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The effect of the 2004 Italian law on outcomes of assisted reproduction technology in severe male factor infertility
Reproductive BioMedicine Online (2010) 20, 2– 10
www.sciencedirect.com www.rbmonline.com
ARTICLE
The effect of the 2004 Italian law on outcomes of assisted reproduction technology in severe male factor infertility GB La Sala *, A Nicoli, F Capodanno, I Rondini, MT Villani, F Iannotti Department of Obstetrics and Gynecology, Arcispedale Santa Maria Nuova, Viale Risorgimento 80, 42100 Reggio Emilia, Italy * Corresponding author. E-mail address: [email protected] (GB La Sala). Dr Giovanni Battista La Sala graduated in Medicine in 1976 from the University of Parma. In 1980 he was awarded a post-graduate qualification in obstetrics and gynecology and reproductive medicine from the same university. Currently he is Professor of Obstetrics and Gynecology at the Nursery School of the University of Modena and Reggio Emilia, Italy. Dr La Sala also directs the Operative Unit of Obstetrics and Gynecology and the Center of Reproductive Medicine of the St Maria Nuova Hospital of Reggio Emilia, Italy. His research interests include oocyte cryopreservation, assisted reproduction follow-up, endometriosis and endoscopic surgery.
Abstract The Italian law regulating assisted reproductive technologies that came into force in 2004 restricts the number of fertil-
ized oocytes per cycle to three, obliges the subsequent transfer of all resulting embryos and prohibits the freezing of surplus embryos. This study evaluates the impact of the law on severe oligozoospermic, cryptozoospermic, obstructive azoospermic and non-obstructive azoospermic patients. Intracytoplasmic sperm injection outcomes of 1066 cycles performed in the 4 years before the passing of the law were compared with 804 cycles performed in the 4 years after the law came to pass. Globally, analysis of clinical and obstetric outcomes showed a significant decrease in terms of pregnancy and delivery rates per cycle (17.8% versus 10.9% and 14.2% versus 8.5%, respectively) and per embryo transfer (18.8% versus 13.8% and 15.0% versus 10.7%, respectively), and a significant drop in multiple deliveries (35.1% versus 17.6%) in the post-law period. Cryptozoospermic and azoospermic couples were affected by the Italian law more than severe oligozoospermic couples. The results showed that the Italian law limits the efficiency of assisted reproduction treatment in couples with severe male factor. It is hoped that the Italian assisted reproductive technologies law is altered as soon as possible, allowing the insemination of more than three oocytes. RBMOnline ª 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: azoospermia, cryptozoospermia, delivery, ICSI, pregnancy, severe oligozoospermia
Introduction On 10 March 2004, the present Italian law regulating assisted reproductive technologies came to pass. In terms of clinical practice, the Italian law restricts the number of fertilized oocytes in each cycle to three, obliges the subse-
quent transfer of all resulting embryos and prohibits the freezing of surplus embryos (Benagiano and Gianaroli, 2004; Clarke, 2003; La Sala et al., 2006a,b,c; Ragni et al., 2005; Turone, 2004). The major problems with this law were immediately identified, involving social issues, human rights, applications of assisted reproductive technologies
1472-6483/$ - see front matter ª 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2009.10.007
The 2004 Italian law and male factor infertility and a respect for the Italian Constitution (La Sala et al., 2006a,b,c; Manna and Nardo, 2005; Robertson, 2004). Several centres evaluated the possible clinical impact on assisted reproduction outcomes of the limitations brought about by the Italian law. Although a multicentric Italian study observed a reduced cumulative success rate per oocyte retrieval, the same authors did not uncover any difference in terms of clinical pregnancy and implantation rate before and after the introduction of the law (Ragni et al., 2005). A comparison of the results from the first 2 years after the Italian reproductive legislation was passed with the results of the 2 years before the law showed that the obligation to transfer all available embryos obtained from up to three inseminated oocytes neither reduced success rates of assisted reproduction treatment nor increased the rate of multiple births (La Sala et al., 2008). Until 2006, almost all data correlating Italian law and assisted reproduction outcomes were obtained by studying the general assisted reproduction population, but of particular interest is the evaluation of the impact of the restrictions brought about by the Italian legislation on specific subpopulations, like severe oligozoospermic, cryptozoospermic, obstructive azoospermic (OA) and non-obstructive azoospermic (NOA) patients. In these specific assisted reproduction subpopulations, spermatozoa are probably more likely to have alterations like meiotic errors (Ferguson et al., 2007; Martin, 2006, 2008), chromosomal abnormalities such as aneuploidy, diploidy, Y chromosome damage (Kovanci et al., 2001; Kirkpatrick et al., 2008; Moemen et al., 2008; Sadeghi-Nejad and Oates, 2008) and DNA fragmentations (Irvine et al., 2000; Practice Committee of American Society for Reproductive Medicine, 2008; Zini et al., 2008). Moreover, testicular spermatozoa obtained from OA and NOA patients are likely to contain a significantly higher proportion of immature spermatozoa (Meseguer et al., 2008). For all of these reasons, data present in literature suggest a positive correlation between lower implantation rate and recurrent pregnancy loss in cases of poor semen quality (Bernardini et al., 2004; Currell et al., 2003; De Vos et al., 2003; Farfalli et al., 2007; Garrido et al., 2008; Larson-Cook et al., 2003; Meseguer et al., 2008; Muriel et al., 2006; Virro et al., 2004). Recently Greco et al. (2006) observed that, in the presence of severe semen alterations (especially in NOA patients), the restriction on the number of oocytes seems to dramatically reduce the chance of achieving a clinical pregnancy. The multicentric analysis conducted by Ciriminna et al. (2007), comparing biological and clinical outcomes obtained 2 years before and after the implementation of the law, showed a negative impact of the Italian reproductive legislation in terms of clinical pregnancy rate and implantation rate, especially in the NOA subgroup. Therefore, recent data seem to suggest that in couples already disadvantaged because of severe semen alterations, the repercussions of the Italian law may have a negative effect in terms of biological and clinical outcomes. The goal of the present study was to investigate this possibility.
Materials and methods The present study was carried out in the non-private infertility clinic at the Arcispedale Santa Maria Nuova, in Reggio Emilia, Italy.
3 Before the introduction of the Italian reproductive law, no more than four oocytes were inseminated forwomen <35 years old, no more than five oocytes for women between 35 and 37 years of age, and all suitable oocytes for women 38 years old and, in all cases, all available embryos were transferred. In azoospermic couples, all suitable oocytes were injected and all available embryos were transferred, regardless of maternal age. This management was due to the fact that the public service has some unique features related to constraints prohibiting disposal and/or freezing of surplus embryos as well as multifetal pregnancy reduction. Moreover, only day 2–3 embryos were transferred without assisted hatching. In contrast, after the passing of the Italian law, non-private clinics are obliged to inseminate no more than three oocytes and to transfer all available embryos per cycle. Out of necessity, surplus oocytes are frozen and used for fertilization in future cycles, with methods previously described (La Sala et al., 2006a,b,c). In order to evaluate the impact of the Italian reproductive law on couples presenting for assisted reproduction treatment with a male infertility factor, laboratory and clinical data on 778 couples submitted to 1066 intracytoplasmic sperm injection (ICSI) cycles performed in the 4 years before the law (from 10 March 2000 to 9 March 2004) were compared with data on 587 couples submitted to 804 ICSI cycles performed in the 4 years after the law (from 10 March 2004 to 9 March 2008). The couples in the post-law sample were never submitted to previous assisted reproduction treatments in the study clinic. In each study group, the same ovarian stimulation, the same oocyte retrieval methods (La Sala et al., 2006a,b,c) and the same sperm preparation (World Health Organization, 1999) and sperm surgical recovery protocols (La Sala et al., 2006a,b,c) were used. The study was approved by the ethics committee of the hospital.
Inclusion criteria The study population included couples affected by severe male factor infertility submitted to ICSI, either only in the pre-law period or only in the post-law period. From 10 March 2000 to 9 March 2008, a total of 1494 couples were submitted to ICSI; 129 couples were excluded because they presented a female infertility cofactor and/or because they were submitted to assisted reproductive technologies both before and after the law. Inclusion criteria for the male partner were severe oligozoospermia (sperm concentration at the time of oocyte retrieval 5 · 106/ml), cryptozoospermia (sperm concentration at the time of oocyte retrieval <1 · 106/ml) and azoospermia. This last group of patients included obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). All severe oligozoospermic patients recruited in the study had a percentage of normal form spermatozoa, <5% (according to strict Kruger’s criteria), and/or a number of activated spermatozoa, <1.5 · 106/ml after capacitation. Before and after the law, respectively, the clinic performed 414 and 304 ICSI cycles in severe oligozoospermic
4 patients, 437 and 397 ICSI cycles in cryptozoospermic patients and 215 and 103 ICSI cycles in azoospermic patients. Before and after the law, in severe oligozoospermic patients, mean maternal age was 34.6 ± 4.2 years and 35.4 ± 4.2 years, respectively, 33.9 ± 4.3 years and 35.3 ± 4.6 years in cryptozoospermic patients and 34.2 ± 4.2 and 34.3 ± 3.6 years in azoospermic patients. Each study group was divided into two subgroups, according to maternal age (<38 and 38 years of age).
