http://informahealthcare.com/gye ISSN: 0951-3590 (print), 1473-0766 (electronic) Gynecol Endocrinol, 2013; 29(11): 997–1001 ! 2013 Informa UK Ltd. DOI: 10.3109/09513590.2013.822063
REPRODUCTIVE AGING AND IVF
Conventional IVF as a laboratory strategy to rescue fertility potential in severe poor responder patients: the impact of reproductive aging Paolo Giovanni Artini1, Maria Elena Rosa Obino1, Elena Carletti1, Sara Pinelli1, Maria Ruggiero1, Giovanna Di Emidio2, Vito Cela1, and Carla Tatone2 Division of Obstetrics and Gynaecology, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy and 2Department of Biomedical Sciences and Technologies, University of L’Aquila, L’Aquila, Italy Abstract
Keywords
Objective: To investigate whether laboratory strategies can improve in vitro fertilization (IVF) outcome in poor responder patients. We compared the effectiveness of conventional IVF and intra cytoplasmic sperm injection (ICSI) in assisted reproductive technologies cycles in which only one or two oocytes were retrieved at ovarian pick up, in the absence of male infertility. Design: Retrospective analysis of 425 cycles in 386 poor responder patients. Intervention(s): Standard stimulation protocol with gonadotropins and gonadotropin releasing hormone (GnRH) antagonist. Main outcome measure(s): Fertilization rate, cleavage rate, good-quality embryo rate, implantation rate, clinical pregnancy rate (PR) and miscarriage rate. Results: IVF was found to be more advantageous for implantation and PR, especially in patients under 35 years and in women aged between 35 and 38 years. No differences were noted in the other parameter evaluated. Patients aged over 38 years showed no difference using the two techniques. Conclusion: The employment of ICSI in the absence of a male factor can reduce reproductive outcome in poor responder. Probably because of aging-related defects overcoming the advantage of sperm selection, the choice of IVF technique is not relevant to reproductive success when oocyte quality is compromised by reproductive aging. Although further randomized trials are needed to confirm our results, we propose that, in absence of male infertility, conventional IVF might be the technique of choice in young patients, especially in those aged below 35 years.
Assisted reproduction, ICSI, IVF, poor responders
Introduction One of the goals of assisted reproduction technologies (ART) is the recruitment of multiple follicles ensuring the recovery of good quality oocytes upon controlled ovarian hyperstimulation (COH). The ovarian response to COH varies substantially among women and is strictly dependent on the size of the ovarian pool of resting follicles, the so-called ovarian reserve [1]. In patients with a low ovarian reserve, a reduced follicular response resulting in a low number of retrieved oocytes is achieved even when a high dose of gonadotropins is administered. Hence, although tests for predicting ovarian reserve are available [2], the parameter that best categorizes a patient as a ‘‘poor responder’’ remains the ovarian response itself. The incidence of poor ovarian response is estimated between 9 and 24% [3–5], values that increase with age [3,5] reaching about 50% in women older than 40 years [6]. Women who respond poorly to COH have poor pregnancy prospects with a pregnancy rate (PR) varying from 7.6 to 17.5% compared with normal responders varying from 25.9 to 36.7%. Among the factors of relevance for prognosis within the poor
Address for correspondence: Paolo Giovanni Artini, Department of Clinical and Experimental Medicine, Division of Obstetrics and Gynecology, University of Pisa, Pisa, Italy. Tel: +39.335.349967. E-mail: [email protected]
History Received 14 May 2013 Revised 1 July 2013 Accepted 1 July 2013 Published online 2 August 2013
responder group, female age appeared to play a distinct role. Overall, older poor responders have lower PRs (ranging between 1.5 and 12.7%) compared with younger poor responders (ranging between 13.0 and 35%) [2]. A second factor predicting pregnancy outcome in poor responders is the degree of poor response. The lower number of retrieved oocytes results in fewer embryos to transfer and a lower prospect for pregnancy, in addition to the expected negative effect of poor ovarian function on oocyte quality. In recent years, there have been an increasing number of in vitro fertilization (IVF) patients in whom few oocytes are obtained in response to COH, a phenomenon mainly related to the postponement of childbearing to the fourth decade of life. In this group of patients where multifollicular response to COH remains a challenge, the optimization of laboratory strategies may help to maximize the chances of pregnancy. Which technique of fertilization should be used in poor responder patients in absence of male factor infertility is still object of controversy. Intra cytoplasmic sperm injection (ICSI) is usually preferred when a male factor associated infertility exists, but sometimes this technique is chosen even in the case of non-male factor indication in order to avoid fertilization failure [7,8]. However, some recent studies suggest that the use of ICSI is not strictly necessary, and its use in the absence of indication is questionable [9–11]. Following the study by Moreno et al. [9] who first proposed that
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the technique of fertilization is not relevant to the reproductive outcome in poor responders, other authors have reported no differences between conventional IVF and ICSI in terms of fertilization and good quality embryo rates even in patients with one [10] or few oocytes inseminated [11]. The aim of this study is to clarify whether natural selection of the fertilizing sperm resulting from conventional IVF may improve reproductive success in poor responder patients with favorable semen quality. To this end, we compared reproductive outcomes following conventional IVF or ICSI in patients in whom only one or two oocytes were retrieved at ovarian pick up by taking into account the impact of reproductive aging.
