Article Influence of spermatogenic profile and meiotic abnormalities on reproductive outcome of infertile patients

RBMOnline - Vol 10. No 6. 2005 735 739 Reproductive BioMedicine Online; www.rbmonline.com/article/1678 on web 13 April 2005 Article Influence of spermatogenic profile and meiotic abnormalities on reproductive outcome of infertile patients Dr Pedro N Barri Dr Pedro N Barri was born in Barcelona in 1949 where he graduated from the Faculty of Medicine in 1971. His training in Obstetrics and Gynecology took place in Barcelona, France and England and he received his doctorate in 1993. He is an active founding member of the Faculty Staff of the Institut Universitari Dexeus in Barcelona where he is currently Head of the Department of Obstetrics and Gynaecology. Dr Barri is Honorary President of the Spanish Fertility Society and a member of the French Society of Sterility and the American Society of Reproductive Medicine. He has been on the Executive Committee of the International Society of Gynecological Endocrinology since 1998. He is on the Editorial Boards of many leading journals in his field. His particular research interests are clinical aspects of Human Infertility and his thesis topic was Respuesta Anómala a la Estimulación de la Maduración Folicular en Fecundación In Vitro. Pedro N Barri 1, Jose M Vendrell, Francisca Martinez, Buenaventura Coroleu, Begoña Arán, Anna Veiga Service of Reproductive Medicine, Department of Obstetrics and Gynecology, Càtedra d Investigació en Obstetrícia i Ginecologia (CIOG), Institut Universitari Dexeus, Paseo Bonanova 67, 08017 Barcelona, Spain 1 Correspondence: e-mail: pbarri@dexeus.com Abstract Genetic aspects of male infertility and the possible risks of new assisted reproduction and their influence on the development of zygotes and children born after intracytoplasmic sperm injection (ICSI) need further research. These patients have an increased risk of diploidy, and disomies are frequent in their spermatozoa. Meiotic disorders are more common in testicular biopsies of patients with severe oligoasthenozoospermia. For these reasons, a detailed andrological study is absolutely mandatory before accepting a couple with these characteristics into an IVF ICSI programme. When an andrological patient has plasma FSH values >10 IU/l and/or very low total motile sperm count <1 10 6, despite a normal karyotype, they clearly need a testicular biopsy and a meiotic study in order to rule out meiotic arrest or synaptic anomalies. Another important aspect to be considered is the possible benefit of applying preimplantation genetic diagnosis in these cases because they normally have a high percentage of chromosomally abnormal embryos, although in the present study this was not evident. All studies agree on the necessity of conducting follow-up studies in the population of children born after IVF ICSI. In this way, it will be possible to find out if these infertile patients and their offspring have a higher risk of suffering epigenetic errors and imprinting disorders. Keywords: congenital malformations, genetic risks, imprinting disorders, male infertility, meiotic studies, preimplantation genetic diagnosis Introduction Classically, it has been male infertility that has produced the worst results from the application of conventional IVF techniques. Not until the development of sperm microinjection (ICSI) and the first pregnancies resulting from it did the prognosis for cases of male infertility increase significantly (Palermo et al., 1992). This technique was much more effective than partial zone dissection or subzonal insemination (Palermo et al. 1993). As of that moment, IVF laboratories became the best therapeutic tool to solve the problems of severe male factor infertility. The results were good and, more importantly, they could be repeated easily in all IVF laboratories throughout the world, thus complying with the first premise that any new technology must meet: it must be reproducible in different scenarios. The high level of efficacy of ICSI techniques had to be accompanied by acceptable levels of safety, and for that reason, a comparative study between the rates of congenital malformations observed in children born by IVF and those who were born following application of ICSI was performed. The results were reassuring in that they did not show any greater incidence of malformations in the group of children born after ICSI (Van Golde et al., 1999). From a strategic point of view, the position was very clear from the start. The incorporation and the efficacy of ICSI should not represent any lesser need for andrological studies, but on the contrary, it seems indispensable that before an infertile couple with male factor are included in an IVF ICSI programme, they must undergo a detailed andrological study 735