Ovarian stimulation, oocyte retrieval and sperm analysis Ovarian down-regulation was achieved by either a long luteal leuprolide acetate protocol (Enantone 3.75 mg; Takeda, Milano, Italy) or by a leuprolide acetate microdose flare protocol (Decapeptyl 0.1 mg; Ipsen Pharma, Barcelona, Spain). Complete ovarian suppression is usually achieved within 14–20 days (serum oestradiol concentrations <50 pg/ml and no follicles [10 mm). Thereafter, recombinant FSH (rFSH) (75 IU FSH, Gonal F; Serono, Rome, Italy) was started, followed by 75–450 IU/day (1–6 ampoules/day) for the next 5–10 days. Ovarian response was monitored daily and the dose of rFSH was individually adjusted according to the ovarian response estimated through serum oestradiol and follicular growth. When one or more follicles [17 mm in diameter and serum oestradiol concentrations of 200 pg/ml per follicle [15 mm in diameter were achieved, 10,000 IU human chorionic gonadotrophin (HCG) (Gonasi; AMSA, Milano, Italy) or recombinant HCG 250 lg (Ovitrelle; Serono) were intramuscularly administered. Oocyte retrieval by ultrasound-guided transvaginal aspiration was performed 34–36 h after HCG administration. All patients scheduled for embryo transfer received intramuscular or transvaginal progesterone supplementation (100 mg/day for 15 days; Prontogest; AMSA; or 600 mg/day for 15 days; Prometrium, Rottapharm, Milano, Italy). Three embryos at the most were transferred 48 h after oocyte retrieval, and the remaining oocytes were cryopreserved (La Sala et al., 2006a,b,c).
GB La Sala et al. of metaphase II oocytes. ICSI was performed according to the method reported by Palermo et al. (1992). For azoospermic patients, sperm surgical extraction by testicular sperm aspiration (TESA) was performed as described elsewhere (La Sala et al., 2006c). All injected oocytes were cultured in 40 ll microdrops under mineral oil (6% CO2, 37C). Oocyte fertilization was assessed 18 20 h after ICSI through confirmation of the presence and location of 2 pronuclei using an inverted microscope with Hoffman modulation contrast at 400· magnification (TE 2000 U; Nikon Corp, Tokyo, Japan). All patients undergoing embryo transfer received supplemental progesterone intramuscularly (100 mg/day for 15 days; Prontogest; AMSA) or vaginally (300 mg/day for 15 days; Prometrium, Rottapharm).
Outcome measures Pregnancy was initially determined 12 days after embryo transfer by a positive quantitative serum bHCG assay [10 IU, followed by another quantitative bHCG after 2– 4 days to ensure appropriately rising levels. A clinical pregnancy was defined as at least one fetus with a positive heart beat revealed by transvaginal sonography at 4–5 weeks after embryo transfer.
Analysis For all ICSI cycles before and after the law, maternal age, sperm concentration, sperm motility percentage and sperm morphology were evaluated. Moreover, the total number of recovered and injected oocytes, cleavage rate and total number of embryos obtained were evaluated. Also, the implantation rate, clinical pregnancy rate, abortion rate, total number of deliveries, singletons, twins and deliveries with three or more than three babies and perinatal outcomes were evaluated. All data were entered in Excel and odds ratios (OR) and Corenfield’s 95% confidence intervals (CI) were derived. Continuous variables were compared with Student’s t-test; P \ 0.05 was considered statistically significant.
Semen preparation, oocyte insemination, and embryo culture
Results
Semen samples were collected after 3–5 days of abstinence and their preparation for ICSI was performed following the WHO standard protocol (World Health Organization, 1999). Briefly, an appropriate volume of fresh semen was diluted with 10 ml of buffer medium (Cook IVF, Australia); after centrifugation (10 min at 800g, at room temperature) the supernatant was removed and replaced with another 5 ml of buffer medium. After a second centrifugation, the supernatant was once again removed and the pellet was overlaid with 1 ml of medium and incubated (37C, 6% CO2 in air) to allow separation by swim up. In cases of a sperm concentration <1 · 106/ml, the sample was concentrated by one centrifugation (1500g) and the pellet was suspended in 1 ml of medium. For ICSI, following removal of the cumulus and corona cells surrounding the oocyte, nuclear maturation was assessed by inverted microscopy to ensure exclusive injection
From 10 March 2000 to 10 March 2008, biological and clinical outcomes obtained in 1066 ICSI cycles performed for 778 couples during the 4 years that preceded the passing of the Italian law were compared with 804 ICSI cycles performed for 587 couples during the 4 years that followed the introduction of the same law. The biological outcomes are shown in Table 1. Comparing the two cohorts of patients, after the law the following statistical significances were observed: (i) an increased maternal age (32.4 ± 4.2 versus 35.2 ± 4.3, P < 0:05); (ii) an increase in the rate of couples, cycles and embryo transfer where the woman was 38 years old (20.7% versus 30.2%, OR 0.604; 95% CI 0.472–0.774; 21.1% versus 31.5%, OR 0.583; 95% CI 0.473–0.718; 20.4% versus 31.0%, OR 0.570; 95% CI 0.454–0.715, respectively); (iii) a decrease in the number of retrieved oocytes (7.6 ± 3.8 versus 6.2 ± 3.7, P < 0:05); (iv) a decrease in the number of
The 2004 Italian law and male factor infertility
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Table 1 Biological outcomes in intracytoplasmic sperm injection cycles with severe male factor performed before and after the introduction of the Italian assisted reproductive technologies law. Parameter
Pre-law
Post-law
Statistics
Mean maternal age in years ± SD No. of couples <38 years of age 38 years of age No. of cycles <38 years of age 38 years of age No. of cycles/couple Embryo transfer <38 years of age 38 years of age No. of oocytes retrieved No. of injected oocytes Cleavage No. of obtained and transferred embryos
34.2 ± 4.2 778 617/778 (79.3) 161/778 (20.7) 1066 841/1066 (78.9) 225/1066 (21.1) 1.4 1009/1066 (94.7) 803/1009 (79.6) 206/1009 (20.4) 8123 (7.6 ± 3.8) 5283 (5.1 ± 2.3) 3438/5283 (65.1) 3438 (3.4 ± 1.9)
35.2 ± 4.3 587 410/587 (69.8) 177/587 (30.2) 804 551/804 (68.5) 253/804 (31.5) 1.4 638/804 (79.4) 440/638 (69.0) 198/638 (31.0) 4930 (6.2 ± 3.7) 2028 (2.6 ± 0.8) 1247/2028 (61.5) 1247 (1.6 ± 1.0)
P < 0:05 – 1.654 (1.292–2.118) 0.604 (0.472–0.774) 1.716 (1.392–2.115) 0.583 (0.473–0.718) – 4.606 (3.359–6.316) 1.754 (1.398–2.201) 0.570 (0.454–0.715) P < 0:05 P < 0:05 1.167 (1.050–1.297) P < 0:05
Values are number/total (percentage) or number (mean ± SD) unless otherwise stated; statistics show P-values, odds ratios and 95% confidence intervals.
injected oocytes (5.1 ± 2.3 versus 2.6 ± 0.8, P < 0:05) and a consequent decrease in the number of obtained and transferred embryos (3.4 ± 1.9 versus 1.6 ± 1.0, P < 0:05); (v) a decreased cleavage rate (65.1% versus 61.5%, OR 1.167; 95% CI 1.050–1.297); and (vi) a decreased rate of embryo transfer per cycle (94.7% versus 79.4%, OR 4.606; 95% CI 3.359–6.316). The mean number of cycles per couple was 1.4 before and after the introduction of the Italian law. Global analysis of clinical and obstetric outcomes (Table 2) showed: (i) in the post-law period, a significant decrease in terms of pregnancy and delivery rates per cycle (17.8% versus 10.9%, OR 1.765; 95% CI 1.346–2.313 and
14.2% versus 8.5%, OR 1.786; 95% CI 1.321–2.414, respectively) and per embryo transfer (18.8% versus 13.8%, OR 1.450; 95% CI 1.102–1.908 and 15.0% versus 10.8%, OR 1.475; 95% CI 1.088–2.000, respectively); (ii) similar implantation and abortion rates between the two periods; and (iii) a significant drop of multiple deliveries after the introduction of the law (35.1% versus 17.6%, OR 2.524; 95% CI 1.254–5.068). Tables 3 and 4 show the clinical and biological outcomes for severe oligozoospermic, cryptozoospermic and azoospermic couples. In the severe oligozoospermic group, in the post-law period there was a non-statistically significant
Table 2 Clinical and obstetrical outcomes in intracytoplasmic sperm injection cycles with severe male factor before and after the introduction of the Italian assisted reproductive technologies law. Parameter
Pre-law
Post-law
Odds ratio (95% CI)
Clinical pregnancies per cycle Clinical pregnancies per transfer Implantation rate Abortion rate Deliveries per cycle Deliveries per transfer Singletons Twins Triplets or greater
190/1066 (17.8) 190/1009 (18.8) 294/3438 (8.6) 36/190 (18.9) 151/1066 (14.2) 151/1009 (15.0) 98/151 (64.9) 42/151 (27.8) 11/151 (7.3)
88/804 (10.9) 88/638 (13.8) 109/1247 (8.7) 20/88 (22.7) 68/804 (8.5) 68/638 (10.7) 56/68 (82.4) 11/68 (16.2) 1/68 (1.5)
1.765 1.450 0.976 0.795 1.786 1.475 0.264 1.997 5.264
Gestational age (weeks) <32 37
7/151 (4.6) 69/151 (45.7)
3/68 (4.4) 20/68 (29.4)
1.053 (0.286–3.852) 2.020 (1.099–3.706)
Birthweight (g) <1500 <2500
29/215 (13.5) 94/215 (43.7)
11/81 (13.6) 20/81 (24.7)
0.992 (0.476–2.066) 2.369 (1.342–4.180)
Values are number/total (percentage) unless otherwise stated.
