The 2004 Italian legislation regulating assisted reproduction technology: a multicentre survey on the results of IVF cycles

G. Ragni1,8, A. Allegra2, P. Anserini3, F. Causio4, A.P. Ferraretti5, E. Greco6, R. Palermo7, E. Somigliana1 on behalf of the Società Italiana della Riproduzione (S.I.d.R.) study group on the impact of the law 40/2004*

1 Infertility Unit, ‘Policlinico–L. Mangiagalli’ Hospital, Milano, 2 Reproductive Medicine Unit, ANDROS Day Surgery, Palermo (Palermo I), 3 Department of Obstetrics–Gynecology, San Martino Hospital, University of Genova, Genova, 4 Centro Medico San Luca, Bari, 5 S.I.S.ME.R., Reproductive Medicine Unit, Bologna, 6 Medicina della Riproduzione Umana, European Hospital, Roma and 7 A.M.B.R.A., Associazione Medici e Biologi per la Riproduzione Assistita, Palermo (Palermo II), Italy

8 To whom correspondence should be addressed at: Infertility Unit, Regina Elena Hospital, Via Manfredo Fanti 6, 20122, Milan, Italy. Email: g.ragni{at}icp.mi.it


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The new Italian law, passed in 2004, regulating assisted reproduction technology imposes that no more than three oocytes can be fertilized at one time and that all embryos obtained must be transferred simultaneously. Oocyte cryopreservation is allowed while embryo cryostorage is banned. The aim of this study was to evaluate the clinical impact of these limitations. METHODS: Seven Italian infertility centres were invited to collect data on IVF cycles performed over the first 4 months of application of the new legislation. As a control, all centres provided data on cycles performed in the same solar period, 1 year before. RESULTS: Data from 1861 cycles were obtained, 961 in the pre-law period and 900 in the post-law period. Pregnancy rate per oocyte retrieval and rate of multiple pregnancies in the pre- and post-law periods were 27.0 and 24.2% (P=0.18) and 25.8 and 20.9% (P=0.11) respectively. However, the prohibition to freeze embryos does appear to have markedly reduced the cumulative rate of success. CONCLUSIONS: The rate of success of IVF–ICSI cycles using fresh embryos is not significantly influenced by the new legislation while the prohibition to freeze embryos seems to result in a more relevant impact.

Key words: embryo transfer/IVF/legislation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Last February, the Italian Parliament gave final approval to a new law regulating assisted reproduction technology (Benagiano and Gianaroli, 2004Go). This law, whose principles became operative on March 10, 2004, imposes many limitations to Italian reproductive specialists. Specifically, no more than three oocytes can be fertilized at one time during an IVF treatment, and subsequently all embryos obtained must be transferred to the woman's uterus simultaneously. The freezing of spare embryos and screening of embryos for genetic and/or chromosomal defects are forbidden. Moreover, the law prohibits oocyte or sperm donation, the use of surrogate mothers, and rules out treatments for gay couples and single people.

This new law, which in fact establishes the rights of the embryo over those of the mother, has been severely criticized (Clarke, 2003Go; Benagiano and Gianaroli, 2004Go; Robertson, 2004Go; Turone, 2004Go). Ethical and clinical aspects of the entire law have recently been exhaustively discussed (Benagiano and Gianaroli, 2004Go). In the present study, we have specifically studied the clinical consequences of one of the most controversial aspects of this new legislation, the limitation of the number of oocytes to be fertilized to no more than three and the obligation to transfer all embryos. This restriction is expected to severely reduce the rate of success while, concomitantly, the obligation to transfer all embryos obtained may expose women to an increased risk of multiple pregnancies. In order to clarify these issues, seven infertility centres were invited to collect data on IVF cycles performed over the first 4 months of application of the new legislation. As a control, all centres provided data on IVF cycles performed in the same solar period, 1 year before. The primary objective of the study was to evaluate the influence of the new law on the pregnancy rate and on the incidence of multiple pregnancies in fresh embryo transfer cycles. The secondary aim was to evaluate the impact of the prohibition to freeze embryos.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An invitation to participate in this collaborative effort was mailed to seven infertility units belonging to the Italian Society of Reproduction (Società Italiana della Riproduzione, S.I.d.R.). All contacted centres (Bari, Bologna, Genova, Milano, Palermo I, Palermo II, Roma) agreed to participate. All participating centres have been engaged for ≥5 years in the field of in vitro reproductive technologies and none of them has reported to have markedly modified their personnel and/or their logistic organization over the last 2 years. Patients selected for IVF or ICSI and who performed oocyte retrieval between March 10 and July 10, 2004 were recruited. Controls were women who underwent oocyte retrieval during the same solar period 1 year before (between March 10, and July 10, 2003). Oocyte retrievals performed on spontaneous cycles were excluded. Centres were also invited to report the number of cancelled cycles during the study periods. Data were provided anonymously by each participating centre. Approval for the study by the local institutional review board was not requested since this was a retrospective study. Patients in the participating Units routinely gave an informed consent for their clinical data to be used for statistical evaluations and/or research purposes.

