Male infertility and increased risk of diseases in future generations

that there was no evidence of an excess of major birth defects among ICSI offspring, whereas the independent reviewers concluded that there was a two-fold excess. Mitchell 3 in an accompanying editorial suggested this discrepancy arose as a result of different definitions and methods of ascertainment of birth defects. Mitchell also discussed apparent differences between an expansion of the original 423 ICSI offspring to 877,4 and a further series of 578 ICSI offspring conceived in New York, and concluded that different methods of ascertainment of birth defects could explain a large element of the apparent discrepancy in the risk estimates of major birth defects. Therefore, methods of ascertainment of birth defects in ICSI and control offspring should be comparable, and the sample sizes should be sufficiently large to ensure results may be interpreted with confidence. Others have strongly advocated further large scale follow-up of ICSI offspring.5 To address the uncertainty that remains, carefully designed epidemiological systems to monitor these offspring should be established so that potential parents may consider the evidence of risk in a more informed way than is possible at present. An important step towards this objective is the establishment of a national register of offspring conceived by ICSI. With the encouragement of the British Fertility Society and the guidance of an Advisory Group we plan to investigate the risks of adverse health outcomes among offspring conceived by ICSI and compare them with those of offspring conceived by conventional IVF, and with those of offspring conceived naturally in the general population. We already have the cooperation of almost every infertility clinic in the UK that carries out ICSI. *M M Hawkins, C L R Barratt, A G Sutcliffe, I D Cooke *Centre for Childhood Cancer Survivor Studies, Department of Public Health and Epidemiology, University of Birmingham, Birmingham B15 2TT, UK; Reproductive Biology and Genetics Group, Department of Obstetrics and Gynaecology, Birmingham Women’s Hospital, Birmingham; Royal Free and University College Medical School, University College London; and Department of Obstetrics and Gynaecology (Jessop), Division of Surgical and Anaesthetic Sciences, Jessop Hospital for Women, Sheffield,UK Dowsing AT, Yong EL, Clark M, et al. Linkage between male infertility and trinucleotide repeat expansion in the androgen-receptor gene. Lancet 1999; 354: 640–43. 2 Kurinczuk JJ, Bower C. Birth defects in infants conceived by intracytoplasmic sperm injection: an alternative interpretation. BMJ 1997; 315: 1260–65. 3 Mitchell AA. Intracytoplasmic sperm injection: offering hope for a term pregnancy

and a healthy child? BMJ 1997; 315: 1245–46. 4 Bonduelle M, Wilikns A, Buysse A, et al. Prospective follow-up study of 877 children born after intracytoplasmic sperm injection (ICSI), with ejaculated epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI. Hum Reprod 1996; 11 (suppl 4): 131–55. 5 te Velde ER, van Baar AL, va Kooij RJ. Concerns about assisted reproduction. Lancet 1998; 351: 1524–25.

Sir—Aneta Dowsing and colleagues1 propose a relation between the length of the poly-glutamine stretch (CAG repeat) in the human androgen receptor and the risk of defective spermatogenesis based on the study of 35 infertile men and 32 controls. They make the assumption that transmission of elongated CAG repeats through assisted reproductive techniques may lead to an increased risk of spinal and bulbar muscular atrophy (Kennedy’s disease) in future generations. We have investigated a cohort of 180 white patients aged 20·9 to 54·4 years who were referred between 1993 and 1997 from the Department of Andrology at the University Hospital Hamburg-Eppendorf. Clinical examinations excluded overt virilisation disorders and other genetic, endocrine, infectious, or obstructive causes of male infertility. Ejaculates were investigated according to WHO guidelines.2 110 patients showed oligoteratozoospermia (<106 sperm/mL ejaculate, <30% normal forms), 57 had teratozoospermia (30% normal sperm), and 13 patients had azoospermia. 53 white men served as controls, all of whom had fathered at least one child. Informed consent was obtained from all individuals and EDTA-blood samples were taken for DNA analysis of the human androgen receptor gene. PCRgenerated amplification products containing the variable CAG-repeat region were electrophoresed on nondenaturing polyacrylamide gels with