(1.346–2.313) (1.102–1.908) (0.776–1.228) (0.431–1.464) (1.321–2.414) (1.088–2.000) (0.119–0.587) (0.965–4.123) (0.850–32.205)
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Table 3 Oocyte retrieval, embryo transfer and biological outcome in severe oligozoospermic, cryptozoospermic and azoospermic patients before and after the introduction of the Italian assisted reproductive technologies law. Severe oligozoospermia
Mean maternal age in years ± SD No. of couples <38 years of age 38 years of age No. of cycles <38 years of age 38 years of age No. of cycles/couple Embryo transfer <38 years of age 38 years of age No. of oocytes retrieved No. of injected oocytes Cleavage
No. of embryos obtained and transferred
Cryptozoospermia
Azoospermia
Pre-law
Post-law
Statistics
Pre-law
Post-law
Statistics
Pre-law
Post-law
Statistics
34.6 ± 4.2
35.4 ± 4.2
P \ 0.05
33.9 ± 4.3
35.3 ± 4.6
P \ 0.05
34.2 ± 4.2
34.3 ± 3.6
NS
334 261/334 (78.1) 73/334 (21.9) 414 322/414 (77.8) 92/414 (22.2) 414/334 (1.2) 392/414 (94.7) 310/322 (96.3) 82/92 (89.1) 3042 (7.3 ± 3.6) 1826 (4.6 ± 2.2) 1305/ 1826 (71.5) 1305 (3.3 ± 1.8)
252 – 168/252 1.788 (1.237– (66.7) 2.583) 84/252 0.559 (0.387– (33.3) 0.808) 304 – 202/304 1.767 (1.269– (66.4) 2.469) 102/304 0.566 (0.406– (33.6) 0.788) 304/252 NS (1.2) 264/304 2.700 (1.576– 4.623) (86.8) 177/202 3.649 (1.810– (87.6) 7.349) 87/102 1.414 (0.611– (85.3) 3.265) 1875 P \ 0.05 (6.2 ± 3.7) 786 P \ 0.05 (2.6 ± 0.7) 542/786 1.128 (0.940– (69.0) 1.353)
317 253/317 (79.8) 64/317 (20.2) 437 352/437 (80.5) 85/437 (19.5) 437/317 (1.4) 406/437 (92.9) 329/352 (93.5) 77/85 (90.6) 3248 (7.4 ± 3.8) 1996 (4.6 ± 2.2) 1340/ 1996 (67.1) 1340 (3.3 ± 1.8)
278 194/278 (69.8) 84/278 (30.2) 397 264/397 (66.5) 133/397 (23.5) 397/278 (1.4) 300/397 (75.6) 200/264 (75.8) 100/133 (75.2) 2428 (6.1 ± 3.5) 996 (2.6 ± 0.7) 581/996 (58.3)
– 1.712 (1.177– 2.488) 0.554 (0.381– 0.807) – 2.086 (1.523– 2.858) 0.479 (0.350– 0.657) NS
127 57 103/127 48/57 (81.1) (84.2) 24/127 9/57 (18.9) (15.8) 215 103 167/215 85/103 (77.7) (82.5) 48/215 18/103 (22.3) (17.5) 215/127 103/57 (1.7) (1.8) 211/215 74/103 (98.1) (71.8) 164/167 63/85 (98.2) (74.1) 47/48 11/18 (97.9) (61.1) 1833 627 (8.5 ± 4.0) (6.3 ± 4.2) 1461 246 (6.8 ± 3.5) (2.8 ± 0.8) 793/1461 124/246 (54.3) (50.4)
– 0.805 (0.354– 1.837) 1.243 (0.544– 2.827) – 0.737 (0.406– 1.338) 1.357 (0.748– 2.462) NS
581 (1.5 ± 1.0)
P \ 0.05
793 (3.8 ± 2.3)
P \ 0.05
542 (1.9 ± 1.0)
P \ 0.05
4.235 (2.758– 6.500) 4.577 (2.765– 7.575) 3.176 (1.410– 7.133) P \ 0.05 P \ 0.05 1.459 (1.247– 1.707)
124 (1.3 ± 0.9)
20.672 (7.317– 58.135) 19.090 (5.856– 61.799) 0.965 (0.347– 2.693) NS P \ 0.05 1.168 (0.892– 1.530)
GB La Sala et al.
Values are number/total (percentage) or number (mean ± SD) unless otherwise stated; statistics show P-values, odds ratios and 95% confidence intervals; NS = not statistically significant.
Parameter
Clinical pregnancies per cycle <38 years of age 38 years of age Clinical pregnancies per embryo transfer <38 years of age 38 years of age Implantation rate Abortion rate Deliveries per cycle <38 years of age 38 years of age Deliveries per embryo transfer <38 years of age 38 years of age
Severe oligozoospermia
Cryptozoospermia
Azoospermia
Pre-law
Post-law
Odds ratio (95% CI)
Pre-law
Post-law
Odds ratio (95% CI)
Pre-law
Post-law
Odds ratio (95% CI)
79/414 (19.1) 69/322 (21.4) 10/92 (10.9) 79 (20.2) 69/310 (22.3) 10/82 (12.2) 122/1305 (9.3) 16/79 (20.3) 60/414 (14.5) 52/322 (16.1) 8/92 (8.7) 60/392 (15.3) 52/310 (16.8) 8/82 (9.8)
43/304 (14.1) 33/202 (16.3) 10/102 (9.8) 43/264 (16.3) 33/177 (18.6) 10/87 (11.5) 58/542 (10.7) 10/43 (23.3) 33/304 (10.9) 27/202 (13.4) 6/102 (5.9) 33/264 (12.5) 27/177 (15.3) 6/87 (6.9)
1.431 (0.956–2.143) 1.397 (0.885–2.203) 1.122 (0.455–2.767) 1.297 (0863–1.950) 1.249 (0.788–1.981) 1.069 (0.430–2.660) 0.861 (0.619–1.196) 0.838 (0.347–2.018) 1.392 (0.887–2.184) 1.320 (0.802–2.170) 1.524 (0.529–4.382) 1.265 (0.803–1.992) 1.014 (0.614–1.674) 1.459 (0.503–4.224)
78/437 (17.8) 67/352 (19.0) 11/85 (12.9) 78/406 (19.2) 67/329 (20.4) 11/77 (14.3) 121/1340 (9.0) 15/78 (19.2) 63/437 (14.4) 55/352 (15.6) 8/85 (9.4) 63/406 (15.5) 55/329 (16.7) 8/77 (10.4)
38/397 (9.6) 33/264 (12.5) 5/133 (3.8) 38/300 (12.7) 33/200 (16.5) 5/100 (5.0) 42/581 (7.2) 7/38 (18.4) 31/397 (7.8) 26/264 (9.8) 5/133 (3.8) 31/300 (10.3) 26/200 (13.0) 5/100 (5.0)
2.622 (1.730– 1.646 (1.050– 3.805 (1.324– 1.640 (1.079– 1.294 (0.819– 3.167 (1.093– 1.621 (1.125– 1.054 (0.398– 1.989 (1.267– 1.695 (1.035– 2.660 (0.881– 1.594 (1.010– 1.343 (0.815– 2.203 (0.724–
33/215 (15.3) 32/167 (19.2) 1/48 (2.1) 33/211 (15.6) 32/164 (19.5) 1/47 (2.1) 51/793 (6.4) 5/33 (15.2) 28/215 (13.0) 28/167 (16.8) 0/48 (0.0) 28/211 (13.3) 28/164 (17.0) 0/47 (0.0)
7/103 (6.8) 6/85 (7.1) 1/18 (5.6) 7/74 (9.5) 6/63 (9.5)
2.487 (1.081– 5.705) 3.121 (1.279– 7.587) 0.362 (0.036– 3.653) 1.774 (0.763– 4.112) 2.303 (0.934– 5.655) 0.217 (0.021– 2.255) 0.878 (0.427– 1.806) 0.238 (0.044– 1.250) 3.706 (1.317–10.390) 4.079 (1.438–11.515) –
3.975) 2.578) 10.894) 2.491) 2.044) 9.135) 2.335) 2.777) 3.121) 2.775) 8.012) 2.514) 2.215) 6.684)
1/11 (9.1) 9/124 (7.3) 3/7 (42.9) 4/103 (3.9) 4/85 (4.7) 0/18 (0.0) 4/74 (5.4) 4/63 (6.3) 0/11 (0.0)
The 2004 Italian law and male factor infertility
Table 4 Clinical results in severe oligozoospermic, cryptozoospermic and azoospermic patients before and after the introduction of the Italian assisted reproductive technologies law.
2.678 (0.944–7.561) 3.037 (1.061–8.640) –
Values are number/total (percentage) unless otherwise stated.
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8
GB La Sala et al.
decrease in the number of deliveries per cycle (14.5% versus 10.9%, OR 1.392; 95% CI 0.887–2.184), both for \38-yearold women (16.2 versus 13.4%, OR 1.320; 95% CI 0.802– 2.170) and 38-year-old women (8.7% versus 5.9%, OR 1.524; 95% CI 0.529–4.382). Similarly, in the cryptozoospermic group there was a statistically significant decrease in the number of deliveries per cycle after the passing of the Italian law (14.4 versus 7.8%, OR 1.989; 95% CI 1.267–3.121), both for \38-year-old women (15.6% versus 9.8%, OR 1.695; 95% CI 1.035–2.775) and 38-year-old women (9.4% versus 3.8%, OR 4.433; 95% CI 0.881–8.012). In the azoospermic population, there was a severe decrease in results in the post-law period in terms of deliveries per cycle (13.0% versus 3.9%, OR 3.706; 95% CI 1.317–10.390). There was also a decrease in deliveries per embryo transfer (13.3% versus 5.4%, OR 2.678; 95% CI 0.944–7.561), but this second difference was not statistically significant. Moreover, a heavy negative impact of the Italian law on NOA couples was observed, with statistically significant decreases in terms of deliveries per cycle (15.5% versus 2.7%, OR 6.713; 95% CI 1.655–26.958) and per embryo transfer (16.0% versus 3.8%, OR 4.726; 95% CI 1.162–19.265) in the post-law period (Table 5). Conversely, in OA couples, despite observing a drop in clinical results after the introduction of the assisted reproductive technologies Italian law, the statistical analysis showed no difference between the two periods.
