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Effect of maternal and paternal age on pregnancy and miscarriage rates after intrauterine insemination
RBMOnline - Vol 17. No 3. 2008 392-397 Reproductive BioMedicine Online; www.rbmonline.com/Article/3526 on web 11 July 2008
Article Effect of maternal and paternal age on pregnancy and miscarriage rates after intrauterine insemination Stéphanie Belloc, graduate of medicine and reproductive biology, Paris VI Tenon Hospital, has trained in reproductive biology since 2002. She is currently working as the head of the male infertility department in Eylau Laboratories, Paris. She researches actively in the area of assisted reproduction technology within the Eylau team, particularly male predictive factors, for example DNA fragmentation, sperm chromatin decondensation and intramorphologically selected sperm injection (IMSI) tests.
Dr Stéphanie Belloc Stéphanie Belloc1,2,3, Paul Cohen-Bacrie1,2,3, Moncef Benkhalifa1,4 Martine Cohen-Bacrie1, Jacques De Mouzon5, André Hazout2, Yves Ménézo1,2,3,4,6 1 Laboratoire d’Eylau, 55 rue Saint Didier, 75116 Paris; 2Unité AMP Eylau La Muette, 46–48 rue Nicolo 75116, Paris; 3 Unité AMP Eylau Cherest, 5 Rue Pierre Cherest 92200 Neuilly sur Seine; 4ATL 78300, La Verriere; 5Unité INSERM 822, 82 rue Général Leclerc, 94276 Le Kremlin Bicetre, France 6 Correspondence: Laboratoire d’Eylau, 55 rue Saint Didier, Paris, France. Fax +33 1 53706491; e-mail: yves.Menezo@ club-internet.fr
Abstract More than 17,000 intrauterine insemination (IUI) cycles were analysed retrospectively with respect to outcome according to differing aetiologies of infertility. The quantity and motility of spermatozoa in the final preparation used for insemination had a positive effect on the outcome, as classically observed in the past. It was found that advanced maternal age had a negative effect on the pregnancy rate and was associated with increased miscarriage rate. More interestingly, an exactly parallel effect was found for paternal age. The impact of increased age on necrospermia and sperm DNA structure is discussed as a probable direct cause of this paternal effect. Keywords: IUI, maternal age, miscarriage, paternal age
Introduction
392
Artificial insemination is currently used to treat infertility for a wide range of indications as it is not traumatic, is easy to perform, and is less expensive than other assisted reproduction treatments. However, as the chances of success in terms of delivery rate per cycle are at best around 10%, the questions that repeatedly arise concern the identification of prognostic factors for success, and how many cycles should be carried out before proceeding to IVF/intracytoplasmic sperm injection (ICSI). Maternal age of 35 years or over is extensively described as a well-known risk factor for decreased fertility. Altered oocyte competence with age impairs chromosome segregation, and this leads to an unbalanced chromosome complement: in addition to an increased incidence of Down syndrome and trisomies, this abnormal segregation also leads to increased miscarriage rates. Moreover, increased age also alters gene expression patterns in the oocyte (Hamatani et
al., 2004), thus leading to impaired cytoplasmic competence and abnormal function of highly important regulatory housekeeping pathways (Ménézo 2006; et al., Ménézo 2007b). However, the possibility of an effect due to paternal age has only recently been considered (Klonoff-Cohen and Natarajan, 2004). Progress in the analysis of sperm DNA structure has clearly demonstrated that sperm DNA fragmentation increases with age, with or without oxidative stress as an additional factor (Duran et al., 2002; Evenson et al., 2002; Aitken et al., 2003; Guérin et al., 2005; Evenson and Wixon, 2006). A high sperm fragmentation index impairs the outcome of IUI and assisted reproduction in general (Duran et al., 2002), and this provides a link to paternal age. However, the impact of paternal age on miscarriage rates has never been clearly analysed. This study analysed the relationship
© 2008 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
Article - Parental age and IUI outcome - S Belloc et al. between paternal and maternal age and IUI outcomes, including miscarriage rates, in a large cohort of patients. Semen characteristics were also included in the analysis.
Materials and methods More than 17,000 intrauterine insemination (IUI) cycles were analysed retrospectively with respect to outcome according to differing aetiologies of infertility. All the sperm treatments were performed in the Laboratoire d’Eylau and all the data were collected in the unit. The period over which data were collected was from January 2003 to December 2006.
Patient cycle management The female patients underwent ovarian stimulation either with clomiphene citrate (60% of patients) or with low-dose FSH treatment [recombinant FSH (Gonal F; Serono and Puregon; Organon) or human menopausal gonadotrophin (Menopur; Ferring)]. For clomiphene-stimulated cycles, 50 mg was given daily from day 2 to day 6. When gonadotrophins were used, a daily injection of 75 or 150 IU was given from day 3 to day 9 of the cycle. Follicle maturation was monitored by ultrasound scan and plasma oestradiol concentration on day 10. When one follicle reached a diameter of 18 mm in the presence of an echogenous endometrium of at least 7 mm in thickness, 10,000 IU of human chorionic gonadotrophin (HCG) were injected. If more than three follicles were present, IUI was cancelled. IUI was performed 34– 36 h post-HCG. A clinical pregnancy was considered as positive when the plasma beta-HCG reached at least 1000 mIU per ml (as requested by the French registry).
