Hum. Reprod. Advance Access originally published online on June 23, 2007
Human Reproduction 2007 22(9):2481-2487; doi:10.1093/humrep/dem180
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Impact of Italian legislation regulating assisted reproduction techniques on ICSI outcomes in severe male factor infertility: a multicentric survey
1 A.M.B.R.A., Associazione Medici e Biologi per la Riproduzione Assistita, Palermo, Italy 2 CRA, Centro Riproduzione Assistita, Catania, Italy 3 Centro di Fisiopatologia della Riproduzione e Procreazione Assistita, Pisa, Italy 4 Struttura Semplice Diagnosi e Terapia dell'Infertilità E.O. Ospedali Galliera, Genova, Italy 5 U.O. Scienze Natalità—Fisiopatologia Riproduzione H.S.Raffaele, Milano (Milano I), Italy 6 ARS Biomedica, Roma (Roma I), Italy 7 GynePro Medicina della Riproduzione, Bologna, Italy 8 Infertility Unit, Fondazione Policlinico-Mangiagalli, Milano (Milano II), Italy 9 Centro di Medicina della Riproduzione Universita' di Torino Az. OIRM-S.Anna, Torino, Italy 10 Medicina e Biologia della Riproduzione, European Hospital, Roma (Roma II), Italy
11 To whom correspondence should be addressed at: A.M.B.R.A, Associazione Medici e Biologi per la Riproduzione Assistita, Via G. De Spuches, 11 90141 Palermo, Italy. Tel: +39 091 6127014; Fax: +39 091 6813313; E-mail: rosanna.ciriminna{at}gmail.com
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Abstract |
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BACKGROUND: In 2004, a law regulating assisted reproduction techniques (ART) was passed in Italy. The new rules allow for the formation and transfer of a maximum of three embryos at one time, whereas embryo selection and embryo storage are prohibited. The aim of this study is to evaluate the impact of these restrictions on ICSI outcome in couples affected by severe male factor infertility.
METHODS: Thirteen Italian ART Units were involved in this study. Data were collected on ICSI cycles performed during 2 years before (control group) and 2 years after (study group) the enforcement of the law. Only cases of obstructive azoospermia (OA), non-obstructive azoospermia (NOA) and severe oligoastenoteratozoospermia (OAT) (sperm count 
1 x 106 per ml; normal forms 5% according to WHO) were included. Laboratory results (fertilization rate, cleavage rate and embryo quality) and clinical outcomes (clinical pregnancy rate, implantation rate, abortion rate) were compared between the two groups.
RESULTS: One thousand six hundred and forty ICSI cycles were examined. The control group included 843 cycles (51.4%), whereas the study group consisted in 797 cycles (48.6%). The restrictions imposed by the law significantly reduced the number of good-morphology embryos available for transfer (57.5 versus 50.1%; P < 0.001). In addition, the clinical pregnancy rate (32.6 versus 22.6%; P < 0.001) and the implantation rate (16.0 versus 12.3%; P< 0.05) per cycle were negatively affected by the enforcement of the law. In particular, dramatic reductions in the pregnancy rate (36.6 versus 15.5%; P < 0.001) and the implantation rate (17.8 versus 9.8%; P < 0.001) were observed in the NOA subgroup.
CONCLUSIONS: Limiting the number of treated oocytes to three per ICSI cycle significantly reduces the chance of transferring good quality embryos and thus achieving a pregnancy in cases of severe male factor infertility. NOA patients are particularly affected by this restriction imposed by the new Italian law.
Key words: legislation/ART/ICSI/azoospermia/pregnancy rate
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Introduction |
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The Italian law regulating assisted reproduction techniques (ART) has been in force since March 2004. Among the restrictions imposed, the number of embryos allowed to form is limited to three; in addition, no embryo selection is possible nor is embryo cryopreservation. In the laboratory practice, this means that not more than three oocytes are allowed to be fertlized per cycle. Moreover, all the obtained embryos must be transferred at once, independently of their morphological aspect and genetic constitution (Benagiano and Gianaroli, 2004
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Recently, an Italian multicentric study (Ragni et al., 2005), comparing 1861 couples undergoing IVF or ICSI cycles before and after the introduction of the law, failed to demonstrate a significant difference in clinical pregnancy and implantation rates of fresh embryo transfers. However, the cumulative success rate per oocyte retrieval, including fresh and frozen-thawed embryos, was reduced in the post-law period. It must be underlined that the population examined in this study was heterogeneous in terms of infertility factors and demographic confounders.