Discussion The Italian law, issued by the legislative authorities, was anticipated to go against the best interests of infertile women (Ragni et al., 2005; Robertson, 2004). Specifically, to defend the rights of human embryos, the restriction to three fertilized oocytes for transfer per cycle, the obligation to transfer all available embryos at the same time, and the prohibition on freezing of surplus embryos, were imposed on assisted reproduction clinicians. Moreover, the law prohibits preimplantation diagnosis, creating the risk of transferring genetically malformed embryos in patients with known genetic disorders. For the above reasons, the Italian law was expected to decrease the likelihood of achieving pregnancy, to increase the number and complexity of procedures per pregnancy and to increase the risk of multiple gestations, infringing upon basic human rights and the proper application of assisted reproductive technologies (Robertson, 2004). Thus, the debate on the Italian law and its effects on assisted reproduction outcomes is still open. The aim of this study was to verify the impact of the 40/2004 law on couples affected by severe male infertility factor (severe oligozoospermia, cryptozoospermia and azoospermia). Overall, this population of patients submitted to IVF, ICSI and ICSI/TESA, 4 years after the introduction of the law, has shown an increasing trend in maternal age compared with the 4 years preceding the law (unpublished data). This trend was confirmed by the present study, which also found a
Table 5 Outcomes in obstructive azoospermic and non-obstructive azoospermic patients before and after the introduction of the Italian assisted reproductive technologies law. Obstructive azoospermic
Mean maternal age in years ± SD No. of couples Cycles/couple Embryo transfer/cycle No. of oocytes retrieved No. of embryos obtained and transferred Clinical pregnancies per cycle Clinical pregnancies per embryo transfer Implantation rate Deliveries per cycle Deliveries per embryo transfer
Non-obstructive azoospermic
Pre-law
Post-law
Statistics
Pre-law
Post-law
Statistics
34.3 ± 4.4
34.2 ± 4.3
NS
34.1 ± 3.9
34.3 ± 3.3
NS
63 1.8 (112/63) 111/112 (99.1) 917 (8.9 ± 4.1) 721 (7.1 ± 3.6)
19 1.5 (28/19) 22/28 (78.6) 160 (6.2 ± 2.6) 32 (1.2 ± 0.8)
– –
64 103/64 (1.6) 100/103 (97.1) 916 (8.2 ± 4.0) 740 (6.6 ± 3.4)
48 75/48 (1.6) 52/75 (69.3) 467 (6.4 ± 4.6) 92 (1.4 ± 1.0)
–
15/112 (13.4) 15/111 (13.5) 26/721 (6.2) 12/112 (10.7) 12/111 (10.8)
2/28 (7.1) 2/22 (9.1) 2/32 (6.3)
2.010 (0.477–8.318) 1.563 (0.365–6.545) 1.92 (0.397–6.314) 1.560 (0.364–6.563) 1.212 (0.278–5.163)
18/103 (17.5) 18/100 (18) 25/740 (6.7) 16/103 (15.5) 16/100 (16.0)
5/75 (6.7)
2.965 (1.082–8.079) 2.063 (0.742–5.697) 0.425 (0.182–0.988) 6.713 (1.655–26.958) 4.762 (1.162–19.265)
2/28 (7.1) 2/22 (9.1)
30.273 (4.467– 198.873) P \ 0.05 P \ 0.05
5/52 (9.6) 7/92 (7.6) 2/75 (2.7) 2/52 (3.8)
14.744 (4.479– 48.098) P \ 0.05 P \ 0.05
Values are number/total (percentage) or number (mean ± SD) unless otherwise stated; statistics show P-values, odds ratios and 95% confidence intervals; NS = not statistically significant.
The 2004 Italian law and male factor infertility decrease in the number of retrieved and injected oocytes, a decrease in the cleavage rate, in the number of transferred embryos and in embryo transfer per cycle. The reason for this decrease in the number of oocytes retrieved at retrieval cannot be attributed to variations in the ovarian stimulation protocol, as this protocol has remained unchanged between the pre- and post-law periods. It can be postulated that this phenomenon is correlated to the increase in maternal age and/or to random events in the examined population, occurring independently from the assisted reproduction treatments. In this population, already at risk because of the severe male infertility factor, the limitations brought about by the law have, of course, caused a decrease in the cleavage rate and in the number of obtained and transferred embryos. In fact, infertile men, especially those with severe male infertility factor, possess substantially more sperm DNA damage and chromosome aneuploidies than do fertile men or infertile men without severe male infertility factor (Rubio et al., 2001) and these sperm genomic characteristics correlate with a poor prognosis in terms of fertilization and cleavage rates (Lopes et al., 1998). In general, in the post-law period, this study observed a significant decrease in the number of clinical pregnancies per cycle (17.8% versus 10.9%) and per embryo transfer (18.8% versus 13.8%), in the number of deliveries per cycle (14.2% versus 8.5%) and per embryo transfer (15.0% versus 10.7%) and a significant decrease in the number of multiple deliveries (35.1% versus 17.6%). Moreover, this study observed a small increase in the abortion rate, even though not statistically significant (18.9% versus 22.7%). This could be correlated with the poor semen characteristics of these populations, as reported in previous studies (Benchaib et al., 2003; Rubio et al., 2001; Zini et al., 2008). Therefore, the results of the single groups were separately analysed to verify which of the three studied categories was more affected by the limitations brought about by the law. In the severe oligozoospermic group, in the postlaw period, there was a decrease in the number of deliveries per cycle both in the \38-year-old women (16.1% versus 13.4%) and in the 38-year-old women (8.7% versus 5.9%), although the differences were not statistically significant. In the cryptozoospermic group, there was a significant decrease in the number of deliveries per cycle (14.4% versus 7.8%), especially in \38-year-old women (15.6% versus 9.8%). In the azoospermic group, finally, there was a significant decrease in the number of deliveries per cycle (13.0% versus 3.9%), especially in couples affected by NOA (15.5% versus 2.7%). On the contrary, in couples affected by OA, this difference was not statistically significant: nevertheless, the OA sample size was too small to show reliable results. Regarding the obstetric–perinatal outcomes, in the postlaw period the study observed a global significant reduction in the number of deliveries \37 gestational weeks (45.7% versus 29.4%) and of babies born with a birthweight \2500 g (43.7% versus 24.7%). In general, if, on one hand, the 40/2004 law by limiting to three the number of inseminated oocytes per cycle has reduced the rate of multiple deliveries (one of the declared aims of the law), on the other hand it has greatly decreased the efficacy of assisted reproductive technologies, espe-
9 cially in couples affected by cryptozoospermia and azoospermia.
References Benagiano, G., Gianaroli, L., 2004. The new Italian IVF legislation. Reprod. BioMed. Online 9, 117–125. Benchaib, M., Braun, V., Lornage, J., et al., 2003. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum. Reprod. 18, 1023–1028. Bernardini, L.M., Costa, M., Bottazzi, C., et al., 2004. Sperm aneuploidy and recurrent pregnancy loss. Reprod. BioMed. Online 9, 312–320. Ciriminna, R., Papale, M.L., Artini, P.G., et al., 2007. Impact of Italian legislation regulating assisted reproduction techniques on ICSI outcomes in severe male factor infertility: a multicentric survey. Hum. Reprod. 22, 2481–2487. Clarke, H., 2003. Italy approves controversial legislation on fertility treatment. Lancet 362, 2076. Currell, D.T., Liu, L., Peterson, C.M., et al., 2003. Sperm DNA fragmentation is increased in couples with an explained recorrent pregnancy loss. Arch. Androl. 49, 49–55. De Vos, A., Van De Velde, H., Joris, H., et al., 2003. Influence of individual sperm morphology on fertilization, embryo morphology, and pregnancy outcome of intracytoplasmic sperm injection. Fertil. Steril. 79, 42–48. Farfalli, V.I., Magli, M.C., Ferraretti, A.P., et al., 2007. Role of aneuploidy on embryo implantation. Gynecol. Obstet. Invest. 64, 161–165. Ferguson, K.A., Wong, E.C., Chow, V., et al., 2007. Abnormal meiotic recombination in infertile men and its association with sperm aneuploidy. Hum. Mol. Genet. 16, 2870–2879. Garrido, N., Remohı´, J., Martı´nez-Conejero, J.A., et al., 2008. Contribution of sperm molecular features to embryo quality and assisted reproduction success. Reprod. BioMed. Online 17, 855– 865. Greco, E., Ubaldi, F., Rienzi, L., et al., 2006. Limiting the number of injected oocytes to three impairs ICSI outcomes in patients with nonobstructive azoospermia. Hum. Reprod. 21, 1521–1524. Irvine, D.S., Twigg, J.P., Gordon, E.L., et al., 2000. DNA integrity in human spermatozoa: relationships with semen quality. J. Androl. 21, 33–44. Kirkpatrick, G., Ferguson, K.A., Gao, H., et al., 2008. A comparison of sperm aneuploidy rates between infertile men with normal and abnormal karyotypes. Hum. Reprod. 23, 167916–167983. Kovanci, E., Kovacs, T., Moretti, E., et al., 2001. FISH assessment of aneuploidy frequencies in mature and immature human spermatozoa classified by the absence or presence of cytoplasmic retention. Hum. Reprod. 16, 1209–1217. Larson-Cook, K.L., Brannian, J.D., Hansen, K.A., et al., 2003. Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertil. Steril. 80, 895– 902. La Sala, G.B., Gallinelli, A., Nicoli, A., et al., 2006a. Pregnancy loss and assisted reproduction: preliminary results after the law 40/ 2004 in Italy. Reprod. BioMed. Online 13, 65–70. La Sala, G.B., Nicoli, A., Villani, M.T., et al., 2006b. Outcome of 518 salvage oocyte-cryopreservation cycles performed as a routine procedure in an in vitro fertilization program. Fertil. Steril. 86, 1423–1427. La Sala, G.B., Valli, B., Leoni, S., et al., 2006c. Testicular sperm aspiration (TESA) in 327 ICSI cycles. Int. J. Fertil. Womens. Med. 51, 177–182. La Sala, G.B., Villani, M.T., Nicoli, A., et al., 2008. The effect of legislation on outcomes of assisted reproduction technology: lessons from the 2004 Italian law. Fertil. Steril. 89, 854–859.