Semen analysis and preparation for insemination Semen analyses were performed for all the ejaculates for each insemination cycle [spermogram, morphology according to World Health Organization criteria, sperm migration and survival test (World Health Organization, 1999 and Cohen-Bacrie et al., 2008)]. Semen preparation was carried out using classical twophase purification (750 g; Suprasperm 45/90%; Medicult, France). The sperm pellet was re-suspended in 0.3 ml of BM1 medium (Eurobio, France). The insemination was cancelled if the total number of living, motile spermatozoa (A + B quality movement: rapid progressive + slow progressive) was below 0.5 × 106 and if >75% abnormal forms were present, irrespective of the final cell concentration in the final sample for insemination. Computerassisted sperm analysis (CASA; Ivos 10.7s; Hamilton Thorne Research, Beverly, MA, USA) was performed at the end of sperm processing, in order to verify the final sperm concentration, taking care of the usual small discrepancy observed between manual and computerized evaluation (overestimation by computer). The amplitude of lateral head displacement (ALH) was recorded for each sample.
Indications A total of 23.4% of the IUI cycles were performed for semen indication only; 12.8% for cervical hostility only, 35.6% for mixed semen and cervical problems, 20.3% for idiopathic infertility, 7.9% for multiple female aetiologies RBMOnline®
Statistics The relationship between the pregnancy rates and the continuous prognosis factors was analysed using variance analysis, and the relationship with categorical variables with the chi-squared test, using continuity corrections or Fisher’s exact testing in case of small numbers. Finally, a multilogistic model was performed to analyse the effect of age and semen characteristics on the pregnancy rates together. In this case, odds ratios (OR) and their 95% confidence intervals (CI) were computed. Analysis was carried out using SAS software, release 9 (SAS Institute Inc., USA).
Results The overall clinical pregnancy rate (PR) was 12.7% per cycle, which might reach a theoretical rate of 64% for six cycles. The mean number of cycles per patient was 2.3. Pregnancy rate decreased with maternal age (Table 1, Figure 1), from 14.4% before 30 years of age to 8.9% after 42 years of age (P < 0.001). The miscarriage rates followed a symmetrical curve, increasing more than four times, from 11.1% before 30 years of age to 46.4% after 42 years of age. Paternal age led to a similar decrease in the PR (Table 1, Figure 2), from 12.3% before 30 years of age to 9.3% after 45 years of age (P < 0.001) and an increase in the miscarriage rate that more than doubled, from 13.7% before 30 years of age, to 32.4% after 45 years of age. The PR was higher in the purely female indications (14.9%) than in male factor (12.6%), mixed (12.6%) or idiopathic (11.6%) aetiologies (P < 0.001). Semen analyses demonstrated that the PR declined when necrospermia reached a threshold of 25% (PR of 12% at ≥25% necrospermia versus PR of 13.6% at <25% necrospermia, P < 0.02). No relationship was observed between PR and sperm concentration. However, the PR increased when motility reached 40% in the ejaculate (PR of 13.7% at 40% motility versus PR of 11.7% at <40% motility, P < 0.01) and the type A motility reached 30% (13.5 versus 12%, P < 0.02). No correlation was found either with the multiple anomaly index (m = 1.7) or with specific morphology anomalies taken individually, except for the flagellum, with a significant threshold at 15%: (PR = 13.9% versus 12.0%, P < 0.01). The percentage of atypical forms increased the miscarriage rate, but not significantly (Figure 3). These observations did not show any impact of abstinence, which was low (less than 5 days), as requested. Regarding the spermatozoa on the day of the attempt, the concentration significantly influenced the chance of success, ranging from 10.4% for fewer than 5 × 106/ml to 13.5% for over 20 × 106/ml, together with the level of motility (P < 0.01 for both). In terms of quantity of motile spermatozoa inseminated, the PR increased from 10% at below 1 × 106/ml to 14% at over 10 × 106/ml (P < 0.01) (Table 2). The mean ALH was 3.17 µm before migration and 4.30 µm after migration (Table 3), and no association was found with the PR (before migration and after migration). When analysing the effect of age and semen characteristics together (Table 4), the pregnancy chance was significantly decreased for women aged 38 years or more (OR = 0.67, 95% CI = 0.56–0.80), and not significantly decreased with male age, reaching almost significant levels for age ≥45
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Article - Parental age and IUI outcome - S Belloc et al. Table 1. Pregnancy and miscarriage rates in intrauterine insemination cycles according to paternal and maternal ages. Cycles n %
Clinical Miscarriages pregnancies n %/cycle n %/pregnancy
Paternal age (years) <30 950 9.5 117 12.3 16 30–34 3551 35.4 484 13.6 81 35–39 2735 27.3 366 13.4 58 40–44 1631 16.3 178 10.9 58 ≥45 1167 11.6 108 9.3 35 Maternal age (years) <30 1502 15.3 216 14.4 24 30–34 3702 37.8 536 14.5 88 35–37 1911 19.5 241 12.6 52 38–41 1731 17.7 173 10.0 49 ≥42 947 9.7 84 8.9 39
13.7 16.7 15.8 32.6 32.4 11.1 16.4 21.6 28.3 46.4
Pregnancy and miscarriage rates were significantly different between the <30 and ≥42 years age groups for both maternal and paternal age (both P < 0.001).