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Materials and methods We retrospectively analyzed a total of 425 cycles (386 patients) attending ART at the Centre of Infertility and Assisted Reproduction of the Department of Clinical and Experimental Medicine of Pisa University between January 2007 and July 2012. In accordance with European Society of Human Reproduction and Embryology (ESHRE) consensus, all patients were classified as poor responder [6] and were included in this study when only one or two oocytes were retrieved during ovarian pick-up and male factor infertility was absent. Both male and female partners signed an informed consent form. The age of patients included in the study ranged from 27 and 47 years (mean age: 38.23 years 3.82 SD). Cycles were divided into two groups based on the technique used. Thirty-nine patients underwent both the procedures in different cycles. IVF and ICSI groups were furthermore divided into three subgroups based on the age of patients (535 years, 35–38 years and438 years), whose results were also compared. COH was carried out with 150–450 UI/d of recombinant follicle stimulating hormone (FSH) (Gonal FÕ, Serono, Rome, Italy), according to basal FSH and anti-mullerian hormone (AMH) levels and age. All patients were administered cetrorelix (CetrotideÕ , Serono), a gonadotropin releasing hormone (GnRH) antagonist, according to a personalized regimen, i.e. when the lead follicle reached 14 mm in diameter, to prevent premature ovulation. Recombinant human chorionic gonadotropin (OvitrelleÕ , Serono) was administered when at least 1–2 follicles reached a mean diameter of 18 mm. After approximately 36 h, transvaginal follicular aspiration was performed for oocyte retrieval. Volume, sperm count, forward motility and morphology were considered according to the World Health Organization criteria [12]. Sperm not meeting the defined threshold was classified as subfertile sperm, possessing questionable fertilization potential, and patients were consequently excluded from the study. Oocytes were retrieved by vaginal ultrasound (US) probe and incubated in oocyte culture medium (Sydney IVF Oocyte Wash Buffer; Cook Ireland Ltd., Limerick, Ireland). Fertilization technique was chosen based on the clinical history of patients and reproductive outcomes in previous ART cycles. In cases where we choose to perform ICSI, we hypothesized that the impairment in fertilization was the cause of the past failures in previous ART cycles. Four to five hours after oocyte retrieval, in cases where IVF was performed, each oocyte was inseminated with 200 000–300 000 motile washed spermatozoa, while ICSI was accomplished as previously described by Artini et al., 1998 [13]. Fertilization was confirmed by the observation of two pronuclei about 16–18 h after fertilization technique. All the fertilized oocytes were transferred into a fresh cleavage medium (Sydney IVF Cleavage Medium; Cook Ireland Ltd.) and cultured until transfer. On day 2, at 46–48 h post-insemination, the embryos were evaluated for cell number and rate of fragmentation and consequently graded as I–IV (best to worst) [14] by a single
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expert embryologist. Embryo transfer (ET) was performed after 48–72 h under the guidance of abdominal US, using a K-Soft 500 Embryo Transfer Catheter (Cook Ireland Ltd.). All patients had luteal phase support with daily vaginal progesterone (Crinone gelÕ 8%, Merck-Serono) and intramuscular progesterone every 72 h (LentogestÕ , AMSA S.r.l., Rome, Italia), starting on the day of ET until both serum pregnancy test result was negative and gestational sac was confirmed on US. For patients with a positive serum pregnancy test, progesterone was continued until the 12th week of pregnancy. Statistical analysis Continuous data were summarized as the mean SD and were analyze by using t-Student test. To compare proportion, we used the Fisher’s exact test.
Results Patients did not differ in both groups in mean age, FSH and AMH values, doses of gonadotropins administered and average number of oocytes retrieved. Fertilization rate, cleavage rate and good quality embryo rate did not differ between IVF and ICSI group when these were not divided by age, while for what concerns implantation rate (13.05 versus 5.26%) and PRs (16.12 versus 6.73%), IVF was found to be more advantageous with a level of significance of p ¼ 0.003 and p ¼ 0.003, respectively. In patients under 35 years, we did not observe any differences in fertilization rate, cleavage rate and good quality embryo rate between IVF and ICSI group, while for what concerns implantation rate (25.92 versus 3.70%; p ¼ 0. 002) and PRs (32.55 versus 4.76%; p ¼ 0.001), we found that IVF was more advantageous. Although miscarriage rate was higher in IVF group, this difference is not significant (Table 1). As noted in younger patients, even in patients aged between 35 and 38 years, we did not observe any significant differences in fertilization rate, cleavage rate and good quality embryo rate, while implantation rate and PRs were 20% versus 6.34% (p ¼ 0.025) and 26.31% versus 7.01% (p ¼ 0.010), respectively. Even in this subgroup, despite a greater percentage of miscarriage in IVF group, this difference was not significant (Table 2). In patients over 38 years, there were no significant differences in fertilization rate, cleavage rate and good quality embryo rate; but in ICSI group the percentage of cycle cancelled, due to fertilization failure or cleavage failure, was significantly higher compared to IVF group (21.1% versus 10.2%; p ¼ 0.27). In this Table 1. Outcomes IVF vs ICSI in patients aged younger than 35 years. IVF-ET535 years No. of patients 40 Age (years) 32.3 1.68 No. of cycles 43 No. of oocytes retrieved 1.67 0.47 No. of oocytes inseminated 1.62 0.48 No. of oocytes fertilized 1.41 0.49 Non transferred cycles, 0% (0/43) % (n) Fertilization rate % (n) 87.1% (61/70) Cleavage rate % (n) 88.5% (54/61) No. of embryos transferred 1.25 0.44 (54) Good quality embryo 75.9% (41/54) rate % (n) Implantation rate % (n) 25.9% (14/54) Clinical pregnancy 32.55% (14/43) rate % (n) Miscarriage rate % (n) 6.97% (3/43)
*p50.05.
ICSI535 years
p Value
42 32.07 1.9 42 1.71 0.45 1.57 0.63 1.35 0.61 7.1% (3/42)
– 0.574 – 0.694 0.645 0.614 0.116
86.3% (57/66) 94.7% (54/57) 1.28 0.59 (54) 77.7% (42/54)
1 0.325 0.793 1
3.7% (2/54) 4.76% (2/42)
0.002* 0.001*
0% (0/42)
0.241
IVF versus ICSI in poor responder
DOI: 10.3109/09513590.2013.822063
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Table 2. Outcomes IVF versus ICSI in patients aged between 35 and 38 years.