10 Lopes, S., Sun, J.G., Jurisicova, A., et al., 1998. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil. Steril. 69, 528–532. Manna, C., Nardo, L.G., 2005. Italian law on assisted conception: clinical and research implications. Reprod. BioMed. Online 11, 532–534. Martin, R.H., 2006. Meiotic chromosome abnormalities in human spermatogenesis. Reprod. Toxicol. 22, 142–147. Martin, R.H., 2008. Meiotic errors in human oogenesis and spermatogenesis. Reprod. BioMed. Online 16, 523–531. Meseguer M, Santiso R, Garrido N et al. 2008. Sperm DNA fragmentation levels in testicular sperm samples from azoospermic males as assessed by the sperm chromatin dispersion (SCD) test. Fertil. Steril. [E-publication ahead of print]. Moemen, M.N., Mostafa, T., Gadalla, A.M., et al., 2008. Sperm disomy in idiopathic severely oligoasthenoteratozoospermic males. Andrologia 40, 381–386. Muriel, L., Garrido, N., Ferna ´ndez, J.L., et al., 2006. Value of the sperm deoxyribonucleic acid fragmentation level, as measured by the sperm chromatin dispersion test, in the outcome of in vitro fertilization and intracytoplasmic sperm injection. Fertil. Steril. 85, 371–383. Palermo, G., Joris, H., Devroey, P., Van-Steirteghem, A.C., 1992. Pregnancies after intracytoplasmic injection of a single spermatozoon into an oocyte. Lancet 340, 17–18. Practice Committee of American Society for Reproductive Medicine 2008. The clinical utility of sperm DNA integrity testing. Fertil. Steril. 90 (suppl. 5), S178–S180. Ragni, G., Allegra, A., Anserini, P., et al., 2005. The 2004 Italian legislation regulating assisted reproduction technology: a mul-
GB La Sala et al. ticentre survey on the results of IVF cycles. Hum. Reprod. 20, 2224–2228. Robertson, J.A., 2004. Protecting embryos and burdening women: assisted reproduction in Italy. Hum. Reprod. 19, 1693–1696. Rubio, C., Gil-Salom, M., Simo ´n, C., et al., 2001. Incidence of sperm chromosomal abnormalities in a risk population: relationship with sperm quality and ICSI outcome. Hum. Reprod. 16, 2084–2092. Sadeghi-Nejad, H., Oates, R.D., 2008. The Y chromosome and male infertility. Curr. Opin. Urol. 18, 628–632. Turone, F., 2004. Italy to pass new law on assisted reproduction. Br. Med. J. 328, 9. Virro, M.R., Larson-Cook, K.L., Evenson, D.P., 2004. Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles. Fertil. Steril. 81, 1289–1295. World Health Organization 1999 WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction. Cambridge University Press, Cambridge. Zini, A., Boman, J.M., Belzile, E., Ciampi, A., 2008. Sperm DNA damage is associated with an increased risk of pregnancy loss after IVF and ICSI: systematic review and meta-analysis. Hum. Reprod. 23, 2663–2668. Declaration: The authors report no financial or commercial conflicts of interest. Received 9 April 2009; refereed 26 May 2009; accepted 23 September 2009.
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ARTICLE
The effect of the 2004 Italian law on outcomes of assisted reproduction technology in severe male factor infertility GB La Sala *, A Nicoli, F Capodanno, I Rondini, MT Villani, F Iannotti Department of Obstetrics and Gynecology, Arcispedale Santa Maria Nuova, Viale Risorgimento 80, 42100 Reggio Emilia, Italy * Corresponding author. E-mail address: [email protected] (GB La Sala). Dr Giovanni Battista La Sala graduated in Medicine in 1976 from the University of Parma. In 1980 he was awarded a post-graduate qualification in obstetrics and gynecology and reproductive medicine from the same university. Currently he is Professor of Obstetrics and Gynecology at the Nursery School of the University of Modena and Reggio Emilia, Italy. Dr La Sala also directs the Operative Unit of Obstetrics and Gynecology and the Center of Reproductive Medicine of the St Maria Nuova Hospital of Reggio Emilia, Italy. His research interests include oocyte cryopreservation, assisted reproduction follow-up, endometriosis and endoscopic surgery.
Abstract The Italian law regulating assisted reproductive technologies that came into force in 2004 restricts the number of fertil-
ized oocytes per cycle to three, obliges the subsequent transfer of all resulting embryos and prohibits the freezing of surplus embryos. This study evaluates the impact of the law on severe oligozoospermic, cryptozoospermic, obstructive azoospermic and non-obstructive azoospermic patients. Intracytoplasmic sperm injection outcomes of 1066 cycles performed in the 4 years before the passing of the law were compared with 804 cycles performed in the 4 years after the law came to pass. Globally, analysis of clinical and obstetric outcomes showed a significant decrease in terms of pregnancy and delivery rates per cycle (17.8% versus 10.9% and 14.2% versus 8.5%, respectively) and per embryo transfer (18.8% versus 13.8% and 15.0% versus 10.7%, respectively), and a significant drop in multiple deliveries (35.1% versus 17.6%) in the post-law period. Cryptozoospermic and azoospermic couples were affected by the Italian law more than severe oligozoospermic couples. The results showed that the Italian law limits the efficiency of assisted reproduction treatment in couples with severe male factor. It is hoped that the Italian assisted reproductive technologies law is altered as soon as possible, allowing the insemination of more than three oocytes. RBMOnline ª 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: azoospermia, cryptozoospermia, delivery, ICSI, pregnancy, severe oligozoospermia
Introduction On 10 March 2004, the present Italian law regulating assisted reproductive technologies came to pass. In terms of clinical practice, the Italian law restricts the number of fertilized oocytes in each cycle to three, obliges the subse-
quent transfer of all resulting embryos and prohibits the freezing of surplus embryos (Benagiano and Gianaroli, 2004; Clarke, 2003; La Sala et al., 2006a,b,c; Ragni et al., 2005; Turone, 2004). The major problems with this law were immediately identified, involving social issues, human rights, applications of assisted reproductive technologies
1472-6483/$ - see front matter ª 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2009.10.007
The 2004 Italian law and male factor infertility and a respect for the Italian Constitution (La Sala et al., 2006a,b,c; Manna and Nardo, 2005; Robertson, 2004). Several centres evaluated the possible clinical impact on assisted reproduction outcomes of the limitations brought about by the Italian law. Although a multicentric Italian study observed a reduced cumulative success rate per oocyte retrieval, the same authors did not uncover any difference in terms of clinical pregnancy and implantation rate before and after the introduction of the law (Ragni et al., 2005). A comparison of the results from the first 2 years after the Italian reproductive legislation was passed with the results of the 2 years before the law showed that the obligation to transfer all available embryos obtained from up to three inseminated oocytes neither reduced success rates of assisted reproduction treatment nor increased the rate of multiple births (La Sala et al., 2008). Until 2006, almost all data correlating Italian law and assisted reproduction outcomes were obtained by studying the general assisted reproduction population, but of particular interest is the evaluation of the impact of the restrictions brought about by the Italian legislation on specific subpopulations, like severe oligozoospermic, cryptozoospermic, obstructive azoospermic (OA) and non-obstructive azoospermic (NOA) patients. In these specific assisted reproduction subpopulations, spermatozoa are probably more likely to have alterations like meiotic errors (Ferguson et al., 2007; Martin, 2006, 2008), chromosomal abnormalities such as aneuploidy, diploidy, Y chromosome damage (Kovanci et al., 2001; Kirkpatrick et al., 2008; Moemen et al., 2008; Sadeghi-Nejad and Oates, 2008) and DNA fragmentations (Irvine et al., 2000; Practice Committee of American Society for Reproductive Medicine, 2008; Zini et al., 2008). Moreover, testicular spermatozoa obtained from OA and NOA patients are likely to contain a significantly higher proportion of immature spermatozoa (Meseguer et al., 2008). For all of these reasons, data present in literature suggest a positive correlation between lower implantation rate and recurrent pregnancy loss in cases of poor semen quality (Bernardini et al., 2004; Currell et al., 2003; De Vos et al., 2003; Farfalli et al., 2007; Garrido et al., 2008; Larson-Cook et al., 2003; Meseguer et al., 2008; Muriel et al., 2006; Virro et al., 2004). Recently Greco et al. (2006) observed that, in the presence of severe semen alterations (especially in NOA patients), the restriction on the number of oocytes seems to dramatically reduce the chance of achieving a clinical pregnancy. The multicentric analysis conducted by Ciriminna et al. (2007), comparing biological and clinical outcomes obtained 2 years before and after the implementation of the law, showed a negative impact of the Italian reproductive legislation in terms of clinical pregnancy rate and implantation rate, especially in the NOA subgroup. Therefore, recent data seem to suggest that in couples already disadvantaged because of severe semen alterations, the repercussions of the Italian law may have a negative effect in terms of biological and clinical outcomes. The goal of the present study was to investigate this possibility.
Materials and methods The present study was carried out in the non-private infertility clinic at the Arcispedale Santa Maria Nuova, in Reggio Emilia, Italy.