Figure 1. Clinical pregnancy rates (PR) and miscarriage rates (MR) in intrauterine insemination cycles according to maternal age.
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Figure 2. Clinical pregnancy rates (PR) and miscarriage rates (MR) in intrauterine insemination cycles according to paternal age.
Figure 3. Clinical pregnancy rates (PR) and miscarriage rates (MR) in intrauterine insemination cycles in relation to morphological anomalies. RBMOnline®
Article - Parental age and IUI outcome - S Belloc et al. Table 2. Clinical pregnancy rates related to sperm characteristics at the time of insemination. Cycles Pregnancies P-value n n %
Sperm concentration (× 106/ml) <5.0 412 43 10.4 5.0–9.9 742 85 11.5 10.0–14.9 916 103 11.2 15.0–19.9 875 108 12.3 20.0–49.9 3784 510 13.5 ≥50.0 2992 395 13.2 Raw motility (A+B+C) (%) <20 641 68 10.6 20–29 1712 184 10.7 30–39 1682 217 12.9 40–59 3339 433 13.0 60–69 1308 204 15.6 ≥70 1089 141 13.0 Type A motility (%) <10 473 48 10.1 10–14 728 87 12.0 15–19 824 94 11.4 20–34 2937 358 12.2 35–49 2186 279 12.8 50–59 1250 194 15.5 ≥60 1342 183 13.6 Number of motile spermatozoa for insemination (×106) <1.0 689 69 10.0 1.0–1.4 450 50 11.1 1.5–3.4 1274 143 11.2 3.5–9.9 2496 328 13.1 10.0–14.9 1866 264 14.1 15.0–19.9 2834 378 13.3 ≥20.0 134 14 10.4 Abstinence (days) 0 234 32 13.7 1 802 94 11.7 2 2320 306 13.2 3 2941 389 13.2 4 1453 160 11.0 5 891 129 14.5 6 341 41 12.0 ≥7 774 96 12.4
0.02
0.01
0.01
0.01
NS
NS = not statistically significant.
Table 3. Characteristics of raw and processed samples at the time of insemination. No pregnancy P-value
Pregnancy
Abstinence (days) Volume (ml) Number of motile spermatozoa in the insemination sample (×106) ALH before processing (µm) ALH after processing (µm)
3.44 ± 2.24 3.48 ± 2.46 3.11 ± 1.62 3.11 ± 2.03 10.4 ± 6.4 9.8 ± 6.8
NS NS 0.01
3.17 ± 0.67 3.16 ± 0.68 4.31 ± 0.82 4.30 ± 0.81
NS NS
Values are mean ± SD, unless otherwise stated. ALH = amplitude of lateral head displacement; NS = not statistically significant.
RBMOnline®
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Article - Parental age and IUI outcome - S Belloc et al. Table 4. Multivariate statistical analysis of the main parameters influencing pregnancy and miscarriage rates in intrauterine insemination cycles (logistic model). Variable
Pregnancy Odds 95% CI ratio
Miscarriage Odds 95% CI ratio
Woman’s age (years) Man’s age (years) Sperm vitality (%) A type motility (%) Flagellum anomalies (%)
1.00 0.88 0.67 1.00 0.90 0.81 0.98 1.00 0.87 0.81 0.89
1.00 1.5 2.31 1.00 1.71 1.75 0.74 1.00 0.94 0.50 0.96
<35 35–37 ≥38 <35 35–44 ≥45 ≥65 ≥30 10–29 <10 ≥15
− 0.75–1.03 0.56–0.80 − 0.75–1.09 0.65–1.02 0.83–1.15 − 0.76–0.99 0.61–1.07 0.79–1.01
− 1.00–2.12 1.58–3.38 − 1.14–2.54 1.06–2.90 0.49–1.12 − 0.68–1.29 0.22–1.13 0.71–1.30
CI = confidence interval.
years (OR = 0.81; 0.65–1.02). There was no significant effect of the semen characteristics entered in the model. The miscarriage risk was increased with age, for both members of the couple, being more than double for women older than 38 years (OR = 2.31, 1.58–3.38) and almost double for men older than 45 years (OR = 1.75, 1.06–2.90), independently of women’s age. Here also, the semen characteristics entered in the model were not significantly associated with an increased risk.
Discussion The data clearly show that paternal and maternal ages affect the outcome in the IUI programme. The impact of female age on miscarriage rate is common knowledge, and it is generally admitted that the deleterious effect is related to the decreased quality of the oocyte (both nuclear and cytoplasmic), rather than the uterine quality. Semen quality, in terms of World Health Organization parameters, has an obvious impact on IUI outcome, as classically observed (Dickey et al., 1999; Badawy et al., 2008). The morphology has a negative although not significant impact, this is due to the severe limit set for morphology in this IUI programme; below a threshold of 25–30% normal forms, IUI has very low chances of success (Badawy et al., 2008).