No. of patients Age (years) No of cycles No of oocytes retrieved No of oocytes inseminated No of oocytes fertilized Non transferred cycles, % (n) Fertilization rate % (n) Cleavage rate % (n) No. of embryos transferred Good quality embryo rate % (n) Implantation rate % (n) Clinical pregnancy rate % (n) Miscarriage rate % (n)
IVF-ET: 35–38 years
ICSI: 35–38 yearss
p Value
54 36.83 1.17 57 1.68 0.46 1.68 0.46 1.36 0.58 5.2% (3/57)
51 36.66 1.27 57 1.61 0.49 1.57 0.49 1.21 0.52 7.0% (4/57)
– 0.487 – 0.436 0.247 0.132 1
81.2% (78/96) 96.1% (75/78) 1.31 0.57 76% (57/75)
76.6% (69/90) 91.3% (63/69) 1.10 0.48 66.6% (42/63)
0.475 0.306 0.036* 0.257
20% (15/75) 26.3% (15/57)
6.3% (4/63) 7.0% (4/57)
0.025* 0.010*
5.26% (3/57)
0% (0/57)
0.243
*p50.05. Table 3. Outcomes IVF versus ICSI in patients older than 38 years.
No. of patients Age (years) No of cycles No of oocytes retrieved No of oocytes inseminated No of oocytes fertilized Non transferred cycles, % (n) Fertilization rate % (n) Cleavage rate % (n) No. of embryos transferred Good quality embryo rate % (n) Implantation rate % (n) Clinical pregnancy rate % (n) Miscarriage rate % (n)
IVF-ET438 years
ICSI438 years
p Value
102 41.35 1.73 117 1.58 0.49 1.56 0.54 1.23 0.62 10.2% (12/117)
97 41.30 1.27 109 1.55 0.49 1.47 0.60 1.09 0.63 21.1% (23/109)
– 0.840 – 0.649 0.256 0.096 0.027*
78.7% (144/183) 96.5% (139/144) 1.188 0.60 73.4% (102/139)
73.9% (119/161) 92.4% (110/119) 1.009 0.65 67.3% (74/110)
0.297 0.172 0.033 0.293
4.3% (6/139) 5.1% (6/117)
7.3% (8/110) 7.3% (8/109)
0.408 0.585
2.6% (3/117)
2.7% (3/109)
1
*p50.05.
subgroup of patients, we did not observe any differences for what concerns implantation, pregnancy and miscarriage rate (Table 3).
Discussion All steps of IVF procedures require more attention when stimulation regimens fail in inducing optimal multifollicular response. Nevertheless, the question whether the choice of fertilization procedure may be relevant to reproductive success in poor responder patients is still debating. Probably, this is why a clear knowledge of both short- and long-term differences between IVF and ICSI fertilization is still lacking. Although ICSI was originally indicated for treating couples with severe male factor infertility [15], some studies indicated that it can provide comparable or higher fertilization rates [16–18] and superior embryo quality even in non-male factor couples [19]. Overall results from this study strongly question the superiority of ICSI over conventional IVF in patients with non-male factor infertility and lead us to hypothesize that under specific conditions, fertilization by ICSI can negatively affect reproductive potential. The analysis of a cohort of poor responders ranging from 27 to 47 years of age carried out in the first part of the study would lead us
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to conclude that the technique of fertilization does not influence reproductive outcome in low responders. This observation supports the general hypothesis that IVF and ICSI in cases of normal semen produce the same results [10,20,21] and is consistent with the finding by Moreno et al. [9] based on the analysis of 96 couples with six or fewer retrieved oocytes. On the other hand, these results are in contrast with Khamsi et al. (2001) [19] showing that ICSI procedure increased fertilization rate and good quality embryo rate even in the absence of male factor. Additionally, in this study, we show that when comparison between IVF and ICSI is conducted on cohorts of poor responder patients with different reproductive age, the analysis reveals that the use of ICSI decreases reproductive potential in women younger than 35 years or aged between 35 and 38 years. Although we found no significant differences in fertilization and cleavage rates in these groups of patients, IVF was significantly more advantageous than ICSI for what concerns implantation and PRs. Our results partially agree with those by Fang et al. [22] who, by retrospectively comparing 196 couples undergoing IVF/ICSI cycles with one or two retrieved oocytes with good-prognosis sperm, found that ICSI patients had higher fertilization rates although no difference in good-quality embryo rate or PR was noted. The discrepancy between this study and our findings in relation to fertilization outcome may be ascribed to differences in the size of the cohorts enrolled in the two studies. Indeed, our results are consistent with those by Xi et al. (2012) who retrospectively analyzed 406 cycles with three or fewer oocytes retrieved from women with similar age undergoing IVF (34.5 4.6 years) or ICSI (36.1 5.5 years) and noted that the PRs and implantation rate were lower in the ICSI group compared with the IVF group [23]. The authors explained the higher PRs and implantation rate after IVF with the lacking of ‘‘natural sperm selection’’ when most steps of the fertilization process are bypassed by sperm injection. In conventional IVF, upon laboratory selection of motile sperm, the sperm that fertilizes is further selected through the biological process of sperm–oocyte interaction beginning at zona pellucida level (ZP) or during sperm penetration through the cumulus matrix [24]. Indeed, the finding that majority of sperm (average 492%) bound to the ZP have normal nuclear chromatin DNA strongly suggest that scientist subjectively selected sperm during ICSI may have a lower quality compared to ZP-bound sperm [25]. Moreover, increased knowledge in the biology of fertilization process has revealed that sperm–oocyte interaction at membrane level involves numerous molecular actors with a possible