3 Before the introduction of the Italian reproductive law, no more than four oocytes were inseminated forwomen <35 years old, no more than five oocytes for women between 35 and 37 years of age, and all suitable oocytes for women 38 years old and, in all cases, all available embryos were transferred. In azoospermic couples, all suitable oocytes were injected and all available embryos were transferred, regardless of maternal age. This management was due to the fact that the public service has some unique features related to constraints prohibiting disposal and/or freezing of surplus embryos as well as multifetal pregnancy reduction. Moreover, only day 2–3 embryos were transferred without assisted hatching. In contrast, after the passing of the Italian law, non-private clinics are obliged to inseminate no more than three oocytes and to transfer all available embryos per cycle. Out of necessity, surplus oocytes are frozen and used for fertilization in future cycles, with methods previously described (La Sala et al., 2006a,b,c). In order to evaluate the impact of the Italian reproductive law on couples presenting for assisted reproduction treatment with a male infertility factor, laboratory and clinical data on 778 couples submitted to 1066 intracytoplasmic sperm injection (ICSI) cycles performed in the 4 years before the law (from 10 March 2000 to 9 March 2004) were compared with data on 587 couples submitted to 804 ICSI cycles performed in the 4 years after the law (from 10 March 2004 to 9 March 2008). The couples in the post-law sample were never submitted to previous assisted reproduction treatments in the study clinic. In each study group, the same ovarian stimulation, the same oocyte retrieval methods (La Sala et al., 2006a,b,c) and the same sperm preparation (World Health Organization, 1999) and sperm surgical recovery protocols (La Sala et al., 2006a,b,c) were used. The study was approved by the ethics committee of the hospital.
Inclusion criteria The study population included couples affected by severe male factor infertility submitted to ICSI, either only in the pre-law period or only in the post-law period. From 10 March 2000 to 9 March 2008, a total of 1494 couples were submitted to ICSI; 129 couples were excluded because they presented a female infertility cofactor and/or because they were submitted to assisted reproductive technologies both before and after the law. Inclusion criteria for the male partner were severe oligozoospermia (sperm concentration at the time of oocyte retrieval 5 · 106/ml), cryptozoospermia (sperm concentration at the time of oocyte retrieval <1 · 106/ml) and azoospermia. This last group of patients included obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). All severe oligozoospermic patients recruited in the study had a percentage of normal form spermatozoa, <5% (according to strict Kruger’s criteria), and/or a number of activated spermatozoa, <1.5 · 106/ml after capacitation. Before and after the law, respectively, the clinic performed 414 and 304 ICSI cycles in severe oligozoospermic
4 patients, 437 and 397 ICSI cycles in cryptozoospermic patients and 215 and 103 ICSI cycles in azoospermic patients. Before and after the law, in severe oligozoospermic patients, mean maternal age was 34.6 ± 4.2 years and 35.4 ± 4.2 years, respectively, 33.9 ± 4.3 years and 35.3 ± 4.6 years in cryptozoospermic patients and 34.2 ± 4.2 and 34.3 ± 3.6 years in azoospermic patients. Each study group was divided into two subgroups, according to maternal age (<38 and 38 years of age).
Ovarian stimulation, oocyte retrieval and sperm analysis Ovarian down-regulation was achieved by either a long luteal leuprolide acetate protocol (Enantone 3.75 mg; Takeda, Milano, Italy) or by a leuprolide acetate microdose flare protocol (Decapeptyl 0.1 mg; Ipsen Pharma, Barcelona, Spain). Complete ovarian suppression is usually achieved within 14–20 days (serum oestradiol concentrations <50 pg/ml and no follicles [10 mm). Thereafter, recombinant FSH (rFSH) (75 IU FSH, Gonal F; Serono, Rome, Italy) was started, followed by 75–450 IU/day (1–6 ampoules/day) for the next 5–10 days. Ovarian response was monitored daily and the dose of rFSH was individually adjusted according to the ovarian response estimated through serum oestradiol and follicular growth. When one or more follicles [17 mm in diameter and serum oestradiol concentrations of 200 pg/ml per follicle [15 mm in diameter were achieved, 10,000 IU human chorionic gonadotrophin (HCG) (Gonasi; AMSA, Milano, Italy) or recombinant HCG 250 lg (Ovitrelle; Serono) were intramuscularly administered. Oocyte retrieval by ultrasound-guided transvaginal aspiration was performed 34–36 h after HCG administration. All patients scheduled for embryo transfer received intramuscular or transvaginal progesterone supplementation (100 mg/day for 15 days; Prontogest; AMSA; or 600 mg/day for 15 days; Prometrium, Rottapharm, Milano, Italy). Three embryos at the most were transferred 48 h after oocyte retrieval, and the remaining oocytes were cryopreserved (La Sala et al., 2006a,b,c).
GB La Sala et al. of metaphase II oocytes. ICSI was performed according to the method reported by Palermo et al. (1992). For azoospermic patients, sperm surgical extraction by testicular sperm aspiration (TESA) was performed as described elsewhere (La Sala et al., 2006c). All injected oocytes were cultured in 40 ll microdrops under mineral oil (6% CO2, 37C). Oocyte fertilization was assessed 18 20 h after ICSI through confirmation of the presence and location of 2 pronuclei using an inverted microscope with Hoffman modulation contrast at 400· magnification (TE 2000 U; Nikon Corp, Tokyo, Japan). All patients undergoing embryo transfer received supplemental progesterone intramuscularly (100 mg/day for 15 days; Prontogest; AMSA) or vaginally (300 mg/day for 15 days; Prometrium, Rottapharm).
Outcome measures Pregnancy was initially determined 12 days after embryo transfer by a positive quantitative serum bHCG assay [10 IU, followed by another quantitative bHCG after 2– 4 days to ensure appropriately rising levels. A clinical pregnancy was defined as at least one fetus with a positive heart beat revealed by transvaginal sonography at 4–5 weeks after embryo transfer.
Analysis For all ICSI cycles before and after the law, maternal age, sperm concentration, sperm motility percentage and sperm morphology were evaluated. Moreover, the total number of recovered and injected oocytes, cleavage rate and total number of embryos obtained were evaluated. Also, the implantation rate, clinical pregnancy rate, abortion rate, total number of deliveries, singletons, twins and deliveries with three or more than three babies and perinatal outcomes were evaluated. All data were entered in Excel and odds ratios (OR) and Corenfield’s 95% confidence intervals (CI) were derived. Continuous variables were compared with Student’s t-test; P \ 0.05 was considered statistically significant.
Semen preparation, oocyte insemination, and embryo culture
Results
Semen samples were collected after 3–5 days of abstinence and their preparation for ICSI was performed following the WHO standard protocol (World Health Organization, 1999). Briefly, an appropriate volume of fresh semen was diluted with 10 ml of buffer medium (Cook IVF, Australia); after centrifugation (10 min at 800g, at room temperature) the supernatant was removed and replaced with another 5 ml of buffer medium. After a second centrifugation, the supernatant was once again removed and the pellet was overlaid with 1 ml of medium and incubated (37C, 6% CO2 in air) to allow separation by swim up. In cases of a sperm concentration <1 · 106/ml, the sample was concentrated by one centrifugation (1500g) and the pellet was suspended in 1 ml of medium. For ICSI, following removal of the cumulus and corona cells surrounding the oocyte, nuclear maturation was assessed by inverted microscopy to ensure exclusive injection
From 10 March 2000 to 10 March 2008, biological and clinical outcomes obtained in 1066 ICSI cycles performed for 778 couples during the 4 years that preceded the passing of the Italian law were compared with 804 ICSI cycles performed for 587 couples during the 4 years that followed the introduction of the same law. The biological outcomes are shown in Table 1. Comparing the two cohorts of patients, after the law the following statistical significances were observed: (i) an increased maternal age (32.4 ± 4.2 versus 35.2 ± 4.3, P < 0:05); (ii) an increase in the rate of couples, cycles and embryo transfer where the woman was 38 years old (20.7% versus 30.2%, OR 0.604; 95% CI 0.472–0.774; 21.1% versus 31.5%, OR 0.583; 95% CI 0.473–0.718; 20.4% versus 31.0%, OR 0.570; 95% CI 0.454–0.715, respectively); (iii) a decrease in the number of retrieved oocytes (7.6 ± 3.8 versus 6.2 ± 3.7, P < 0:05); (iv) a decrease in the number of
The 2004 Italian law and male factor infertility
5
Table 1 Biological outcomes in intracytoplasmic sperm injection cycles with severe male factor performed before and after the introduction of the Italian assisted reproductive technologies law. Parameter
Pre-law
Post-law
Statistics
Mean maternal age in years ± SD No. of couples <38 years of age 38 years of age No. of cycles <38 years of age 38 years of age No. of cycles/couple Embryo transfer <38 years of age 38 years of age No. of oocytes retrieved No. of injected oocytes Cleavage No. of obtained and transferred embryos
34.2 ± 4.2 778 617/778 (79.3) 161/778 (20.7) 1066 841/1066 (78.9) 225/1066 (21.1) 1.4 1009/1066 (94.7) 803/1009 (79.6) 206/1009 (20.4) 8123 (7.6 ± 3.8) 5283 (5.1 ± 2.3) 3438/5283 (65.1) 3438 (3.4 ± 1.9)
35.2 ± 4.3 587 410/587 (69.8) 177/587 (30.2) 804 551/804 (68.5) 253/804 (31.5) 1.4 638/804 (79.4) 440/638 (69.0) 198/638 (31.0) 4930 (6.2 ± 3.7) 2028 (2.6 ± 0.8) 1247/2028 (61.5) 1247 (1.6 ± 1.0)
P < 0:05 – 1.654 (1.292–2.118) 0.604 (0.472–0.774) 1.716 (1.392–2.115) 0.583 (0.473–0.718) – 4.606 (3.359–6.316) 1.754 (1.398–2.201) 0.570 (0.454–0.715) P < 0:05 P < 0:05 1.167 (1.050–1.297) P < 0:05
Values are number/total (percentage) or number (mean ± SD) unless otherwise stated; statistics show P-values, odds ratios and 95% confidence intervals.