When a specific subpopulation was analysed, namely non-obstructive azoospermic (NOA) patients, a negative impact of the law was clearly reported (Greco et al., 2006). In this study, the quality of transferred embryos and clinical pregnancy rate were significantly reduced in the post-law period. In NOA patients, evidence for reduced developmental competence of the embryos obtained are widely reported in the literature, even with no restriction on the number of oocytes treated (Mansour et al., 1997; Ghazzawi et al., 1998; Ubaldi et al., 1999). The possibility to choose the best embryos for transfer from a large cohort is therefore, not surprisingly, particularly important in this situation.
A reduced sperm competence may also be present in other subpopulations, such as severe oligoastenoteratozoospermic (OAT) and obstructive azoospermic (OA) patients submitted to ICSI. First of all, it is not always possible to find a normally appearing spermatozoon in these patients. Morphologically abnormal sperm injection has been associated with a lower implantation rate (De Vos et al., 2003). Furthermore, only major morphological defects can be assessed at x 400 magnification. Minor sperm nuclei abnormalities (such as vacuoles), normally not identifiable during the ICSI procedure, have been associated with a lower pregnancy rate (Bartoov et al., 2002). Sperm retrieved from severe OAT patients are probably more likely to have such undetectable abnormalities. Moreover, a correlation has been found between higher incidence of meiotic errors and lower sperm counts (Pang et al., 1999). Sperm immaturity has also been correlated with increased rates of chromosomal abnormalities (Kovanci et al., 2001; Rodrigo et al., 2004). Testicular and epididymal spermatozoa obtained from OA and NOA patients are likely to contain a significantly higher proportion of immature spermatozoa. For the above reasons, adverse effects on ICSI outcome has been suggested when OAT, epididymal and testicular sperm are used for fertlization (Pang et al., 1999; Pfeffer et al., 1999; Bernardini et al., 2000; Calogero et al., 2001; Rubio et al., 2001; Rodrigo et al., 2004).
Therefore, we hypothesize that in all cases of severe male factor infertility, the restriction of the number of fertilizable oocytes has a negative impact on ICSI outcome.
Consequently, a retrospective multicentric study was designed to compare ICSI outcome in severe OAT, OA and NOA patients before and after the application of oocyte restriction. Only couples in which the female partner was potentially fertile were included in the study. Cycles performed in the same time interval (2 years) before and after the enforcement of the law were considered. Laboratory results (fertilization rate, cleavage rate and embryo quality) and clinical outcome (clinical pregnancy rate, implantation rate, abortion rate) were compared between the two groups.
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Materials and Methods |
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Study design
Thirteen Italian ART Units [Palermo, Roma, Pisa, Lecce, Torino, S. Rossore (PI), Catania, Vicenza, Bologna, Roma II, Milano, Genova, Milano II] participated in this study. All the Units had more than 5 years experience in ART practice.
The study involves sequential ICSI cycles performed during a 4-year period: from 10 March 2004 to 10 March 2006 and, as a control, from March 2002 to February 2004. Data were provided anonymously by each participating Unit. Laboratory and clinical data were retrospectively obtained from patients’ charts.
Inclusion criteria
Inclusion criteria for female partner were: age 38 years, normal ovarian reserve (basal FSH < 10 mIU/ml) (Ubaldi et al., 2005), no uterine pathologies, nor endometriosis. Only tubal obstruction had been admitted as a cause of female infertility. However, patients with mono or bilateral hydrosalpinx have been excluded (Camus et al., 1999).
Inclusion criteria for the male partners were: sperm concentration at the time of oocyte retrieval 1 x 106 ml, normal forms 5% (according to WHO), OA and NOA. Presence of motile sperm in the ejaculatate was also requested.
Laboratory data
For each cycle, the following laboratory items were recorded: sperm recovery modality (ejaculation, surgical sperm extraction), sperm concentration, total sperm motility and morphology, number of oocytes retrieved, number of oocytes injected, number of fertilized oocytes, number of abnormally fertilized oocytes, number of embryos obtained, number of embryos transferred, number of good morphology embryos obtained and number of good morphology embryos transferred.
In agreement with all the embryologists participating in the study, oocytes were considered normally fertilized only when two clearly distinct, equally sized, pronuclei were present. The presence of a single pronucleous, of asymmetric pronuclei as well as the presence of three or more pronuclei in the oocyte cytoplasm were classified as abnormal fertilization. Good-morphology embryos were defined as follows: appropriate cell number for developmental stage (4–6 cells on day 2, 8–10 cells on day 3), even blastomeres, fragmentation <10% and no multinucleation.