role in sperm fusion and gamete selection [24]. Overall, these mechanisms may act against sperm carrying chromosomal anomaly from paternal origin as well as against chromosomally abnormal oocytes generating developmentally defective embryos leading to pregnancy failure or miscarriage [23,26]. In addition, decreased chances for successful fertilization and pregnancy could result from the possible mechanical damage to the oocyte after ICSI [27] as well as to the introduction of foreign material such as culture medium or exogenous DNA and infectious material [28]. Finally, our results in reproductively young women can be well explained by considering the relevance of early events of oocyte activation in promoting successful implantation. These include the sperminduced calcium signal that drives meiosis resumption and embryo development, as well as implantation and postimplantation events, through a specific spatiotemporal pattern of Ca2þ oscillation [29]. Beyond the delivery of sperm DNA, the main reason for ICSI to succeed is that it allows the delivery of phospholipase C-zeta (PLC z), the sperm component that is capable of generating the fertilization calcium signal upon sperm fusion [30]. Nevertheless, the finding that abnormal calcium signals were observed in ICSI oocytes [31,32] suggest that
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missing gamete interaction at surface level in ICSI fertilization would result in missing or abnormal signaling pathways with a role in later embryonic development. An additional factor with a negative influence on reproductive outcome is ICSI-related risk of parthenogenetic activation caused by oocyte manipulation [33– 35]. This may cause asynchrony between the timing of oocyte activation and sperm injection leading to cleavage abnormalities and early embryonic arrest [27]. Our finding that reproductive outcome in patients aged over 38 years undergoing IVF or ICSI were comparable for all parameters analyzed strongly indicates that the advantage of IVF over ICSI tends to disappear with the increasing of age. This result can be ascribed to the phenomenon of ovarian ageing responsible for the production of oocytes with a reduced developmental competence related to defective molecular storage, mitochondrial dysfunctions and poor control of chromosome segregation during meiosis [36,37]. These defects represent a reliable reason why aged oocytes do not benefit from fertilization mechanisms preserved in IVF and lost in ICSI. Nevertheless, a further reason for that could be found in the low activation competence of aged oocytes suggested by the observation of abnormal signaling upon exposure to parthenogenetic agents [38].
Conclusions A constant effort of reproductive medicine is to apply the simplest, most economical and least invasive method to ensure a positive outcome. In this context, we provide evidence that obtaining one or two oocytes in one cycle is not an indication for ICSI when the sperm sample is apparently normal. Indeed, a relevant factor to the choice of IVF technique under these conditions is represented by female age. In spite of the effects of a reduced ovarian reserve, oocytes from young poor responder patients, probably counting on biological resources definitively lost with aging, can still benefit of the advantage of IVF. We suggest that IVF could be used as a technique of choice in young poor responder patients in absence of male factor. However, since ours is a retrospective study, only further randomized trials will be able to confirm our results. Finally, we conclude that, in addition to optimization of stimulation regimens, further biological knowledge of IVF techniques will be helpful in tailoring the best IVF treatment to individual patients in order to give infertile couples the best chance of conceiving a healthy baby.
Declaration of interest The authors reports no declaration of interest.
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7. Oehninger S, Gosden RG. Should ICSI be the treatment of choice for all cases of in-vitro conception? No, not in light of the scientific data. Hum Reprod 2002;17:2237–42. 8. Van der Westerlaken L, Helmerhorst F, Dieben S, Naaktgeboren N. Intracytoplasmic sperm injection as a treatment for unexplained total fertilization failure or low fertilization after conventional in vitro fertilization. Fertil Steril 2005;83:612–7. 9. Moreno C, Ruiz A, Simo´n C, et al. Intracytoplasmic sperm injection as a routine indication in low responder patients. Hum Reprod 1998; 13:2126–9. 10. Gozlan I, Dor A, Farber B, et al. Comparing intracytoplasmic sperm injection and in vitro fertilization in patients with single oocyte retrieval. Fertil Steril 2007;87:515–8. 11. Borini A, Gambardella A, Bonu MA, et al. Comparison of IVF and ICSI when only few oocytes are available for insemination. Reprod Biomed Online 2009;19:270–5. 12. Cooper TG, Noonan E, von Eckardstein S, et al. World Health Organization reference values for human semen characteristics. Hum Reprod Update 2010;16:231–45. 13. Artini PG, Fasciani A, Monti M, et al. Changes in vascular endothelial growth factor levels and the risk of ovarian hyperstimulation syndrome in women enrolled in an in vitro fertilization program. Fertil Steril 1998;70:560–4. 14. Borini A, Sciajno R, Bianchi V, et al. Clinical outcome of oocyte cryopreservation after slow cooling with a protocol utilizing a high sucrose concentration. Hum Reprod 2006;21:512–7. 15. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17–8. 16. Payne D, Flaherty SP, Jeffrey R, et al. Successful treatment of severe male factor infertility in 100 consecutive cycles using intracytoplasmic sperm injection. Hum Reprod 1994;9:2051–7. 