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More surprising is the highly significant influence of male age on miscarriage rates. The number of attempts that should be made before moving to more sophisticated assisted reproduction techniques is always a question mark (Custers et al., 2008); the present observations are in favour of shortening the time spent on IUI cycles. It surely depends on maternal age, but also paternal age as demonstrated here. It is always tempting after IUI failures to move on to IVF or ICSI (Oehninger and Gosden, 2002). The paternal effect seems to be overcome by ICSI (Aboulgar et al., 2007). There is some evidence that ICSI may help in resolving the problem of sperm DNA fragmentation (Bungum et al., 2004, 2007). If one considers DNA decay, ‘normal’, in-vitro or in-vivo fertilization implies a burst of reactive oxygen species (ROS) concomitant with the capacitacion process (De Lamirande and Gagnon, 1995);
this may increase decay in sperm DNA. ICSI bypasses this specific risk, but first, not all the decay is fragmentation related (Badouard et al., 2008) and second, ICSI is not totally without risk (Morozumi and Yanagimachi, 2005; Ménézo, 2006; Fernández-Gonzalez et al., 2008) and is certainly not a panacea (Oehninger and Gosden, 2002). In fact, the human oocyte has a complete, but finite, capacity for DNA repair (Ménézo et al., 2007b), even in the best maternal conditions (Frydman et al., 2007). Moreover, mRNA expression of the genes involved decreases with maternal age (Hamatani et al., 2004). Incorrect DNA repair, i.e. tolerance of sperm DNA damage by the oocyte, may have serious deleterious effects in the offspring, including childhood cancers (Aitken and Baker, 2006; Fernández-Gonzalez et al., 2008). It is thus important to minimize sperm DNA decay if possible. It has therefore been tempting to try to reduce ROS-linked DNA fragmentation by treatment of patients with anti-oxidant (Agarwal et al., 2004). However, the wide discrepancy observed in the scientific literature with regard to antioxidant treatments is linked to the negative effect of antioxidants on sperm condensation; an excessively high level of antioxidants can impair the normal reduction of cysteine to cystine, which prevents the protamine cross-linking required for DNA condensation. This results in weak, abnormal sperm DNA condensation, which increases the risk of DNA damage, leading to an increased risk of chromosomal anomalies in the embryo after fertilization (Ménézo et al., 2007a) In conclusion, a strict check of the spermatozoa is mandatory before all assisted reproduction treatment. Passage from IUI to ICSI must not be automatic. Sperm DNA fragmentation and decondensation, which have only a weak correlation with classical sperm analysis, must be performed after failed IUI cycles, before ICSI. Intracytoplasmic injection of morphologically selected sperm (IMSI), based on motile sperm organellar morphology examination (MSOME) is perhaps an alternative way to avoid the deleterious effect of injecting highly fragmented spermatozoa (Berkovitz et al., 2005; Hazout et al., 2006). A better selection of ICSI indications is needed (Oehninger and Gosden, 2002), taking into account that paternal DNA decay, RBMOnline®
Article - Parental age and IUI outcome - S Belloc et al. increasing with age, has to be repaired by the oocyte, which has a finite capacity for repair, and whose quality decreases with age.
References Aboulghar M, Mansour R, Al-Inany H et al. 2007 Paternal age and outcome of intracytoplasmic sperm injection. Reproductive BioMedicine Online 14, 588–592. Agarwal A, Nallela KP, Allamaneni SS et al. 2004 Role of antioxidant treatment of male infertility: an overview of literature. Reproductive BioMedicine Online 8, 616–628. Aitken RJ, Baker MA 2006 Oxidative stress, sperm survival and fertility control. Molecular and Cellular Endocrinology 250, 66–69. Badawy A, Elnashar A, Eltotongy M 2008 Effect of sperm morphology and number on success of intrauterine insemination. Fertility and Sterility [Epub ahead of print February 25]. Badouard C, Ménézo Y, Panteix G et al. 2008 Determination of new types of DNA lesions in human sperm. Zygote 16, 9–13. Berkovitz A, Eltes F, Yaari S, et al. 2005 The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm. Human Reproduction 20, 185–190. Bungum M, Humaidan P, Axmon A et al. 2007 Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome. Human Reproduction 22, 174–179. Bungum M, Humaidan P, Spano M et al. 2004 The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI. Human Reproduction 19, 1401–1408. Cohen-Bacrie P, Belloc S, Ménézo Y et al. 2008 Correlation between DNA damages and sperm parameters: a prospective study involving 1633 patients. Fertility and Sterility [Epub ahead of print April 24]. Custers IM, Steures P, Hompes P et al. 2008 Intrauterine insemination: how many cycles should we perform? Human Reproduction 23, 885–888. de Lamirande E, Gagnon C 1995 Capacitation-associated production of super oxide anion by human spermatozoa. Free Radical Biology and Medicine 18, 487–95. Dickey RP, Pyrzak R, Lu PY et al. 1999 Comparison of the sperm quality necessary for successful intrauterine insemination with World Health Organization threshold values for normal sperm. Fertility and Sterility 71, 684–689. Duran EH, Morshedi M, Taylor S et al. 2002 Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study. Human Reproduction 17, 3122–3128. Evenson DP, Wixon R 2006 Clinical aspects of sperm DNA fragmentation detection and male infertility. Theriogenology 65, 979–991. Evenson DP, Larson KL, Jost LK 2002 Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. Journal of Andrology 23, 25–43. Fernández-Gonzalez R, Moreira P, Perez Crespo M et al. 2008 Longterm effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring. Biology of Reproduction 78, 761–772. Frydman N, Prisant N, Hesters L et al. 2008 Adequate ovarian follicular status does not prevent the decrease in pregnancy rates associated with high sperm DNA fragmentation. Fertility and Sterility 89, 92–97. Guérin P, Matillon C, Bleau G et al. 2005 Impact of sperm DNA fragmentation on ART outcome. Gynecologie, Obstetrique & Fertilité 33, 665–668. Hamatani T, Falco G, Carter MG et al. 2004 Age-associated alteration of gene expression patterns in mouse oocytes. Human Molecular Genetics 13, 2263–2278. Hazout A, Dumont-Hassan M, Junca AM et al. 2006 Highmagnification ICSI overcomes paternal effect to conventional RBMOnline®