Clinical data
The following clinical items were recorded for each included cycle: age, previous IVF-ICSI attempts, indication for treatment, ovarian stimulation protocol, therapeutic regimen, total IU of FSH used, duration of stimulation, clinical pregnancy, outcome of pregnancy and number of implanted embryos.
Clinical pregnancy was defined as ultrasonographic demonstration of an intrauterine gestational sac at least 4 weeks after embryo transfer.
Statistical analysis
Continuous variables are presented as mean and standard deviation (SD). Categorical variables are presented as percentage.
Comparisons of continuous variables observed in groups were performed using the t-test for independent samples. The chi-square test was used for the comparisons of categorical data. To verify the influence of potential confounding variables (female age, stimulation length and FSH dose) on clinical pregnancy rate, several multiple logistic regression analyses were conducted. For all analyses, P< 0.05 were considered significant.
The a posteriori power analysis of statistically significant outcomes showed that the test used in all comparisons displayed a sufficient statistical power (
> 0.8).
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Results |
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The present study included 1160 infertile couples who underwent 1640 ICSI cycles (1.29 cycles/patient); among them, 843 (51.4%) cycles were performed before (control group), and 797 (48.6%) were after (study group), the introduction of the new legislation.
Characterisitcs of patients and cycles are presented in Table 1. The mean age of women was 33.0 ± 3.4 and 32.4 ± 3.6 years in the study and control groups, respectively (P < 0.001). The cause of infertility was purely male in 90% of couples, and 'combined' (male plus tubal infertility) in 10% of cases, with a similar distribution in the two groups.
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Different protocols for ovarian stimulation were performed both in the study and control groups. The use of GnRH antagonist and short protocols has increased, whereas the use of long protocol regimens has decreased in the study group. Moreover, stimulation length and total gonadotropin dose per cycle were also significantly lower in the study group.
Laboratory results are presented in Table 2. The mean number of retrieved oocytes per cycle was significantly lower in the study group when compared with the control group (8.7 ± 4.6 versus 11.5 ± 6.1, respectively; P < 0.001). According to the law restrictions, the mean number of injected oocytes per cycle was significantly reduced (2.9 ± 0.8 versus 8.7 ± 4.6 respectively; P < 0.001). On the other hand, normal fertilization was significantly higher in the study group (70.3 versus 62.6%; P < 0.001). The percentage of abnormal fertilization was significantly decreased in the post-law treatments (4.9 versus 6.7%; P < 0.005). The percentages of good-quality embryos obtained in the study and in the control group were significantly different (50.1 and 40.8%, respectively; P < 0.001). However, because of the possibility of embryo selection, the proportion of good-quality embryos transferred in the control group was significantly higher when compared with that in the study group (57.5 versus 50.1%, respectively; P < 0.001).
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Clinical outcomes are presented in Table 3. The percentage of treatments with no embryos available for transfer significantly increased after introduction of the law (5.5 versus 3.2%; P < 0.05). The overall clinical pregnancy rate per oocyte pick-up dropped from 32.6 to 22.6% (P < 0.001), and the implantation rate was also significantly decreased in the study group (16.0 versus 12.3%; P < 0.05). The abortion rate increased, from 17.2% in the pre-law period to 22.5% in the post-law peroid, although the difference was not significant. The multiple pregnancy rate was comparable in the two subgroups.
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The difference in clinical pregnancy rate observed between the study and the control group was only due to the number of treated oocytes per cycles and not to other clinical parameters, as shown by the multivariate logistic regression presented in Table 4. The difference remained highly significant when female age, stimulation length and FSH dose were included as adjusting variables in the analysis. On the other hand, no difference was observed after the inclusion in the regression equation of the number of treated oocytes.
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The distribution of the number of transferred embryos and the relationship between clinical pregnancy rate and number of transferred embryo were analysed in Table 5. Statistically significant differences were found between the two groups. In the pre-law period, the number of embryos transferred was decided according to the patient characteristics and embryo quality. Conversely, in the post-law period, the number of transferred embryos was linked to the fertilization rate. In addition, transferring two selected embryos in the pre-law period was more successful in terms of pregnancy rate than transferring two unselected embryos in the post-law peroid.
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The results obtained in the three different subpopulations of patients involved in this study are described in Table 6. In 1023 ICSI cycles, spermatozoa were retrieved from the ejaculate of severe OAT patients (536 in the control and 487 in the study group, respectively). Among these patients, the clinical pregnancy and implantation rates were 30.9 and 14.8% in the control group and 25.4 and 13.0% in the study group, respectively. The differences did not reach statistical significances probably due to the number of cases analysed. Conversely, a significant difference was observed in the ongoing pregnancy rates between the two groups.