Clinical and biological data were retrospectively obtained from patients' charts. Specifically the following items were requested: age, body mass index (BMI), duration of infertility, previous pregnancies, previous IVF–ICSI cycles, indication for treatment, therapeutic regimen, total IU of FSH used, duration of stimulation, type of assisted reproduction technique (IVF, ICSI, MESA–TESE), sperm concentration at the time of oocyte retrieval, number of oocytes retrieved, number of oocytes used, number of embryos obtained, number of embryos transferred, clinical pregnancy rate, number of implanted embryos, occurrence of severe ovarian hyperstimulation syndrome (OHSS) requiring hospitalization. Centres were also invited to provide data on the cumulative pregnancy rate per oocyte retrieval using frozen oocytes and frozen embryos. Clinical pregnancy was defined as ultrasonographic demonstration of an intrauterine gestational sac 4 weeks after embryo transfer. Patients could be included in both study groups. Data were adjourned to December 2004.

Data were analysed using {chi}2-test, {chi}2-test for trend, Fisher's exact test, Student's t-test and non-parametric Wilcoxon test as appropriate (SPSS/Windows, USA).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Data from 1861 cycles were obtained, 961 in the pre-law period (March 10 and July 10, 2003) and 900 in the post-law period (March 10, and July 10, 2004). Distribution according to study centre was as follows: Bari (83 + 93), Bologna (179 + 156), Genova (61 + 60), Milano (241 + 242), Palermo I (113 + 110), Palermo II (44 + 49), Roma (240 + 190). Baseline clinical characteristics of patients in the pre- and post-law periods were similar (Table I). Stimulation protocols were significantly different in the two study periods: the use of protocols with GnRH antagonist has increased whereas the use of long protocol regimens has decreased (Table II). Total amount of FSH used was significantly reduced after the advent of the new legislation (mean reduction: 7%, 95% CI: 2–12) (Table II). Biological data and clinical outcome of recruited cycles are shown in Table III. Introduction of the new legislation was associated with an increase in number of ICSI procedures and a decrease in number of classical IVF. The following variables were also significantly reduced in the post-law period: number of oocytes retrieved, number of oocytes used, number of embryos obtained and number of embryos transferred. In particular, a significant increase in the rate of single embryo transfer was observed (13.1 and 21.3% in the pre- and post-law periods respectively). Fertilization rate was increased in the post-law period. Percentage of cycles when transfer of embryos could not be performed was similar (Table III). Pregnancy rate per oocyte retrieval and per transfer in the pre- and post-law periods was 27.0 and 24.2% and 30.5 and 27.2% respectively. These differences were not statistically significant (P=0.18 and P=0.14 respectively). Numbers of cancelled cycles were 169 (15.0) and 187 (17.2) in the pre- and post-law periods respectively (P=0.16). Pregnancy rates per cycle started were thus 22.9 and 20.1% respectively (P=0.11). In none of the participating centres was a statistically significant difference in pregnancy rate detected (data not shown). Implantation rate was similar. Severe OHSS was a rare event in both groups. A slightly lower rate of triplets was observed in the post-law period (5.0% before versus 2.8% after); this difference was not statistically significant (Table IV).