ICSI. Reproductive BioMedicine Online 12, 19–25. Klonoff-Cohen HS, Natarajan L 2004 The effect of advancing paternal age on pregnancy and live birth rates in couples undergoing in vitro fertilization or gamete intrafallopian transfer. American Journal of Obstetrics and Gynecology 191, 507–514. Ménézo Y 2006 Paternal and maternal factors in preimplantation embryogenesis: interaction with the biochemical environment. Reproductive BioMedicine Online 12, 616–621. Ménézo YJ, Hazout A, Panteix G et al. 2007a Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect. Reproductive BioMedicine Online 14, 418–421. Ménézo Y JR, Russo G, Tosti E et al. 2007b Expression profile of genes coding for DNA repair in human oocytes using pangenomic microarrays, with a special focus on ROS linked decays. Journal of Assisted Reproduction and Genetics 24, 513–520. Morozumi K, Yanagimachi R 2005 Incorporation of the acrosome into the oocyte during intracytoplasmic sperm injection could be potentially hazardous to embryo development. Proceedings of the National Academy of Sciences of the USA 102, 14209–14214. Oehninger S, Gosden RG 2002 Should ICSI be the treatment of choice for all cases of in-vitro conception? No, not in light of the scientific data. Human Reproduction 17, 2237–2242. World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and Semen–Cervical Mucus Interaction, 4th edn. Cambridge University Press, Cambridge, p. 17.
Declaration: The authors report no financial or commercial conflicts of interest. Received 6 March 2008; refereed 6 May 2008; accepted 9 July 2008.
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Article Effect of maternal and paternal age on pregnancy and miscarriage rates after intrauterine insemination Stéphanie Belloc, graduate of medicine and reproductive biology, Paris VI Tenon Hospital, has trained in reproductive biology since 2002. She is currently working as the head of the male infertility department in Eylau Laboratories, Paris. She researches actively in the area of assisted reproduction technology within the Eylau team, particularly male predictive factors, for example DNA fragmentation, sperm chromatin decondensation and intramorphologically selected sperm injection (IMSI) tests.
Dr Stéphanie Belloc Stéphanie Belloc1,2,3, Paul Cohen-Bacrie1,2,3, Moncef Benkhalifa1,4 Martine Cohen-Bacrie1, Jacques De Mouzon5, André Hazout2, Yves Ménézo1,2,3,4,6 1 Laboratoire d’Eylau, 55 rue Saint Didier, 75116 Paris; 2Unité AMP Eylau La Muette, 46–48 rue Nicolo 75116, Paris; 3 Unité AMP Eylau Cherest, 5 Rue Pierre Cherest 92200 Neuilly sur Seine; 4ATL 78300, La Verriere; 5Unité INSERM 822, 82 rue Général Leclerc, 94276 Le Kremlin Bicetre, France 6 Correspondence: Laboratoire d’Eylau, 55 rue Saint Didier, Paris, France. Fax +33 1 53706491; e-mail: yves.Menezo@ club-internet.fr
Abstract More than 17,000 intrauterine insemination (IUI) cycles were analysed retrospectively with respect to outcome according to differing aetiologies of infertility. The quantity and motility of spermatozoa in the final preparation used for insemination had a positive effect on the outcome, as classically observed in the past. It was found that advanced maternal age had a negative effect on the pregnancy rate and was associated with increased miscarriage rate. More interestingly, an exactly parallel effect was found for paternal age. The impact of increased age on necrospermia and sperm DNA structure is discussed as a probable direct cause of this paternal effect. Keywords: IUI, maternal age, miscarriage, paternal age
Introduction
392
Artificial insemination is currently used to treat infertility for a wide range of indications as it is not traumatic, is easy to perform, and is less expensive than other assisted reproduction treatments. However, as the chances of success in terms of delivery rate per cycle are at best around 10%, the questions that repeatedly arise concern the identification of prognostic factors for success, and how many cycles should be carried out before proceeding to IVF/intracytoplasmic sperm injection (ICSI). Maternal age of 35 years or over is extensively described as a well-known risk factor for decreased fertility. Altered oocyte competence with age impairs chromosome segregation, and this leads to an unbalanced chromosome complement: in addition to an increased incidence of Down syndrome and trisomies, this abnormal segregation also leads to increased miscarriage rates. Moreover, increased age also alters gene expression patterns in the oocyte (Hamatani et
al., 2004), thus leading to impaired cytoplasmic competence and abnormal function of highly important regulatory housekeeping pathways (Ménézo 2006; et al., Ménézo 2007b). However, the possibility of an effect due to paternal age has only recently been considered (Klonoff-Cohen and Natarajan, 2004). Progress in the analysis of sperm DNA structure has clearly demonstrated that sperm DNA fragmentation increases with age, with or without oxidative stress as an additional factor (Duran et al., 2002; Evenson et al., 2002; Aitken et al., 2003; Guérin et al., 2005; Evenson and Wixon, 2006). A high sperm fragmentation index impairs the outcome of IUI and assisted reproduction in general (Duran et al., 2002), and this provides a link to paternal age. However, the impact of paternal age on miscarriage rates has never been clearly analysed. This study analysed the relationship
© 2008 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
Article - Parental age and IUI outcome - S Belloc et al. between paternal and maternal age and IUI outcomes, including miscarriage rates, in a large cohort of patients. Semen characteristics were also included in the analysis.