injected oocytes (5.1 ± 2.3 versus 2.6 ± 0.8, P < 0:05) and a consequent decrease in the number of obtained and transferred embryos (3.4 ± 1.9 versus 1.6 ± 1.0, P < 0:05); (v) a decreased cleavage rate (65.1% versus 61.5%, OR 1.167; 95% CI 1.050–1.297); and (vi) a decreased rate of embryo transfer per cycle (94.7% versus 79.4%, OR 4.606; 95% CI 3.359–6.316). The mean number of cycles per couple was 1.4 before and after the introduction of the Italian law. Global analysis of clinical and obstetric outcomes (Table 2) showed: (i) in the post-law period, a significant decrease in terms of pregnancy and delivery rates per cycle (17.8% versus 10.9%, OR 1.765; 95% CI 1.346–2.313 and
14.2% versus 8.5%, OR 1.786; 95% CI 1.321–2.414, respectively) and per embryo transfer (18.8% versus 13.8%, OR 1.450; 95% CI 1.102–1.908 and 15.0% versus 10.8%, OR 1.475; 95% CI 1.088–2.000, respectively); (ii) similar implantation and abortion rates between the two periods; and (iii) a significant drop of multiple deliveries after the introduction of the law (35.1% versus 17.6%, OR 2.524; 95% CI 1.254–5.068). Tables 3 and 4 show the clinical and biological outcomes for severe oligozoospermic, cryptozoospermic and azoospermic couples. In the severe oligozoospermic group, in the post-law period there was a non-statistically significant
Table 2 Clinical and obstetrical outcomes in intracytoplasmic sperm injection cycles with severe male factor before and after the introduction of the Italian assisted reproductive technologies law. Parameter
Pre-law
Post-law
Odds ratio (95% CI)
Clinical pregnancies per cycle Clinical pregnancies per transfer Implantation rate Abortion rate Deliveries per cycle Deliveries per transfer Singletons Twins Triplets or greater
190/1066 (17.8) 190/1009 (18.8) 294/3438 (8.6) 36/190 (18.9) 151/1066 (14.2) 151/1009 (15.0) 98/151 (64.9) 42/151 (27.8) 11/151 (7.3)
88/804 (10.9) 88/638 (13.8) 109/1247 (8.7) 20/88 (22.7) 68/804 (8.5) 68/638 (10.7) 56/68 (82.4) 11/68 (16.2) 1/68 (1.5)
1.765 1.450 0.976 0.795 1.786 1.475 0.264 1.997 5.264
Gestational age (weeks) <32 37
7/151 (4.6) 69/151 (45.7)
3/68 (4.4) 20/68 (29.4)
1.053 (0.286–3.852) 2.020 (1.099–3.706)
Birthweight (g) <1500 <2500
29/215 (13.5) 94/215 (43.7)
11/81 (13.6) 20/81 (24.7)
0.992 (0.476–2.066) 2.369 (1.342–4.180)
Values are number/total (percentage) unless otherwise stated.
(1.346–2.313) (1.102–1.908) (0.776–1.228) (0.431–1.464) (1.321–2.414) (1.088–2.000) (0.119–0.587) (0.965–4.123) (0.850–32.205)
6
Table 3 Oocyte retrieval, embryo transfer and biological outcome in severe oligozoospermic, cryptozoospermic and azoospermic patients before and after the introduction of the Italian assisted reproductive technologies law. Severe oligozoospermia
Mean maternal age in years ± SD No. of couples <38 years of age 38 years of age No. of cycles <38 years of age 38 years of age No. of cycles/couple Embryo transfer <38 years of age 38 years of age No. of oocytes retrieved No. of injected oocytes Cleavage
No. of embryos obtained and transferred
Cryptozoospermia
Azoospermia
Pre-law
Post-law
Statistics
Pre-law
Post-law
Statistics
Pre-law
Post-law
Statistics
34.6 ± 4.2
35.4 ± 4.2
P \ 0.05
33.9 ± 4.3
35.3 ± 4.6
P \ 0.05
34.2 ± 4.2
34.3 ± 3.6
NS
334 261/334 (78.1) 73/334 (21.9) 414 322/414 (77.8) 92/414 (22.2) 414/334 (1.2) 392/414 (94.7) 310/322 (96.3) 82/92 (89.1) 3042 (7.3 ± 3.6) 1826 (4.6 ± 2.2) 1305/ 1826 (71.5) 1305 (3.3 ± 1.8)
252 – 168/252 1.788 (1.237– (66.7) 2.583) 84/252 0.559 (0.387– (33.3) 0.808) 304 – 202/304 1.767 (1.269– (66.4) 2.469) 102/304 0.566 (0.406– (33.6) 0.788) 304/252 NS (1.2) 264/304 2.700 (1.576– 4.623) (86.8) 177/202 3.649 (1.810– (87.6) 7.349) 87/102 1.414 (0.611– (85.3) 3.265) 1875 P \ 0.05 (6.2 ± 3.7) 786 P \ 0.05 (2.6 ± 0.7) 542/786 1.128 (0.940– (69.0) 1.353)
317 253/317 (79.8) 64/317 (20.2) 437 352/437 (80.5) 85/437 (19.5) 437/317 (1.4) 406/437 (92.9) 329/352 (93.5) 77/85 (90.6) 3248 (7.4 ± 3.8) 1996 (4.6 ± 2.2) 1340/ 1996 (67.1) 1340 (3.3 ± 1.8)
278 194/278 (69.8) 84/278 (30.2) 397 264/397 (66.5) 133/397 (23.5) 397/278 (1.4) 300/397 (75.6) 200/264 (75.8) 100/133 (75.2) 2428 (6.1 ± 3.5) 996 (2.6 ± 0.7) 581/996 (58.3)
– 1.712 (1.177– 2.488) 0.554 (0.381– 0.807) – 2.086 (1.523– 2.858) 0.479 (0.350– 0.657) NS
127 57 103/127 48/57 (81.1) (84.2) 24/127 9/57 (18.9) (15.8) 215 103 167/215 85/103 (77.7) (82.5) 48/215 18/103 (22.3) (17.5) 215/127 103/57 (1.7) (1.8) 211/215 74/103 (98.1) (71.8) 164/167 63/85 (98.2) (74.1) 47/48 11/18 (97.9) (61.1) 1833 627 (8.5 ± 4.0) (6.3 ± 4.2) 1461 246 (6.8 ± 3.5) (2.8 ± 0.8) 793/1461 124/246 (54.3) (50.4)
– 0.805 (0.354– 1.837) 1.243 (0.544– 2.827) – 0.737 (0.406– 1.338) 1.357 (0.748– 2.462) NS
581 (1.5 ± 1.0)
P \ 0.05
793 (3.8 ± 2.3)
P \ 0.05
542 (1.9 ± 1.0)
P \ 0.05
4.235 (2.758– 6.500) 4.577 (2.765– 7.575) 3.176 (1.410– 7.133) P \ 0.05 P \ 0.05 1.459 (1.247– 1.707)
124 (1.3 ± 0.9)
20.672 (7.317– 58.135) 19.090 (5.856– 61.799) 0.965 (0.347– 2.693) NS P \ 0.05 1.168 (0.892– 1.530)
GB La Sala et al.
Values are number/total (percentage) or number (mean ± SD) unless otherwise stated; statistics show P-values, odds ratios and 95% confidence intervals; NS = not statistically significant.
Parameter
Clinical pregnancies per cycle <38 years of age 38 years of age Clinical pregnancies per embryo transfer <38 years of age 38 years of age Implantation rate Abortion rate Deliveries per cycle <38 years of age 38 years of age Deliveries per embryo transfer <38 years of age 38 years of age
Severe oligozoospermia
Cryptozoospermia
Azoospermia
Pre-law
Post-law
Odds ratio (95% CI)
Pre-law
Post-law
Odds ratio (95% CI)
Pre-law
Post-law
Odds ratio (95% CI)
79/414 (19.1) 69/322 (21.4) 10/92 (10.9) 79 (20.2) 69/310 (22.3) 10/82 (12.2) 122/1305 (9.3) 16/79 (20.3) 60/414 (14.5) 52/322 (16.1) 8/92 (8.7) 60/392 (15.3) 52/310 (16.8) 8/82 (9.8)
43/304 (14.1) 33/202 (16.3) 10/102 (9.8) 43/264 (16.3) 33/177 (18.6) 10/87 (11.5) 58/542 (10.7) 10/43 (23.3) 33/304 (10.9) 27/202 (13.4) 6/102 (5.9) 33/264 (12.5) 27/177 (15.3) 6/87 (6.9)
1.431 (0.956–2.143) 1.397 (0.885–2.203) 1.122 (0.455–2.767) 1.297 (0863–1.950) 1.249 (0.788–1.981) 1.069 (0.430–2.660) 0.861 (0.619–1.196) 0.838 (0.347–2.018) 1.392 (0.887–2.184) 1.320 (0.802–2.170) 1.524 (0.529–4.382) 1.265 (0.803–1.992) 1.014 (0.614–1.674) 1.459 (0.503–4.224)
78/437 (17.8) 67/352 (19.0) 11/85 (12.9) 78/406 (19.2) 67/329 (20.4) 11/77 (14.3) 121/1340 (9.0) 15/78 (19.2) 63/437 (14.4) 55/352 (15.6) 8/85 (9.4) 63/406 (15.5) 55/329 (16.7) 8/77 (10.4)
38/397 (9.6) 33/264 (12.5) 5/133 (3.8) 38/300 (12.7) 33/200 (16.5) 5/100 (5.0) 42/581 (7.2) 7/38 (18.4) 31/397 (7.8) 26/264 (9.8) 5/133 (3.8) 31/300 (10.3) 26/200 (13.0) 5/100 (5.0)
2.622 (1.730– 1.646 (1.050– 3.805 (1.324– 1.640 (1.079– 1.294 (0.819– 3.167 (1.093– 1.621 (1.125– 1.054 (0.398– 1.989 (1.267– 1.695 (1.035– 2.660 (0.881– 1.594 (1.010– 1.343 (0.815– 2.203 (0.724–
33/215 (15.3) 32/167 (19.2) 1/48 (2.1) 33/211 (15.6) 32/164 (19.5) 1/47 (2.1) 51/793 (6.4) 5/33 (15.2) 28/215 (13.0) 28/167 (16.8) 0/48 (0.0) 28/211 (13.3) 28/164 (17.0) 0/47 (0.0)
7/103 (6.8) 6/85 (7.1) 1/18 (5.6) 7/74 (9.5) 6/63 (9.5)
2.487 (1.081– 5.705) 3.121 (1.279– 7.587) 0.362 (0.036– 3.653) 1.774 (0.763– 4.112) 2.303 (0.934– 5.655) 0.217 (0.021– 2.255) 0.878 (0.427– 1.806) 0.238 (0.044– 1.250) 3.706 (1.317–10.390) 4.079 (1.438–11.515) –
3.975) 2.578) 10.894) 2.491) 2.044) 9.135) 2.335) 2.777) 3.121) 2.775) 8.012) 2.514) 2.215) 6.684)
1/11 (9.1) 9/124 (7.3) 3/7 (42.9) 4/103 (3.9) 4/85 (4.7) 0/18 (0.0) 4/74 (5.4) 4/63 (6.3) 0/11 (0.0)
The 2004 Italian law and male factor infertility
Table 4 Clinical results in severe oligozoospermic, cryptozoospermic and azoospermic patients before and after the introduction of the Italian assisted reproductive technologies law.