736 to rule out additional genetic risk factors. For this reason, a number of andrological screening studies have been carried out which have made it possible to learn about fundamental aspects of andrological physiopathology such as the sperm chromosome component, analysed by means of fluorescence in-situ hybridization (FISH) techniques (Aran et al., 1999), and studies of meiosis in tissue material obtained by testicular biopsy (Egozcue et al., 2000a,b). With this knowledge, it has been possible to establish diagnostic protocols that should be applied to all patients who suffer severe male factor infertility that requires IVF ICSI. Materials and methods In a first study, 103 andrological patients were included who presented severe oligoasthenozoospermia (motile sperm concentration 1.5 10 6 spermatozoa/ml) presumably of idiopathic origin, with normal karyotype, which had to be treated with ICSI. These patients were studied through two spermograms, testicular volume, concentration of basal plasmatic FSH and testicular biopsy for histological and meiotic study (Vendrell et al., 1999). Next, the influence of the different spermatogenic patterns was studied on the early development of embryos obtained in the later IVF ICSI cycle that these same patients underwent (Vendrell et al., 2003). In addition, the final result of the cycle in relation to the pregnancy rates obtained was considered (Aran et al., 2003). Finally, the possible benefit that could be offered by preimplantation genetic diagnosis (PGD) for those andrological patients with greater genetic risk when they underwent an IVF ICSI cycle was considered (Aran et al., 2004). Results Motile sperm concentration ranged between 0 and 1.5 10 6 /ml, being <1 10 6 /ml in 88 patients (85.4%). Testicular volume was <15 ml (Tanner, 1978) in 73 patients (70.9%) and increased concentrations of basal FSH (>10 IU/l) were observed in 34.8% of patients. Quantitative analysis of testicular biopsy, based on Silber s criteria (Silber and Rodriguez Rigau, 1981), showed a mean number of mature spermatids per tubule that ranged between 0.15 and 21.5. Histological diagnosis was incomplete maturation arrest in 91.3% of cases. Despite the normal karyotypes, meiotic studies were normal in only 62.1% of patients and severe arrest and synaptic anomalies were found in 20.4 and 17.5% of patients respectively. In patients with sperm concentration <1 10 6 /ml (P < 0.001), motile sperm concentration <0.5 10 6 /ml (P = 0.001) and serum FSH concentration >10 IU/l (P < 0.05), meiotic abnormalities were significantly more frequent (Table 1). After multivariate analysis, sperm concentration and serum FSH concentration were the only independent predictive factors of abnormal meiotic pattern (Table 2). When these patients completed an IVF ICSI cycle, it was possible to study fertilization and cleavage as well as 4-cell stage embryo division rate on day 2, and to compare the results with those obtained in a group of normozoospermic patients. The first interesting observation was that the fertilization rates were normal regardless of the spermatogenic pattern, except in cases with sperm concentration 1 10 6 / ml. However, the percentage of 4-cell embryos on day 2 was significantly lower in the group of patients with oligoasthenozoospermia (P < 0.01, Table 3). The same thing happened in the group of patients with meiotic anomalies and with sperm counts lower than 1 10 6 /ml, who also presented a significant reduction in the number of 4-cell embryos on day 2. Once again, multivariate analysis showed that the the two