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Table I. Characteristics of cycles according to study period: clinical data

 

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Table II. Characteritics of cycles according to study period: stimulation protocols

 

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Table III. Characteritics of cycles according to study period: biological data and clinical outcome

 

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Table IV. Incidence of extrauterine and multiple pregnancy according to study period

 
Overall, the following baseline characteristics of cycles resulted in significant differences in the two study periods: protocol of stimulation, amount of FSH used, type of assisted reproduction technique, number of oocytes retrieved. In order to control for these potentially confounding factors, subgroup analyses were performed (Table V). No significant difference according to study period emerged. The documentation that pregnancy rate is not markedly influenced by the advent of the new legislation does not mean that some subgroups of patients may be more severely hampered. In particular, older women and/or couples with a severe male cause of infertility might represent two categories of patients that may be mostly damaged by the new legislation. A subgroup analysis was thus performed to investigate these possibilities; in both groups of patients success rate was not found to be significantly reduced (Table VI).


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Table V. Pregnancy rate per oocyte retrieval in the two study periods according to stimulation protocol, type of assisted reproduction technique and amount of FSH used

 

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Table VI. Pregnancy rate per oocyte retrieval in the two study periods according to womens age and semen concentration

 
As previously mentioned, the new legislation prohibits embryo freezing while allowing oocyte cryostorage. In order to give insights on this aspect of the law, data from the four participating centers which froze embryos in the pre-law period and froze oocytes in the post-law period were analysed (Bologna, Palermo I, Palermo II, Roma) (Table VII). The number of frozen procedures and use of stored material have dropped in the post-law period. Cumulative pregnancy rate per oocyte retrieval was significantly higher in the pre-law period (33.7 and 26.3%, P=0.009).


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Table VII. Impact of the prohibition of embryo freezing

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Approval of the new Italian legislation has been followed by severe criticisms since it was univocally hypothesized that the rate of success of IVF techniques would have been markedly reduced while, concomitantly, the necessity to transfer all embryos obtained would have increased the rate of multiple pregnancies (Clarke, 2003Go; Benagiano and Gianaroli, 2004Go; Robertson, 2004Go; Turone, 2004Go). To the best of our knowledge, this is the first study documenting the clinical effects of this legislation. Our data have shown that a strategy of no more than three fertilized oocytes and transfer of all obtained embryos has a limited impact on the success rate of IVF cycles using fresh embryos. Moreover, we failed to observe a relevant increase in the rate of multiple pregnancies. Rate of triplets appears even to be decreased although this difference was not statistically significant. Interestingly, the number of single embryo transfers has increased while, obviously, no patients received four or more embryos in the post-law period. Conversely, the prohibition to freeze embryos may markedly reduce the chance of conception per stimulation cycle, in particular in women retrieving a high number of oocytes. Oocyte cryopreservation is an alternative technique that is allowed by the new legislation and that holds promise for the future. However, the technique cannot currently be considered as valid as embryo cryostorage (Borini et al., 2004Go; Tucker et al., 2004Go; Practice Committee of the American Society for Reproductive Medicine, 2004Go). In line with these findings, the cumulative pregnancy rate per oocyte retrieval was significantly lower in the post-law period (when frozen oocytes were used) compared to the pre-law period. Conversely, pregnancy rate using frozen oocytes and frozen embryos did not significantly differ. Two major points have to be kept in mind when considering these results. First, residual oocytes were not systematically stored in the post-law period. Cryopreservation (embryos and oocytes in the pre- and post-law periods respectively) was hence less frequent after the advent of the law (11.3 versus 33.5% of retrieving cycles respectively: P<0.001). The low number of cycles using frozen oocytes does not allow reliable comparisons of the two frozen techniques. Second, length of follow-up is different in the two groups. All patients in the pre-law period are expected to have already used their material whereas many women in the post-law period may have not yet used their frozen oocytes. In line with this hypothesis, a lower rate of use was documented in the post-law period.