Materials and methods More than 17,000 intrauterine insemination (IUI) cycles were analysed retrospectively with respect to outcome according to differing aetiologies of infertility. All the sperm treatments were performed in the Laboratoire d’Eylau and all the data were collected in the unit. The period over which data were collected was from January 2003 to December 2006.
Patient cycle management The female patients underwent ovarian stimulation either with clomiphene citrate (60% of patients) or with low-dose FSH treatment [recombinant FSH (Gonal F; Serono and Puregon; Organon) or human menopausal gonadotrophin (Menopur; Ferring)]. For clomiphene-stimulated cycles, 50 mg was given daily from day 2 to day 6. When gonadotrophins were used, a daily injection of 75 or 150 IU was given from day 3 to day 9 of the cycle. Follicle maturation was monitored by ultrasound scan and plasma oestradiol concentration on day 10. When one follicle reached a diameter of 18 mm in the presence of an echogenous endometrium of at least 7 mm in thickness, 10,000 IU of human chorionic gonadotrophin (HCG) were injected. If more than three follicles were present, IUI was cancelled. IUI was performed 34– 36 h post-HCG. A clinical pregnancy was considered as positive when the plasma beta-HCG reached at least 1000 mIU per ml (as requested by the French registry).
Semen analysis and preparation for insemination Semen analyses were performed for all the ejaculates for each insemination cycle [spermogram, morphology according to World Health Organization criteria, sperm migration and survival test (World Health Organization, 1999 and Cohen-Bacrie et al., 2008)]. Semen preparation was carried out using classical twophase purification (750 g; Suprasperm 45/90%; Medicult, France). The sperm pellet was re-suspended in 0.3 ml of BM1 medium (Eurobio, France). The insemination was cancelled if the total number of living, motile spermatozoa (A + B quality movement: rapid progressive + slow progressive) was below 0.5 × 106 and if >75% abnormal forms were present, irrespective of the final cell concentration in the final sample for insemination. Computerassisted sperm analysis (CASA; Ivos 10.7s; Hamilton Thorne Research, Beverly, MA, USA) was performed at the end of sperm processing, in order to verify the final sperm concentration, taking care of the usual small discrepancy observed between manual and computerized evaluation (overestimation by computer). The amplitude of lateral head displacement (ALH) was recorded for each sample.
Indications A total of 23.4% of the IUI cycles were performed for semen indication only; 12.8% for cervical hostility only, 35.6% for mixed semen and cervical problems, 20.3% for idiopathic infertility, 7.9% for multiple female aetiologies RBMOnline®
Statistics The relationship between the pregnancy rates and the continuous prognosis factors was analysed using variance analysis, and the relationship with categorical variables with the chi-squared test, using continuity corrections or Fisher’s exact testing in case of small numbers. Finally, a multilogistic model was performed to analyse the effect of age and semen characteristics on the pregnancy rates together. In this case, odds ratios (OR) and their 95% confidence intervals (CI) were computed. Analysis was carried out using SAS software, release 9 (SAS Institute Inc., USA).