findings, meiotic anomalies and low sperm count, were independent risk variables for a low rate of embryo division (Table 4). A retrospective study analysed the outcome of IVF ICSI cycles in terms of pregnancy in 44 andrological patients with meiotic anomalies who underwent 66 cycles of treatment (51 meiotic arrest, 15 synaptic anomalies). The control group included 93 andrological patients with normal meiotic studies who underwent 158 cycles during the same period of time. No statistical differences were found in pregnancy, implantation and miscarriage rates among the three groups (normal meiosis, meiotic arrest or synaptic anomalies) (Table 5). Finally, this study investigated whether it was possible to improve the results by applying PGD techniques in the 27 IVF ICSI cycles undergone by 25 couples infertile for male factor in which the male partner presented meiotic anomalies. On comparing the results with those obtained in 66 IVF ICSI cycles without PGD performed in 44 couples also infertile for male factor whose meiotic studies were also abnormal, it was shown that 42.5% of the embryos in the altered meiosis group in which PGD was performed were abnormal. There were no significant differences in the rates of fertilization, pregnancy, implantation or miscarriage between the two groups (Table 6), although this was probably due to the small size of the sample included so far in this preliminary evaluation. Discussion It is obvious that new assisted reproduction techniques have given possibility of becoming fathers to men whose only alternative would have been to resort to artificial insemination with donated semen. Moreover, clinical observations derived from the records of congenital malformations are important in casting a shadow of caution and of safety over the unquestionable efficacy of these techniques. Initial studies showed that the children born after IVF or ICSI did not present a higher rate of congenital malformations than that observed in the general population (Van Golde et al., 1999). However, these were preliminary studies and wider series were necessary to confirm these initial data; soon some worrying studies appeared on rates of congenital malformations that were significantly increased with the use of either IVF or ICSI techniques (Hansen et al. 2002; Koivurova et al. 2002). Nevertheless, at the same time more reassuring opinions were published, which made it possible to continue applying these techniques without fear (Steinkampf and Grifo, 2002). More wide-ranging studies with a longer follow-up period were necessary in order to be able to state that in general, children conceived after ICSI did not have a higher rate of malformations than children conceived naturally (Van Steirteghem et al., 2002). In addition, studies are now

Table 1. Frequency distribution of meiotic patterns (%) in different subgroups of patients. Meiotic pattern N B S B + S (n = 64) (n = 21) (n = 18) (n = 39) Sperm concentration ( 10 6 /ml) a,b (n = 45) 19 (29.7) 14 (66.7) 12 (66.7) 26 (66.7) Motile concentration ( 0.5 10 6 /ml) (n = 76) 42 (65.6) 18 (85.7) 16 (88.9) 34 (87.2) Testicular volume (<15 ml) (n = 73) 44 (68.8) 15 (71.4) 14 (77.8) 29 (74.4) FSH (>10 IU/l) c,d,e (n = 31) 14 (25.5) 10 (55.6) 7 (43.8) 17 (50.0) N = normal meiosis, B = meiotic arrest, S = synaptic anomalies, B + S = meiotic abnormalities. a P < 0.001 versus B + S. b P < 0 001 versus B and S. c P < 0.05 versus B + S. d P < 0.05 versus B and S. e Number of patients studied: normal meiosis, 55; meiotic arrest, 18; synaptic anomalies, 16; meiotic abnormalities, 34. Table 2. Logistic regression analysis. Risk factors for normal or abnormal meiosis. Variables β SE Wald gl P-value Sperm concentration 1.3032 0.4734 7.5799 1 0.0059 FSH 1.0436 0.4858 4.6154 1 0.0317 Constant 0.4587 0.4442 1.0663 1 0.3018 β = coefficient, SE = standard error. Table 3. Overall embryo outcomes (%). Normozoospermia Oligoasthenozoospermia P-value Oocytes inseminated 850 809 Oocytes fertilized 625 (73) 575 (71) NS a Cleaved embryos 587 (93) 538 (93) NS a Embryos 4 cells 367 (58) 290 (50) <0.01 a a Chi-squared test. Table 4. Logistic regression. Relative risk of slow embryo development. Spermatogenic pattern OR (95%CI) SE P-value Meiotic abnormalities 1.49 (1.03 2.15) 0.28 0.035 1 10 6 /ml 1.53 (1.09 2.13) 0.26 0.013 1 10 6 AN 2.00 (1.28 3.12) 0.46 0.002 AN = meiotic abnormalities, OR = odds ratio, Cl = confidence interval, SE = standard error. 737