17. Caldero´n G, Belil I, Aran B, et al. Intracytoplasmic sperm injection versus conventional in-vitro fertilization: first results. Hum Reprod 1995;10:2835–9. 18. Aboulghar MA, Mansour RT, Serour GI, et al. Intracytoplasmic sperm injection and conventional in vitro fertilization for sibling oocytes in cases of unexplained infertility and borderline semen. J Assist Reprod Genet 1996;13:38–42. 19. Khamsi F, Yavas Y, Roberge S, et al. Intracytoplasmic sperm injection increased fertilization and good-quality embryo formation in patients with non-male factor indications for in vitro fertilization: a prospective randomized study. Fertil Steril 2001;75:342–7. 20. Staessen C, Camus M, Clasen K, et al. Conventional in-vitro fertilization versus intracytoplasmic sperm injection in sibling oocytes from couples with tubal infertility and normozoospermic semen. Hum Reprod 1999;14:2474–9. 21. Kim HH, Bundorf MK, Behr B, McCallum SW. Use and outcomes of intracytoplasmic sperm injection for non-male factor infertility. Fertil Steril 2007;88:622–8. 22. Fang C, Tang J, Huang R, et al. Comparison of IVF outcomes using conventional insemination and ICSI in ovarian cycles in which only one or two oocytes are obtained. J Gynecol Obstet Biol Reprod 2012;41:650–6. 23. Xi Q, Zhu L, Hu J, et al. Should few retrieved oocytes be as an indication for intracytoplasmic sperm injection? J Zhejiang Univ Sci B 2012;13:717–22. 24. Ikawa M, Inoue N, Benham AM, Okabe M. Fertilization: a sperm’s journey to and interaction with the oocyte. J Clin Invest 2010;120: 984–94. 25. Liu F, Qiu Y, Zou Y, et al. Use of zona pellucida-bound sperm for intracytoplasmic sperm injection produces higher embryo quality and implantation than conventional intracytoplasmic sperm injection. Fertil Steril 2011;95:815–8. 26. Kunathikom S, Makemaharn O, Suksompong S, Laokirkkiat P. Chromosomal analysis of ‘‘failed-fertilized’’ human oocytes resulting from in-vitro fertilization and intracytoplasmic sperm injection. J Med Assoc Thai 2001;84:532–8. 27. Miller JE, Smith TT. The effect of intracytoplasmic sperm injection and semen parameters on blastocyst development in vitro. Hum Reprod 2001;16:918–24. 28. Chan AW, Luetjens CM, Dominko T, et al. Foreign DNA transmission by ICSI: injection of spermatozoa bound with exogenous DNA results in embryonic GFP expression and live rhesus monkey births. Mol Hum Reprod 2000;6:26–33.
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29. Ozil J-P, Banrezes B, To´th S, et al. Ca2þ oscillatory pattern in fertilized mouse eggs affects gene expression and development to term. Dev Biol 2006;300:534–44. 30. Swann K, Lai FA. PLCz and the initiation of Ca(2þ) oscillations in fertilizing mammalian eggs. Cell Calcium 2013;53:55–62. 31. Tesarik J, Sousa M. Comparison of Ca2þ responses in human oocytes fertilized by subzonal insemination and by intracytoplasmic sperm injection. Fertil Steril 1994;62:1197–204. 32. Ciapa B, Arnoult C. Could modifications of signalling pathways activated after ICSI induce a potential risk of epigenetic defects? Int J Dev Biol 2011;55:143–52. 33. Fishel S, Timson J, Lisi F, Rinaldi L. Evaluation of 225 patients undergoing subzonal insemination for the procurement of fertilization in vitro. Fertil Steril 1992;57:840–9. 34. Palermo G, Joris H, Derde MP, et al. Sperm characteristics and outcome of human assisted fertilization by subzonal insemination
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REPRODUCTIVE AGING AND IVF
Conventional IVF as a laboratory strategy to rescue fertility potential in severe poor responder patients: the impact of reproductive aging Paolo Giovanni Artini1, Maria Elena Rosa Obino1, Elena Carletti1, Sara Pinelli1, Maria Ruggiero1, Giovanna Di Emidio2, Vito Cela1, and Carla Tatone2 Division of Obstetrics and Gynaecology, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy and 2Department of Biomedical Sciences and Technologies, University of L’Aquila, L’Aquila, Italy Abstract
Keywords
Objective: To investigate whether laboratory strategies can improve in vitro fertilization (IVF) outcome in poor responder patients. We compared the effectiveness of conventional IVF and intra cytoplasmic sperm injection (ICSI) in assisted reproductive technologies cycles in which only one or two oocytes were retrieved at ovarian pick up, in the absence of male infertility. Design: Retrospective analysis of 425 cycles in 386 poor responder patients. Intervention(s): Standard stimulation protocol with gonadotropins and gonadotropin releasing hormone (GnRH) antagonist. Main outcome measure(s): Fertilization rate, cleavage rate, good-quality embryo rate, implantation rate, clinical pregnancy rate (PR) and miscarriage rate. Results: IVF was found to be more advantageous for implantation and PR, especially in patients under 35 years and in women aged between 35 and 38 years. No differences were noted in the other parameter evaluated. Patients aged over 38 years showed no difference using the two techniques. Conclusion: The employment of ICSI in the absence of a male factor can reduce reproductive outcome in poor responder. Probably because of aging-related defects overcoming the advantage of sperm selection, the choice of IVF technique is not relevant to reproductive success when oocyte quality is compromised by reproductive aging. Although further randomized trials are needed to confirm our results, we propose that, in absence of male infertility, conventional IVF might be the technique of choice in young patients, especially in those aged below 35 years.