Some limits of the present study have to be considered. First, since this is not a randomized study, it cannot be excluded that some selection biases may have influenced the observed results. Baseline clinical characteristics of cycles in the two study periods are similar, suggesting that this potential bias should not have played a major role. In particular, number of cancelled cycles and rates of poor responders and hyper-responders did not differ. Conversely, significant differences have been found for some variables strictly related to the stimulation regimen (protocol used, amount of FSH administered, type of assisted reproduction technique and number of oocytes retrieved). The increased use of protocols with GnRH antagonist in the post-law period may be of particular concern. There are at least two possible explanations for these differences. (i) The historical context: GnRH antagonists have become part of the armamentarium of stimulation drugs in recent years. Of note, lower doses of FSH administered and a reduced number of oocytes have been associated with this therapeutic regimen (Borm and Mannaerts, 2000Go; Olivennes et al., 2000Go; Al-Inany and Aboulghar, 2002Go). (ii) The new legislation: the limit of three oocytes to be fertilized may have led to reduced doses of FSH and consequently to a reduced number of retrieved oocytes. To exclude a possible confounding role of these factors, data were analysed controlling for these variables. This analysis still failed to document any relevant influence of the new legislation on the chances of success (Table V). A second limitation of our study is related to the sample size. Setting probabilities of type I and type II error at 0.05 and 0.20 respectively, the sample size herein recruited allowed us to detect absolute differences in pregnancy rate of ≥6% (Mittendorf et al., 1995Go). Differences below this limit may still be considered clinically relevant.

On the basis of the present results, other aspects deserve consideration. The limit of no more than three oocytes to be fertilized implies that oocytes have to be better selected. In this regard, oocyte denudation, a required step during ICSI procedures, allows optimal evaluation of the cell. This may explain why the number of ICSI procedures has increased in the post-law period even though the quality of semen samples has remained unchanged. The enhanced fertilization rate observed with the new legislation may also be consequent to the selection of higher quality oocytes. Although the increased percentage of ICSI procedures was not detrimental to the results, this phenomenon may have significant clinical implications considering that the safety of this technique is a matter of discussion (Retzloff and Hornstein, 2003Go; Katalinic et al., 2004Go; Devroey and Van Steirteghem, 2004Go; Bonduelle et al., 2005Go; Hansen et al., 2005Go). Another interesting aspect emerging from this survey is the reduction of the amount of FSH used. The observation that a milder stimulation is not associated with a reduced pregnancy rate is intriguing. This represents an economically positive aspect of the law. However, as previously mentioned, it cannot be excluded that the increased use of protocols with GnRH antagonist may also have played a role. Despite this reduction, the rate of severe OHSS requiring hospitalization was unchanged. This observation is not surprising considering that this syndrome typically arises in women known to be at risk and, thus, typically treated with low doses of FSH. Finally, it is interesting to note that pregnancy rate similarly increased with the number of oocytes retrieved in both study periods even though the advent of the new legislation imposed a limit on the number of oocytes to be used. There are two possible explanations for this observation: (i) retrieving a high number of oocytes may be per se an indicator of oocyte quality; (ii) retrieving a higher number of oocytes gives biologists the opportunity to select those of higher quality.

In conclusion, the impact of the new legislation on the rate of success of IVF cycles using fresh embryos is limited, at most. Considering the sample size recruited, it can be inferred that the absolute reduction in pregnancy rate per oocyte retrieval is <6%. The impact of the prohibition on freezing embryos remains to be determined.


    Notes
 
* Collaborators in the Società Italiana della Riproduzione (S.I.d.R.) study group on the impact of the law 40/2004 were: I.Caliari and A.E.Nicolosi (Infertility Unit, Regina Elena Hospital, Milano); F.Sammartano and A.Volpes (Reproductive Medicine Unit, ANDROS Day Surgery, Palermo); P.Lanera (Department of Obstetrics–Gynecology, San Martino Hospital, University of Genova, Genova); E.Sarcina (Centro Medico San Luca, Bari); A.D'Angelo and M.Toschi (S.I.S.ME.R., Reproductive Medicine Unit, Bologna); L.Rienzi and F.Ubaldi (Medicina della Riproduzione Umana, European Hospital, Roma); R.Ciriminna, V.Agrifoglio and P.Napoli (A.M.B.R.A., Associazione Medici e Biologi per la Riproduzione Assistita, Palermo). Back


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 Materials and methods
 Results
 Discussion
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Submitted on December 29, 2004; resubmitted on March 1, 2005; accepted on March 8, 2005.