Results The overall clinical pregnancy rate (PR) was 12.7% per cycle, which might reach a theoretical rate of 64% for six cycles. The mean number of cycles per patient was 2.3. Pregnancy rate decreased with maternal age (Table 1, Figure 1), from 14.4% before 30 years of age to 8.9% after 42 years of age (P < 0.001). The miscarriage rates followed a symmetrical curve, increasing more than four times, from 11.1% before 30 years of age to 46.4% after 42 years of age. Paternal age led to a similar decrease in the PR (Table 1, Figure 2), from 12.3% before 30 years of age to 9.3% after 45 years of age (P < 0.001) and an increase in the miscarriage rate that more than doubled, from 13.7% before 30 years of age, to 32.4% after 45 years of age. The PR was higher in the purely female indications (14.9%) than in male factor (12.6%), mixed (12.6%) or idiopathic (11.6%) aetiologies (P < 0.001). Semen analyses demonstrated that the PR declined when necrospermia reached a threshold of 25% (PR of 12% at ≥25% necrospermia versus PR of 13.6% at <25% necrospermia, P < 0.02). No relationship was observed between PR and sperm concentration. However, the PR increased when motility reached 40% in the ejaculate (PR of 13.7% at 40% motility versus PR of 11.7% at <40% motility, P < 0.01) and the type A motility reached 30% (13.5 versus 12%, P < 0.02). No correlation was found either with the multiple anomaly index (m = 1.7) or with specific morphology anomalies taken individually, except for the flagellum, with a significant threshold at 15%: (PR = 13.9% versus 12.0%, P < 0.01). The percentage of atypical forms increased the miscarriage rate, but not significantly (Figure 3). These observations did not show any impact of abstinence, which was low (less than 5 days), as requested. Regarding the spermatozoa on the day of the attempt, the concentration significantly influenced the chance of success, ranging from 10.4% for fewer than 5 × 106/ml to 13.5% for over 20 × 106/ml, together with the level of motility (P < 0.01 for both). In terms of quantity of motile spermatozoa inseminated, the PR increased from 10% at below 1 × 106/ml to 14% at over 10 × 106/ml (P < 0.01) (Table 2). The mean ALH was 3.17 µm before migration and 4.30 µm after migration (Table 3), and no association was found with the PR (before migration and after migration). When analysing the effect of age and semen characteristics together (Table 4), the pregnancy chance was significantly decreased for women aged 38 years or more (OR = 0.67, 95% CI = 0.56–0.80), and not significantly decreased with male age, reaching almost significant levels for age ≥45
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Article - Parental age and IUI outcome - S Belloc et al. Table 1. Pregnancy and miscarriage rates in intrauterine insemination cycles according to paternal and maternal ages. Cycles n %
Clinical Miscarriages pregnancies n %/cycle n %/pregnancy
Paternal age (years) <30 950 9.5 117 12.3 16 30–34 3551 35.4 484 13.6 81 35–39 2735 27.3 366 13.4 58 40–44 1631 16.3 178 10.9 58 ≥45 1167 11.6 108 9.3 35 Maternal age (years) <30 1502 15.3 216 14.4 24 30–34 3702 37.8 536 14.5 88 35–37 1911 19.5 241 12.6 52 38–41 1731 17.7 173 10.0 49 ≥42 947 9.7 84 8.9 39
13.7 16.7 15.8 32.6 32.4 11.1 16.4 21.6 28.3 46.4
Pregnancy and miscarriage rates were significantly different between the <30 and ≥42 years age groups for both maternal and paternal age (both P < 0.001).
Figure 1. Clinical pregnancy rates (PR) and miscarriage rates (MR) in intrauterine insemination cycles according to maternal age.
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Figure 2. Clinical pregnancy rates (PR) and miscarriage rates (MR) in intrauterine insemination cycles according to paternal age.
Figure 3. Clinical pregnancy rates (PR) and miscarriage rates (MR) in intrauterine insemination cycles in relation to morphological anomalies. RBMOnline®
Article - Parental age and IUI outcome - S Belloc et al. Table 2. Clinical pregnancy rates related to sperm characteristics at the time of insemination. Cycles Pregnancies P-value n n %
Sperm concentration (× 106/ml) <5.0 412 43 10.4 5.0–9.9 742 85 11.5 10.0–14.9 916 103 11.2 15.0–19.9 875 108 12.3 20.0–49.9 3784 510 13.5 ≥50.0 2992 395 13.2 Raw motility (A+B+C) (%) <20 641 68 10.6 20–29 1712 184 10.7 30–39 1682 217 12.9 40–59 3339 433 13.0 60–69 1308 204 15.6 ≥70 1089 141 13.0 Type A motility (%) <10 473 48 10.1 10–14 728 87 12.0 15–19 824 94 11.4 20–34 2937 358 12.2 35–49 2186 279 12.8 50–59 1250 194 15.5 ≥60 1342 183 13.6 Number of motile spermatozoa for insemination (×106) <1.0 689 69 10.0 1.0–1.4 450 50 11.1 1.5–3.4 1274 143 11.2 3.5–9.9 2496 328 13.1 10.0–14.9 1866 264 14.1 15.0–19.9 2834 378 13.3 ≥20.0 134 14 10.4 Abstinence (days) 0 234 32 13.7 1 802 94 11.7 2 2320 306 13.2 3 2941 389 13.2 4 1453 160 11.0 5 891 129 14.5 6 341 41 12.0 ≥7 774 96 12.4
0.02
0.01
0.01
0.01
NS
NS = not statistically significant.
Table 3. Characteristics of raw and processed samples at the time of insemination. No pregnancy P-value
Pregnancy
Abstinence (days) Volume (ml) Number of motile spermatozoa in the insemination sample (×106) ALH before processing (µm) ALH after processing (µm)
3.44 ± 2.24 3.48 ± 2.46 3.11 ± 1.62 3.11 ± 2.03 10.4 ± 6.4 9.8 ± 6.8
NS NS 0.01
3.17 ± 0.67 3.16 ± 0.68 4.31 ± 0.82 4.30 ± 0.81
NS NS
Values are mean ± SD, unless otherwise stated. ALH = amplitude of lateral head displacement; NS = not statistically significant.