Assisted reproduction, ICSI, IVF, poor responders
Introduction One of the goals of assisted reproduction technologies (ART) is the recruitment of multiple follicles ensuring the recovery of good quality oocytes upon controlled ovarian hyperstimulation (COH). The ovarian response to COH varies substantially among women and is strictly dependent on the size of the ovarian pool of resting follicles, the so-called ovarian reserve [1]. In patients with a low ovarian reserve, a reduced follicular response resulting in a low number of retrieved oocytes is achieved even when a high dose of gonadotropins is administered. Hence, although tests for predicting ovarian reserve are available [2], the parameter that best categorizes a patient as a ‘‘poor responder’’ remains the ovarian response itself. The incidence of poor ovarian response is estimated between 9 and 24% [3–5], values that increase with age [3,5] reaching about 50% in women older than 40 years [6]. Women who respond poorly to COH have poor pregnancy prospects with a pregnancy rate (PR) varying from 7.6 to 17.5% compared with normal responders varying from 25.9 to 36.7%. Among the factors of relevance for prognosis within the poor
Address for correspondence: Paolo Giovanni Artini, Department of Clinical and Experimental Medicine, Division of Obstetrics and Gynecology, University of Pisa, Pisa, Italy. Tel: +39.335.349967. E-mail: [email protected]
History Received 14 May 2013 Revised 1 July 2013 Accepted 1 July 2013 Published online 2 August 2013
responder group, female age appeared to play a distinct role. Overall, older poor responders have lower PRs (ranging between 1.5 and 12.7%) compared with younger poor responders (ranging between 13.0 and 35%) [2]. A second factor predicting pregnancy outcome in poor responders is the degree of poor response. The lower number of retrieved oocytes results in fewer embryos to transfer and a lower prospect for pregnancy, in addition to the expected negative effect of poor ovarian function on oocyte quality. In recent years, there have been an increasing number of in vitro fertilization (IVF) patients in whom few oocytes are obtained in response to COH, a phenomenon mainly related to the postponement of childbearing to the fourth decade of life. In this group of patients where multifollicular response to COH remains a challenge, the optimization of laboratory strategies may help to maximize the chances of pregnancy. Which technique of fertilization should be used in poor responder patients in absence of male factor infertility is still object of controversy. Intra cytoplasmic sperm injection (ICSI) is usually preferred when a male factor associated infertility exists, but sometimes this technique is chosen even in the case of non-male factor indication in order to avoid fertilization failure [7,8]. However, some recent studies suggest that the use of ICSI is not strictly necessary, and its use in the absence of indication is questionable [9–11]. Following the study by Moreno et al. [9] who first proposed that
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the technique of fertilization is not relevant to the reproductive outcome in poor responders, other authors have reported no differences between conventional IVF and ICSI in terms of fertilization and good quality embryo rates even in patients with one [10] or few oocytes inseminated [11]. The aim of this study is to clarify whether natural selection of the fertilizing sperm resulting from conventional IVF may improve reproductive success in poor responder patients with favorable semen quality. To this end, we compared reproductive outcomes following conventional IVF or ICSI in patients in whom only one or two oocytes were retrieved at ovarian pick up by taking into account the impact of reproductive aging.
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Materials and methods We retrospectively analyzed a total of 425 cycles (386 patients) attending ART at the Centre of Infertility and Assisted Reproduction of the Department of Clinical and Experimental Medicine of Pisa University between January 2007 and July 2012. In accordance with European Society of Human Reproduction and Embryology (ESHRE) consensus, all patients were classified as poor responder [6] and were included in this study when only one or two oocytes were retrieved during ovarian pick-up and male factor infertility was absent. Both male and female partners signed an informed consent form. The age of patients included in the study ranged from 27 and 47 years (mean age: 38.23 years 3.82 SD). Cycles were divided into two groups based on the technique used. Thirty-nine patients underwent both the procedures in different cycles. IVF and ICSI groups were furthermore divided into three subgroups based on the age of patients (535 years, 35–38 years and438 years), whose results were also compared. COH was carried out with 150–450 UI/d of recombinant follicle stimulating hormone (FSH) (Gonal FÕ, Serono, Rome, Italy), according to basal FSH and anti-mullerian hormone (AMH) levels and age. All patients were administered cetrorelix (CetrotideÕ , Serono), a gonadotropin releasing hormone (GnRH) antagonist, according to a personalized regimen, i.e. when the lead follicle reached 14 mm in diameter, to prevent premature ovulation. Recombinant human chorionic gonadotropin (OvitrelleÕ , Serono) was administered when at least 1–2 follicles reached a mean diameter of 18 mm. After approximately 36 h, transvaginal follicular aspiration was performed for oocyte retrieval. Volume, sperm count, forward motility and morphology were considered according to the World Health Organization criteria [12]. Sperm not meeting the defined threshold was classified as subfertile sperm, possessing questionable fertilization potential, and patients were consequently excluded from the study. Oocytes were retrieved by vaginal ultrasound (US) probe and incubated in oocyte culture medium (Sydney IVF Oocyte Wash Buffer; Cook Ireland Ltd., Limerick, Ireland). Fertilization technique was chosen based on the clinical history of patients and reproductive outcomes in previous ART cycles. In cases where we choose to perform ICSI, we hypothesized that the impairment in fertilization was the cause of the past failures in previous ART cycles. Four to five hours after oocyte retrieval, in cases where IVF was performed, each oocyte was inseminated with 200 000–300 000 motile washed spermatozoa, while ICSI was accomplished as previously described by Artini et al., 1998 [13]. Fertilization was confirmed by the observation of two pronuclei about 16–18 h after fertilization technique. All the fertilized oocytes were transferred into a fresh cleavage medium (Sydney IVF Cleavage Medium; Cook Ireland Ltd.) and cultured until transfer. On day 2, at 46–48 h post-insemination, the embryos were evaluated for cell number and rate of fragmentation and consequently graded as I–IV (best to worst) [14] by a single
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expert embryologist. Embryo transfer (ET) was performed after 48–72 h under the guidance of abdominal US, using a K-Soft 500 Embryo Transfer Catheter (Cook Ireland Ltd.). All patients had luteal phase support with daily vaginal progesterone (Crinone gelÕ 8%, Merck-Serono) and intramuscular progesterone every 72 h (LentogestÕ , AMSA S.r.l., Rome, Italia), starting on the day of ET until both serum pregnancy test result was negative and gestational sac was confirmed on US. For patients with a positive serum pregnancy test, progesterone was continued until the 12th week of pregnancy. Statistical analysis Continuous data were summarized as the mean SD and were analyze by using t-Student test. To compare proportion, we used the Fisher’s exact test.