Table 5. IVF ICSI outcome in relation to meiotic results. Group A Group B Group C Normal meiosis Meiotic arrest Synaptic anomalies No. cycles 158 51 15 2PN oocytes (%) 69.0 66.4 71.3 Embryos replaced (mean) 2.7 2.8 2.9 Pregnancies (%) 55 (34.8) 23 (45.1) 6 (40.0) Miscarriages (%) 2 (3.6) 3 (13.0) 0 Implantation rate (%) 19.6 25 18.6 Table 6. General characteristics and clinical outcome of couples with meiotic abnormalities after ICSI cycles with and without PGD. Meiotic abnormalities with PGD Meiotic abnormalities without PGD No. cycles 27 66 Mean oocytes microinjected 14.6 11.1 2PN oocytes (%) 74.5 67.5 No. replacements 25 66 Mean no. embryos/transfer 2.1 2.8 No. pregnancies 13 29 Pregnancy rate/transfer 52.0 43.9 Implantation rate (%) 32.1 23.5 Miscarriage rate (%) 15.4 10.3 738 available that show that the psychomotor development of ICSI children at 15 and 24 months is no worse than that of naturally conceived children (Bonduelle et al., 2003; Sutcliffe et al., 2003). It is important to point out that the high degree of efficacy of ICSI techniques must not represent a lessening in the need for andrological study of the patients before submitting them to an IVF ICSI cycle. In contrast, studies show that patients affected by severe male factor and with very poor sperm counts present more chromosomal anomalies in spermatozoids analysed with FISH (Aran et al., 1999) and a greater incidence of meiotic anomalies in the testicular biopsy (Vendrell et al., 1999). However, though the pace of embryo division that is obtained when these patients undergo an IVF ICSI cycle is slower (Vendrell et al., 2003), it seems that the final result of the cycle with regard to ongoing pregnancy rate is not affected (Aran et al., 2003). For these reasons, the greater diagnostic possibilities that are now available must help in identifying the cases of chromosomal anomalies in the aetiology of male infertility. As these anomalies are especially common in patients with severe oligoasthenozoospermia, FISH studies in spermatozoa and/or meiotic studies performed in material obtained from testicular biopsy should be recommended. The groups with greater experience in ICSI who have followed up children born more than 10 years ago refer to the fact that in these cases there is a slight but significant increase in denovo and sex and autosomal chromosomal abnormalities. It is also important to note their observation that although the rate of malformations and the psychomotor development of these children at 2 years of age is normal, there is a greater risk of imprinting disorders in the ICSI children which must be followed up with caution (Devroey and Van Steirteghem, 2004). References Aran B, Blanco J, Vidal F et al. 1999 Screening for abnormalities of chromosomes X, Y, and 18 and for diploidy in spermatozoa from infertile men participating in an in vitro fertilization intracytoplasmic sperm injection program. Fertility and Sterility 72, 699 701. Aran B, Vidal F, Vendrell JM et al. 2003 Outcome of intracytoplasmic sperm injection injection in relation to the meiotic pattern in patients with severe oligoasthenozoospermia. Fertility and Sterility 80, 91 95. Aran B, Veiga A, Vidal F et al. 2004 Preimplantation genetic diagnosis in patients with male meiotic abnormalities. Reproductive BioMedicine Online 8, 470 476. Bonduelle M, Ponjaert I, Van Steirteghem A et al. 2003 Developmental outcome at 2 years of age for children born after ICSI compared with children born after IVF. Human Reproduction 18, 342 350. Devroey P, Van Steirteghem A 2004 A review of ten years experience of ICSI. Human Reproduction Update 10, 19 28. Egozcue S, Blanco J, Vendrell JM et al. 2000a Human male infertility: chromosome anomalies, meiotic disorders, abnormal spermatozoa and recurrent abortion. Human Reproduction Update 6, 93 105. Egozcue S, Vendrell JM, Garcia F et al. 2000b Increased incidence

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