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Article - Parental age and IUI outcome - S Belloc et al. Table 4. Multivariate statistical analysis of the main parameters influencing pregnancy and miscarriage rates in intrauterine insemination cycles (logistic model). Variable
Pregnancy Odds 95% CI ratio
Miscarriage Odds 95% CI ratio
Woman’s age (years) Man’s age (years) Sperm vitality (%) A type motility (%) Flagellum anomalies (%)
1.00 0.88 0.67 1.00 0.90 0.81 0.98 1.00 0.87 0.81 0.89
1.00 1.5 2.31 1.00 1.71 1.75 0.74 1.00 0.94 0.50 0.96
<35 35–37 ≥38 <35 35–44 ≥45 ≥65 ≥30 10–29 <10 ≥15
− 0.75–1.03 0.56–0.80 − 0.75–1.09 0.65–1.02 0.83–1.15 − 0.76–0.99 0.61–1.07 0.79–1.01
− 1.00–2.12 1.58–3.38 − 1.14–2.54 1.06–2.90 0.49–1.12 − 0.68–1.29 0.22–1.13 0.71–1.30
CI = confidence interval.
years (OR = 0.81; 0.65–1.02). There was no significant effect of the semen characteristics entered in the model. The miscarriage risk was increased with age, for both members of the couple, being more than double for women older than 38 years (OR = 2.31, 1.58–3.38) and almost double for men older than 45 years (OR = 1.75, 1.06–2.90), independently of women’s age. Here also, the semen characteristics entered in the model were not significantly associated with an increased risk.
Discussion The data clearly show that paternal and maternal ages affect the outcome in the IUI programme. The impact of female age on miscarriage rate is common knowledge, and it is generally admitted that the deleterious effect is related to the decreased quality of the oocyte (both nuclear and cytoplasmic), rather than the uterine quality. Semen quality, in terms of World Health Organization parameters, has an obvious impact on IUI outcome, as classically observed (Dickey et al., 1999; Badawy et al., 2008). The morphology has a negative although not significant impact, this is due to the severe limit set for morphology in this IUI programme; below a threshold of 25–30% normal forms, IUI has very low chances of success (Badawy et al., 2008).
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More surprising is the highly significant influence of male age on miscarriage rates. The number of attempts that should be made before moving to more sophisticated assisted reproduction techniques is always a question mark (Custers et al., 2008); the present observations are in favour of shortening the time spent on IUI cycles. It surely depends on maternal age, but also paternal age as demonstrated here. It is always tempting after IUI failures to move on to IVF or ICSI (Oehninger and Gosden, 2002). The paternal effect seems to be overcome by ICSI (Aboulgar et al., 2007). There is some evidence that ICSI may help in resolving the problem of sperm DNA fragmentation (Bungum et al., 2004, 2007). If one considers DNA decay, ‘normal’, in-vitro or in-vivo fertilization implies a burst of reactive oxygen species (ROS) concomitant with the capacitacion process (De Lamirande and Gagnon, 1995);
this may increase decay in sperm DNA. ICSI bypasses this specific risk, but first, not all the decay is fragmentation related (Badouard et al., 2008) and second, ICSI is not totally without risk (Morozumi and Yanagimachi, 2005; Ménézo, 2006; Fernández-Gonzalez et al., 2008) and is certainly not a panacea (Oehninger and Gosden, 2002). In fact, the human oocyte has a complete, but finite, capacity for DNA repair (Ménézo et al., 2007b), even in the best maternal conditions (Frydman et al., 2007). Moreover, mRNA expression of the genes involved decreases with maternal age (Hamatani et al., 2004). Incorrect DNA repair, i.e. tolerance of sperm DNA damage by the oocyte, may have serious deleterious effects in the offspring, including childhood cancers (Aitken and Baker, 2006; Fernández-Gonzalez et al., 2008). It is thus important to minimize sperm DNA decay if possible. It has therefore been tempting to try to reduce ROS-linked DNA fragmentation by treatment of patients with anti-oxidant (Agarwal et al., 2004). However, the wide discrepancy observed in the scientific literature with regard to antioxidant treatments is linked to the negative effect of antioxidants on sperm condensation; an excessively high level of antioxidants can impair the normal reduction of cysteine to cystine, which prevents the protamine cross-linking required for DNA condensation. This results in weak, abnormal sperm DNA condensation, which increases the risk of DNA damage, leading to an increased risk of chromosomal anomalies in the embryo after fertilization (Ménézo et al., 2007a) In conclusion, a strict check of the spermatozoa is mandatory before all assisted reproduction treatment. Passage from IUI to ICSI must not be automatic. Sperm DNA fragmentation and decondensation, which have only a weak correlation with classical sperm analysis, must be performed after failed IUI cycles, before ICSI. Intracytoplasmic injection of morphologically selected sperm (IMSI), based on motile sperm organellar morphology examination (MSOME) is perhaps an alternative way to avoid the deleterious effect of injecting highly fragmented spermatozoa (Berkovitz et al., 2005; Hazout et al., 2006). A better selection of ICSI indications is needed (Oehninger and Gosden, 2002), taking into account that paternal DNA decay, RBMOnline®
Article - Parental age and IUI outcome - S Belloc et al. increasing with age, has to be repaired by the oocyte, which has a finite capacity for repair, and whose quality decreases with age.
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Declaration: The authors report no financial or commercial conflicts of interest. Received 6 March 2008; refereed 6 May 2008; accepted 9 July 2008.
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