Results Patients did not differ in both groups in mean age, FSH and AMH values, doses of gonadotropins administered and average number of oocytes retrieved. Fertilization rate, cleavage rate and good quality embryo rate did not differ between IVF and ICSI group when these were not divided by age, while for what concerns implantation rate (13.05 versus 5.26%) and PRs (16.12 versus 6.73%), IVF was found to be more advantageous with a level of significance of p ¼ 0.003 and p ¼ 0.003, respectively. In patients under 35 years, we did not observe any differences in fertilization rate, cleavage rate and good quality embryo rate between IVF and ICSI group, while for what concerns implantation rate (25.92 versus 3.70%; p ¼ 0. 002) and PRs (32.55 versus 4.76%; p ¼ 0.001), we found that IVF was more advantageous. Although miscarriage rate was higher in IVF group, this difference is not significant (Table 1). As noted in younger patients, even in patients aged between 35 and 38 years, we did not observe any significant differences in fertilization rate, cleavage rate and good quality embryo rate, while implantation rate and PRs were 20% versus 6.34% (p ¼ 0.025) and 26.31% versus 7.01% (p ¼ 0.010), respectively. Even in this subgroup, despite a greater percentage of miscarriage in IVF group, this difference was not significant (Table 2). In patients over 38 years, there were no significant differences in fertilization rate, cleavage rate and good quality embryo rate; but in ICSI group the percentage of cycle cancelled, due to fertilization failure or cleavage failure, was significantly higher compared to IVF group (21.1% versus 10.2%; p ¼ 0.27). In this Table 1. Outcomes IVF vs ICSI in patients aged younger than 35 years. IVF-ET535 years No. of patients 40 Age (years) 32.3 1.68 No. of cycles 43 No. of oocytes retrieved 1.67 0.47 No. of oocytes inseminated 1.62 0.48 No. of oocytes fertilized 1.41 0.49 Non transferred cycles, 0% (0/43) % (n) Fertilization rate % (n) 87.1% (61/70) Cleavage rate % (n) 88.5% (54/61) No. of embryos transferred 1.25 0.44 (54) Good quality embryo 75.9% (41/54) rate % (n) Implantation rate % (n) 25.9% (14/54) Clinical pregnancy 32.55% (14/43) rate % (n) Miscarriage rate % (n) 6.97% (3/43)
*p50.05.
ICSI535 years
p Value
42 32.07 1.9 42 1.71 0.45 1.57 0.63 1.35 0.61 7.1% (3/42)
– 0.574 – 0.694 0.645 0.614 0.116
86.3% (57/66) 94.7% (54/57) 1.28 0.59 (54) 77.7% (42/54)
1 0.325 0.793 1
3.7% (2/54) 4.76% (2/42)
0.002* 0.001*
0% (0/42)
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Table 2. Outcomes IVF versus ICSI in patients aged between 35 and 38 years.
No. of patients Age (years) No of cycles No of oocytes retrieved No of oocytes inseminated No of oocytes fertilized Non transferred cycles, % (n) Fertilization rate % (n) Cleavage rate % (n) No. of embryos transferred Good quality embryo rate % (n) Implantation rate % (n) Clinical pregnancy rate % (n) Miscarriage rate % (n)
IVF-ET: 35–38 years
ICSI: 35–38 yearss
p Value
54 36.83 1.17 57 1.68 0.46 1.68 0.46 1.36 0.58 5.2% (3/57)
51 36.66 1.27 57 1.61 0.49 1.57 0.49 1.21 0.52 7.0% (4/57)
– 0.487 – 0.436 0.247 0.132 1
81.2% (78/96) 96.1% (75/78) 1.31 0.57 76% (57/75)
76.6% (69/90) 91.3% (63/69) 1.10 0.48 66.6% (42/63)
0.475 0.306 0.036* 0.257
20% (15/75) 26.3% (15/57)
6.3% (4/63) 7.0% (4/57)
0.025* 0.010*
5.26% (3/57)
0% (0/57)
0.243
*p50.05. Table 3. Outcomes IVF versus ICSI in patients older than 38 years.
No. of patients Age (years) No of cycles No of oocytes retrieved No of oocytes inseminated No of oocytes fertilized Non transferred cycles, % (n) Fertilization rate % (n) Cleavage rate % (n) No. of embryos transferred Good quality embryo rate % (n) Implantation rate % (n) Clinical pregnancy rate % (n) Miscarriage rate % (n)
IVF-ET438 years
ICSI438 years
p Value
102 41.35 1.73 117 1.58 0.49 1.56 0.54 1.23 0.62 10.2% (12/117)
97 41.30 1.27 109 1.55 0.49 1.47 0.60 1.09 0.63 21.1% (23/109)
– 0.840 – 0.649 0.256 0.096 0.027*
78.7% (144/183) 96.5% (139/144) 1.188 0.60 73.4% (102/139)
73.9% (119/161) 92.4% (110/119) 1.009 0.65 67.3% (74/110)
0.297 0.172 0.033 0.293
4.3% (6/139) 5.1% (6/117)
7.3% (8/110) 7.3% (8/109)
0.408 0.585
2.6% (3/117)
2.7% (3/109)
1
*p50.05.
subgroup of patients, we did not observe any differences for what concerns implantation, pregnancy and miscarriage rate (Table 3).