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Significantly decreased clinical pregnancy and implantation rates were obtained in the study group when only azoospermic patients (307 in the control and 310 in the study group) were considered (35.5 and 17.9% versus 18.5 and 8.9%; respectively; P < 0.001). This subgroup was composed of 181 subjects affected by OA (95 pre-law period and 86 post-law period) and 436 patients affected by NOA (216 pre-law period and 220 post-law period). The clinical outcomes obtained in only OA patients were not significantly different between the two groups. On the other hand, when only NOA patients were considered, the restrictions imposed by the law have clearly affected the clinical results in terms of clinical pregnancy rate, ongoing pregnancy rate and implantation rate.
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Discussion |
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The Italian legislation on ART, passed in March 2004, reduced the number of oocytes that can be fertilized per cycle to a maximum of three in order to avoid the creation of supernumerary embryos. The new law is inspired by the desire to protect every newly produced human life (Benagiano and Gianaroli, 2004
). The aim of our study is to evaluate the potential impact of the law restriction by comparing a study group (post-law) to a control group (pre-law), in a population of patients characterized by severe male factor infertility. The results of the present study show that the chance to obtain a pregnancy, after the enforcement of the law, is generally reduced in this population of patients.
Fertilization rate and overall embryo quality were higher after the introduction of the law. This observation may be explained by the different ovarian stimulation strategies adopted in the two groups. It has been recently reported (Baart et al., 2007) that mild stimulation regimen using gonadotrophin-releasing hormone and antagonist co-treatment, which does not disrupt secondary follicle recruitment, is associated with better embryo quality. Moreover, a strict selection of oocytes, prior to the ICSI procedure, has been performed in the post-law group. This represents the only option for the embryologist to try to minimize the negative effect of the restrictions imposed. Detailed morphological observation of the oocytes (which include extracytoplasmic and cytoplasmic assessment) (De Santis et al., 2005; Chamayou et al., 2006) and, in some cases, also non-invasive meiotic spindle visualization (Rienzi et al., 2003; De Santis et al., 2005; Chamayou et al., 2006; Greco et al. 2006) have been routinely introduced in Italian laboratories. Despite all these efforts, the quality of the transferred embryos was significantly lower in the post-law group. This suggests that oocyte assessment alone does not perform as well as pronuclear and embryo evaluation. In fact, embryo competence may be largely due to the quality of both originating gametes. Adverse paternal effects on embryo development have been suggested, especially in cases of severe male factor infertility. The mechanisms involved are reported to be: centrosome dysfunction or deficiency of oocyte-activating factors (Palermo et al., 1994), sperm DNA fragmentation (Lopes et al., 1998; Filatov et al., 1999; Host et al., 2000a,b; Larson et al., 2000, Morris et al., 2002; Tomsu et al., 2002; Benchaib et al., 2003; Tesarik et al., 2004, Greco et al., 2005a,b; Borini et al., 2006), sperm chromosomal abnormalities and sperm-related aneuploidies in the deriving embryos (Moosani et al., 1995; Colombero et al., 1999; Pang et al., 1999; Pfeffer et al., 1999; Bernardini et al., 2000; Gianaroli et al., 2000; Silber et al., 2003; Rodrigo et al., 2004; Ferlin et al., 2007). In particular, the biological impact of an abnormal sperm chromatin structure depends on the combined effects of the extent of DNA or chromatin damage in the spermatozoa and the capacity of the oocyte to repair that damage (Gandini et al., 2004). The ability of standard semen evaluation (including concentration, motility and morphology) to identify these sperms pathologies is limited (Host et al., 2001) and new assays for sperm competence evaluation are therefore needed.
Several methods are available to detect sperm DNA fragmentation [such as terminaldeoxynucleotidyltransferase-mediated dUTP nick-end labelling and single-cell gel electrophoresis (COMET) assay]. Evidence of a negative effect of DNA fragmentation on pregnancy progression has been recently reported in the Italian situation (Borini et al., 2006; Greco et al., 2006). The evaluation of sperm apoptotic stage in ejaculates of severe OAT patients is thus recommended in order to identify the proportion of spermatozoa with this deficiency responsible for embryo demise (Greco et al., 2005a,b; Borini et al., 2006). Oral antioxidant treatments have been proposed to reduce the percentage of damaged chromatin in sperm cells in these cases (Greco et al., 2005b).
