 4.3, 95% CI 0.8±23.7). Major malformations were present in 4% of the liveborn children (n = 2 out of 54 with a known result) obtained with testicular sperm of NOA men versus in 3% in children of OA men (n = 5 out of 188 with a known result) (RR: 1.4, 95% CI 0.19±7.8). One polymalformative syndrome was present in a stillborn child of the NOA group. The minor malformation rate in live born children in the NOA and OA group was 2% (n =1 out of 54 with a known result) versus 4% (n = 8 out of 188 with a known result) (RR: 0.4, 95% CI 0.02±3.27) (Table III). Fifteen of the 61 fetuses had a prenatal karyotype in the NOA group; one trisomy 18 was observed (7%) and the pregnancy was terminated. Two karyotypes were performed on miscarriage tissue and one trisomy 22 was observed. Seventy 2095

5 V.Vernaeve et al. of the 196 OA fetuses were tested prenatally (36%); an abnormal karyotype was obtained in two (3%): one de-novo 46,XY inv7(q22;q34) abnormality (1%) and one inherited karyotype abnormality [45,XX der(13,14)(q10;q10)] (RR: 4.7, 95% CI 0.31±70.45). Discussion At present, ICSI with testicular sperm from NOA patients is possible but results in lower fertilization and pregnancy rates than with sperm from men with OA (Vernaeve et al., 2003). There are some concerns when spermatozoa from men with severe testicular failure are used for ICSI. These spermatozoa are known to show a higher chromosomal aneuploidy rate (Bernardini et al., 2000; Martin et al., 2000; Levron et al., 2001; Mateizel et al., 2002). It is assumed that genomic imprinting may be less complete when immature gametes are used (Tesarik and Mendoza, 1996). Incomplete imprinting is unlikely to impair fertilization and early development, but developmental anomalies might become manifest at birth or even later in life. Furthermore, immature gametes may contain dysfunctional sperm centrosome and may show abnormal decondensation of the male pronucleus (Hewitson et al., 2002). As a result of all these concerns, the use of testicular spermatozoa from men with NOA has been banned in The Netherlands. Few publications so far have addressed the obstetric and neonatal outcome of children born after ICSI using testicular sperm. The study by Ludwig et al. (2002) analysed the outcome of 229 pregnancies in which testicular sperm was used and found no additional risk of major malformations in children born after the use of testicular spermatozoa. The major malformation rate, up to 8 weeks after birth, in this study was 9% based on live born and stillborn children and including spontaneous and induced abortions, compared wth 8.4% in ICSI with ejaculated spermatozoa. Bonduelle et al. (2002a) examined the outcome of malformation rates and found no differences, up to 8 weeks after birth, between ejaculated (3.4%, n = 2477) and testicular sperm (2.9%, n = 206). The study performed by Wennerholm et al. (2000b) found no major malformations in the 31 children born after the use of testicular sperm for ICSI. In these studies, the subgroups of testicular sperm are small and no discrimination is made between OA and NOA. Only the study by Palermo et al. (1999) differentiated between these two subgroups, although it was not clear whether it was done on a clinical or a histopathological basis. In the 34 pregnancies obtained after the use of testicular sperm, eight were classi ed as obstructive and 26 as non-obstructive. Similarly, in this study, the prevalence of congenital malformation did not vary in relation to the sample origin (or the cause of azoospemia). In our study, we found a malformation rate of 4% after the use of testicular sperm of NOA patients and 3% after the use of testicular sperm of OA patients. These rates are comparable with the rates observed in the study by Bonduelle et al. (2000a) where a 3.4% major malformation rate was found in ICSI children after the use of ejaculated sperm, using the same methodology and de nitions as in this study. With regards to the pregnancy outcome, we observed a strong tendency towards lower gestational age among the singletons and a higher percentage of premature twins in the NOA group, when comparing two different subgroups of azoospermic patients. In this study we did not include a historical control group such as oligoasthenoteratozoospermic patients or spontaneous conceptions. The sample size of our current data set would be too small to draw any valid conclusion. However, given our ndings and the concerns about the use of immature testicular spermatozoa from men with spermatogenetic failure, further study is recommended. In view of the low pregnancy rate after ICSI in NOA, a multicentre study in which a distinction is made between azoospermic patients with OA and those with NOA is suggested. Acknowledgements We would like to thank the clinical, paramedical and laboratory staff of the Centre for Reproductive Medicine and the Centre for Medical Genetics, Professor Collins and Dr. Efstratios Kolibianakis for statistical help, and Mrs. Ines Devolder of the Language Education Centre of our University for editing our manuscript. The work was supported by grants of the Fund for Scienti c Research-Flanders (FWO-Vlaanderen) and an unrestricted educational grant from Organon International. References Aytoz, A., Camus, M., Tournaye, H., Bonduelle, M., Van Steirteghem, A. and Devroey, P. (1998) Outcome of pregnancies after intracytoplasmic sperm injection and the effect of sperm origin and quality on this outcome. Fertil. Steril., 70, 5000±5005. Bernardini, L., Gianaroli, L., Fortini, D., Conte, N., Magli, C., Cavani, S., Gaggero, G., Tindiglia, C., Ragni, N. and Venturini, P.L. (2000) Frequency of hyper-, hypohaploidy and diploidy in ejaculated, epididymal and testicular germ cells of infertile patients. Hum. Reprod., 15, 2165±2172. Bonduelle, M., Liebaers, I., Deketelaere, V., Derde, M.P., Camus, M., Devroey, P. and Van Steirteghem, A. (2002a) Neonatal data on a cohort of 2889 infants born after ICSI (1991±1999) and of 2995 infants born after IVF (1983±1999). Hum. Reprod., 17, 671±694. Bonduelle, M., Van Assche, E., Joris, H., Keymolen, K., Devroey, P., Van Steirteghem. A. and Liebaers, I. 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