2.678 (0.944–7.561) 3.037 (1.061–8.640) –
Values are number/total (percentage) unless otherwise stated.
7
8
GB La Sala et al.
decrease in the number of deliveries per cycle (14.5% versus 10.9%, OR 1.392; 95% CI 0.887–2.184), both for \38-yearold women (16.2 versus 13.4%, OR 1.320; 95% CI 0.802– 2.170) and 38-year-old women (8.7% versus 5.9%, OR 1.524; 95% CI 0.529–4.382). Similarly, in the cryptozoospermic group there was a statistically significant decrease in the number of deliveries per cycle after the passing of the Italian law (14.4 versus 7.8%, OR 1.989; 95% CI 1.267–3.121), both for \38-year-old women (15.6% versus 9.8%, OR 1.695; 95% CI 1.035–2.775) and 38-year-old women (9.4% versus 3.8%, OR 4.433; 95% CI 0.881–8.012). In the azoospermic population, there was a severe decrease in results in the post-law period in terms of deliveries per cycle (13.0% versus 3.9%, OR 3.706; 95% CI 1.317–10.390). There was also a decrease in deliveries per embryo transfer (13.3% versus 5.4%, OR 2.678; 95% CI 0.944–7.561), but this second difference was not statistically significant. Moreover, a heavy negative impact of the Italian law on NOA couples was observed, with statistically significant decreases in terms of deliveries per cycle (15.5% versus 2.7%, OR 6.713; 95% CI 1.655–26.958) and per embryo transfer (16.0% versus 3.8%, OR 4.726; 95% CI 1.162–19.265) in the post-law period (Table 5). Conversely, in OA couples, despite observing a drop in clinical results after the introduction of the assisted reproductive technologies Italian law, the statistical analysis showed no difference between the two periods.
Discussion The Italian law, issued by the legislative authorities, was anticipated to go against the best interests of infertile women (Ragni et al., 2005; Robertson, 2004). Specifically, to defend the rights of human embryos, the restriction to three fertilized oocytes for transfer per cycle, the obligation to transfer all available embryos at the same time, and the prohibition on freezing of surplus embryos, were imposed on assisted reproduction clinicians. Moreover, the law prohibits preimplantation diagnosis, creating the risk of transferring genetically malformed embryos in patients with known genetic disorders. For the above reasons, the Italian law was expected to decrease the likelihood of achieving pregnancy, to increase the number and complexity of procedures per pregnancy and to increase the risk of multiple gestations, infringing upon basic human rights and the proper application of assisted reproductive technologies (Robertson, 2004). Thus, the debate on the Italian law and its effects on assisted reproduction outcomes is still open. The aim of this study was to verify the impact of the 40/2004 law on couples affected by severe male infertility factor (severe oligozoospermia, cryptozoospermia and azoospermia). Overall, this population of patients submitted to IVF, ICSI and ICSI/TESA, 4 years after the introduction of the law, has shown an increasing trend in maternal age compared with the 4 years preceding the law (unpublished data). This trend was confirmed by the present study, which also found a
Table 5 Outcomes in obstructive azoospermic and non-obstructive azoospermic patients before and after the introduction of the Italian assisted reproductive technologies law. Obstructive azoospermic
Mean maternal age in years ± SD No. of couples Cycles/couple Embryo transfer/cycle No. of oocytes retrieved No. of embryos obtained and transferred Clinical pregnancies per cycle Clinical pregnancies per embryo transfer Implantation rate Deliveries per cycle Deliveries per embryo transfer
Non-obstructive azoospermic
Pre-law
Post-law
Statistics
Pre-law
Post-law
Statistics
34.3 ± 4.4
34.2 ± 4.3
NS
34.1 ± 3.9
34.3 ± 3.3
NS
63 1.8 (112/63) 111/112 (99.1) 917 (8.9 ± 4.1) 721 (7.1 ± 3.6)
19 1.5 (28/19) 22/28 (78.6) 160 (6.2 ± 2.6) 32 (1.2 ± 0.8)
– –
64 103/64 (1.6) 100/103 (97.1) 916 (8.2 ± 4.0) 740 (6.6 ± 3.4)
48 75/48 (1.6) 52/75 (69.3) 467 (6.4 ± 4.6) 92 (1.4 ± 1.0)
–
15/112 (13.4) 15/111 (13.5) 26/721 (6.2) 12/112 (10.7) 12/111 (10.8)
2/28 (7.1) 2/22 (9.1) 2/32 (6.3)
2.010 (0.477–8.318) 1.563 (0.365–6.545) 1.92 (0.397–6.314) 1.560 (0.364–6.563) 1.212 (0.278–5.163)
18/103 (17.5) 18/100 (18) 25/740 (6.7) 16/103 (15.5) 16/100 (16.0)
5/75 (6.7)
2.965 (1.082–8.079) 2.063 (0.742–5.697) 0.425 (0.182–0.988) 6.713 (1.655–26.958) 4.762 (1.162–19.265)
2/28 (7.1) 2/22 (9.1)
30.273 (4.467– 198.873) P \ 0.05 P \ 0.05
5/52 (9.6) 7/92 (7.6) 2/75 (2.7) 2/52 (3.8)
14.744 (4.479– 48.098) P \ 0.05 P \ 0.05
Values are number/total (percentage) or number (mean ± SD) unless otherwise stated; statistics show P-values, odds ratios and 95% confidence intervals; NS = not statistically significant.
The 2004 Italian law and male factor infertility decrease in the number of retrieved and injected oocytes, a decrease in the cleavage rate, in the number of transferred embryos and in embryo transfer per cycle. The reason for this decrease in the number of oocytes retrieved at retrieval cannot be attributed to variations in the ovarian stimulation protocol, as this protocol has remained unchanged between the pre- and post-law periods. It can be postulated that this phenomenon is correlated to the increase in maternal age and/or to random events in the examined population, occurring independently from the assisted reproduction treatments. In this population, already at risk because of the severe male infertility factor, the limitations brought about by the law have, of course, caused a decrease in the cleavage rate and in the number of obtained and transferred embryos. In fact, infertile men, especially those with severe male infertility factor, possess substantially more sperm DNA damage and chromosome aneuploidies than do fertile men or infertile men without severe male infertility factor (Rubio et al., 2001) and these sperm genomic characteristics correlate with a poor prognosis in terms of fertilization and cleavage rates (Lopes et al., 1998). In general, in the post-law period, this study observed a significant decrease in the number of clinical pregnancies per cycle (17.8% versus 10.9%) and per embryo transfer (18.8% versus 13.8%), in the number of deliveries per cycle (14.2% versus 8.5%) and per embryo transfer (15.0% versus 10.7%) and a significant decrease in the number of multiple deliveries (35.1% versus 17.6%). Moreover, this study observed a small increase in the abortion rate, even though not statistically significant (18.9% versus 22.7%). This could be correlated with the poor semen characteristics of these populations, as reported in previous studies (Benchaib et al., 2003; Rubio et al., 2001; Zini et al., 2008). Therefore, the results of the single groups were separately analysed to verify which of the three studied categories was more affected by the limitations brought about by the law. In the severe oligozoospermic group, in the postlaw period, there was a decrease in the number of deliveries per cycle both in the \38-year-old women (16.1% versus 13.4%) and in the 38-year-old women (8.7% versus 5.9%), although the differences were not statistically significant. In the cryptozoospermic group, there was a significant decrease in the number of deliveries per cycle (14.4% versus 7.8%), especially in \38-year-old women (15.6% versus 9.8%). In the azoospermic group, finally, there was a significant decrease in the number of deliveries per cycle (13.0% versus 3.9%), especially in couples affected by NOA (15.5% versus 2.7%). On the contrary, in couples affected by OA, this difference was not statistically significant: nevertheless, the OA sample size was too small to show reliable results. Regarding the obstetric–perinatal outcomes, in the postlaw period the study observed a global significant reduction in the number of deliveries \37 gestational weeks (45.7% versus 29.4%) and of babies born with a birthweight \2500 g (43.7% versus 24.7%). In general, if, on one hand, the 40/2004 law by limiting to three the number of inseminated oocytes per cycle has reduced the rate of multiple deliveries (one of the declared aims of the law), on the other hand it has greatly decreased the efficacy of assisted reproductive technologies, espe-
9 cially in couples affected by cryptozoospermia and azoospermia.
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