Furthermore, in order to improve the clinical outcome in patients affected by severe male factor infertility, sperm cell selection of those with a strictly normal nucleus assessed at a higher magnification (inverted light microscope equipped with high-power Nomarski optics enhanced by digital imaging to achieve a magnification up to x 6300) (Bartoov et al., 2001) might be considered. The application of this methodology is of particular interest since individual sperm head morphology was shown to correlate with chromosomal constitution (Lee et al., 1996).
Some differences observed between the control and study groups regarding patient's and cycle's characteristics, deserve some clarifications. Because of the restrictions in the number of oocytes that can be inseminated, patients were generally treated with milder stimulation protocols. Significant differences between the study and the control groups were therefore found in variables mainly related to the stimulation regimen, namely length and dose of FSH and number of oocytes retrieved, as well as in female age. To exclude a possible confounding role of this therapeutic choice, data were analysed to control for these variables. The multivariate analysis documented a relevant influence of the new legislation on pregnancy success even when data were corrected for female age and/or stimulation regimen. This observation is not surprising, since only young and potentially fertile female partners were included in the study.
It is relevant that in this study the subpopulation of NOA patients is shown to be mostly affected by the new Italian legislation, as previously suggested (Greco et al., 2006). Various mechanisms may determine an impairment to reproductive performance in spermatozoa recovered by testicular biopsy once injected into the oocyte. Pathological conditions of the testis associated with NOA may lead to impaired spermiogenesis predisposing sperm cells to DNA fragmentation, defective cytoplasmic development, functional immaturity (Tesarik and Medoza, 2003; Greco et al., 2006) and abnormal chromosomal constitution of the available spermatozoa (Bernardini et al., 2000; Kovanci et al., 2001; Levron et al., 2001; Burrello et al., 2002; Mateizel et al., 2002; Rodrigo et al., 2004). For all these reasons, the chance of accidentally selecting a genetically abnormal spermatozoon during the ICSI procedure is higher in these cases.
Our study reports an overall reduction in clinical pregnancy rates from 36.2 to 22.6% (OR 1.000 –0.635 = 0.365; P < 0.001) during the post-law period, in the population of patients affected by severe male factor infertility. However, OAT and OA patients seem to be less affected from the law limitation. Increased laboratory and clinical efficiency may be able to partly compensate the negative effect of the restrictions imposed. Particular efforts in gamete selection are thus strongly recommended. On the other hand, a dramatic reduction in clinical pregnancy and implantation rates are observed in NOA patients. In this specific situation, because of the reduced presence of healthy spermatozoa, the efforts of embryologists do not seem to be sufficient. The use of a higher number of spermatozoa (and consequently of oocytes) seems to us the only option to improve the results. An exception to the current Italian legislation is thus requested for patients affected by NOA.
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Footnotes |
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* Collaborators in the Italian Society of Embriology, Reproduction and Research (SIERR) were: R. Barbaro (A.M.B.R.A., Associazione Medici e Biologi per la Riproduzione Assistita, Palermo, Italy); V. Cela (Centro di Fisiopatologia della Riproduzione e Procreazione Assistita, Pisa, Italy); I. Cino (U.O. Scienze Natalitá - Fisiopatologia Riproduzione H.S. Raffaele, Milano, Italy); D. Colia (Struttura Semplice Diagnosi e Terapia dell'Infertilitá E.O. Ospedali Galliera, Genova, Italy); G. D'Ambrogio (Centro di Fisiopatologia della Riproduzione e Procreazione Assistita, Lecce, Italy); L. Diotallevi (Centro Medico Palladio, Vicenza, Italy); M. Dusi (Centro Medico Palladio, Vicenza, Italy); M. Filicori (GynePro Medicina della Riproduzione, Bologna, Italy); A.R. Genazzani (Centro di Fisiopatologia della Riproduzione e Procreazione Assistita, Pisa, Italy); G. Giuffrida (CRA Centro Riproduzione Assistita, Catania, Italy); F. Lombardo (ARS Biomedica, Roma, Italy); A. Paffoni (Infertility Unit, Fondazione Policlinico-Mangiagalli, Milano, Italy); C. Racca (Centro di Medicina della Riproduzione Universita' di Torino Az. OIRM-S.Anna, Torino, Italy); E. Greco (Medicina e Biologia della Riproduzione, European Hospital, Roma, Italy).
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Submitted on April 3, 2007; resubmitted on May 21, 2007; accepted on May 23, 2007.
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