Discussion All steps of IVF procedures require more attention when stimulation regimens fail in inducing optimal multifollicular response. Nevertheless, the question whether the choice of fertilization procedure may be relevant to reproductive success in poor responder patients is still debating. Probably, this is why a clear knowledge of both short- and long-term differences between IVF and ICSI fertilization is still lacking. Although ICSI was originally indicated for treating couples with severe male factor infertility [15], some studies indicated that it can provide comparable or higher fertilization rates [16–18] and superior embryo quality even in non-male factor couples [19]. Overall results from this study strongly question the superiority of ICSI over conventional IVF in patients with non-male factor infertility and lead us to hypothesize that under specific conditions, fertilization by ICSI can negatively affect reproductive potential. The analysis of a cohort of poor responders ranging from 27 to 47 years of age carried out in the first part of the study would lead us
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to conclude that the technique of fertilization does not influence reproductive outcome in low responders. This observation supports the general hypothesis that IVF and ICSI in cases of normal semen produce the same results [10,20,21] and is consistent with the finding by Moreno et al. [9] based on the analysis of 96 couples with six or fewer retrieved oocytes. On the other hand, these results are in contrast with Khamsi et al. (2001) [19] showing that ICSI procedure increased fertilization rate and good quality embryo rate even in the absence of male factor. Additionally, in this study, we show that when comparison between IVF and ICSI is conducted on cohorts of poor responder patients with different reproductive age, the analysis reveals that the use of ICSI decreases reproductive potential in women younger than 35 years or aged between 35 and 38 years. Although we found no significant differences in fertilization and cleavage rates in these groups of patients, IVF was significantly more advantageous than ICSI for what concerns implantation and PRs. Our results partially agree with those by Fang et al. [22] who, by retrospectively comparing 196 couples undergoing IVF/ICSI cycles with one or two retrieved oocytes with good-prognosis sperm, found that ICSI patients had higher fertilization rates although no difference in good-quality embryo rate or PR was noted. The discrepancy between this study and our findings in relation to fertilization outcome may be ascribed to differences in the size of the cohorts enrolled in the two studies. Indeed, our results are consistent with those by Xi et al. (2012) who retrospectively analyzed 406 cycles with three or fewer oocytes retrieved from women with similar age undergoing IVF (34.5 4.6 years) or ICSI (36.1 5.5 years) and noted that the PRs and implantation rate were lower in the ICSI group compared with the IVF group [23]. The authors explained the higher PRs and implantation rate after IVF with the lacking of ‘‘natural sperm selection’’ when most steps of the fertilization process are bypassed by sperm injection. In conventional IVF, upon laboratory selection of motile sperm, the sperm that fertilizes is further selected through the biological process of sperm–oocyte interaction beginning at zona pellucida level (ZP) or during sperm penetration through the cumulus matrix [24]. Indeed, the finding that majority of sperm (average 492%) bound to the ZP have normal nuclear chromatin DNA strongly suggest that scientist subjectively selected sperm during ICSI may have a lower quality compared to ZP-bound sperm [25]. Moreover, increased knowledge in the biology of fertilization process has revealed that sperm–oocyte interaction at membrane level involves numerous molecular actors with a possible role in sperm fusion and gamete selection [24]. Overall, these mechanisms may act against sperm carrying chromosomal anomaly from paternal origin as well as against chromosomally abnormal oocytes generating developmentally defective embryos leading to pregnancy failure or miscarriage [23,26]. In addition, decreased chances for successful fertilization and pregnancy could result from the possible mechanical damage to the oocyte after ICSI [27] as well as to the introduction of foreign material such as culture medium or exogenous DNA and infectious material [28]. Finally, our results in reproductively young women can be well explained by considering the relevance of early events of oocyte activation in promoting successful implantation. These include the sperminduced calcium signal that drives meiosis resumption and embryo development, as well as implantation and postimplantation events, through a specific spatiotemporal pattern of Ca2þ oscillation [29]. Beyond the delivery of sperm DNA, the main reason for ICSI to succeed is that it allows the delivery of phospholipase C-zeta (PLC z), the sperm component that is capable of generating the fertilization calcium signal upon sperm fusion [30]. Nevertheless, the finding that abnormal calcium signals were observed in ICSI oocytes [31,32] suggest that
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missing gamete interaction at surface level in ICSI fertilization would result in missing or abnormal signaling pathways with a role in later embryonic development. An additional factor with a negative influence on reproductive outcome is ICSI-related risk of parthenogenetic activation caused by oocyte manipulation [33– 35]. This may cause asynchrony between the timing of oocyte activation and sperm injection leading to cleavage abnormalities and early embryonic arrest [27]. Our finding that reproductive outcome in patients aged over 38 years undergoing IVF or ICSI were comparable for all parameters analyzed strongly indicates that the advantage of IVF over ICSI tends to disappear with the increasing of age. This result can be ascribed to the phenomenon of ovarian ageing responsible for the production of oocytes with a reduced developmental competence related to defective molecular storage, mitochondrial dysfunctions and poor control of chromosome segregation during meiosis [36,37]. These defects represent a reliable reason why aged oocytes do not benefit from fertilization mechanisms preserved in IVF and lost in ICSI. Nevertheless, a further reason for that could be found in the low activation competence of aged oocytes suggested by the observation of abnormal signaling upon exposure to parthenogenetic agents [38].
Conclusions A constant effort of reproductive medicine is to apply the simplest, most economical and least invasive method to ensure a positive outcome. In this context, we provide evidence that obtaining one or two oocytes in one cycle is not an indication for ICSI when the sperm sample is apparently normal. Indeed, a relevant factor to the choice of IVF technique under these conditions is represented by female age. In spite of the effects of a reduced ovarian reserve, oocytes from young poor responder patients, probably counting on biological resources definitively lost with aging, can still benefit of the advantage of IVF. We suggest that IVF could be used as a technique of choice in young poor responder patients in absence of male factor. However, since ours is a retrospective study, only further randomized trials will be able to confirm our results. Finally, we conclude that, in addition to optimization of stimulation regimens, further biological knowledge of IVF techniques will be helpful in tailoring the best IVF treatment to individual patients in order to give infertile couples the best chance of conceiving a healthy baby.
Declaration of interest The authors reports no declaration of interest.
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