adequate size markers and visualised with silver staining. 3 Additionally, selected samples were sequenced to assess the exact number of CAG repeats, and these control samples were included in electrophoresis. CAG repeat sizes were determined by a computerised analysis system (ImageMaster, Pharmacia, Freiburg, Germany). In the patient group, the mean CAG repeat length was 23 repeats (range 13–30), whereas in the control group the mean was 24 repeats (17–39; figure). Wilcoxon and Mann-Whitney tests were used for statistical analysis and showed no significant differences between the two groups (p=0·471). This finding agrees with Giwercman and colleagues’ report on the CAG repeat length in a Swedish population of 33 infertile men.4 Thus, we cannot confirm the assumptions of Dowsing and colleagues and conclude that polyglutamine repeat expansion within the androgen-receptor gene in infertile men does not play a significant part in male infertility. This study was supported by grants from the German Ministry of Education and Research (BMBF 01KY9301/1 to OH and GHGS) and German Research Foundation (DFG Hi 497/3-3 to OH).

*Olaf Hiort, Thorsten Horter, Wolfgang Schulze, Britta Kremke, Gernot H G Sinnecker *Departments of Paediatrics, Medical University of Lübeck, D-23538 Lübeck, Germany; City Hospital, Wolfsburg; Children’s Hospital, Hamburg-Altona; and Department of Andrology, University Hospital, Hamburg-Eppendorf (e-mail: [email protected]) 1

2

Dowsing AT, Young EL, Clark M, McLachlan RI, de Kretser DM, Trounson AO. Linkage between male infertility and trinucleotide repeat expansion in the androgen-receptor gene. Lancet 1999; 354: 640–43. WHO. WHO laboratory manual for the examination of human semen and spermcervial mucus interaction, 3rd edn. Cambridge: Cambridge University Press, 1992.

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Hiort O, Sinnecker GHG, Holterhus PM, Nitsche EM, Kruse K. The clinical and molecular spectrum of androgen insensitivity. Am J Med Genet 1996; 63: 218–22. Giwercman YL, Xu C, Arver S, Pousette A, Reneland R. No association between the androgen receptor gene CAG repeat and impaired sperm production in Swedish men. Clin Genet 1998; 54: 435–36.

Hormone replacement therapy and C-reactive protein Sir—Naveed Sattar and colleagues (Aug 7, 487)1 report a reduction in Creactive protein (CRP) concentrations after 6 months of combined hormone replacement therapy (HRT) in postmenopausal women with type 2 diabetes mellitus. Several aspects of their study need to be addressed. The investigators choose a combined rather than an oestrogen-only HRT preparation, which is surprising since there is substantial evidence that progestagens attenuate the beneficial cardiovascular effects of oestrogen.2 This may be the reason why the beneficial effects on coronary heart disease events in observational studies were not confirmed in the HERS trial 3 in postmenopausal women. Furthermore, Sattar and colleagues did not correlate concentrations of each hormonal component with that of CRP, which may have been more valuable. Measurement of serum hormonal concentrations is important, particularly because absorption of oestrogen is more variable by the transdermal route compared with the oral route. No data on body mass index were given. In vivo, adipose tissue secretes interleukin 6,4 a cytokine that plays a key part in the regulation of CRP production.4 Matching body mass index of women with type-2 diabetes with that of controls is therefore essential. To state disease duration is misleading because type-2 diabetes commonly has an unclear onset. It would be much more valuable to state the presence or absence of diabetic microangiopathy, which correlates closely with increase in CRP and other acute-phase proteins.5 N N Chan EURODIAB, University College London, London WC1E 6BT, UK (e-mail: [email protected]) 1

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Sattar N, Perera M, Small M, Lumsden M-A. Hormone replacement therapy and sensitive C-reactive protein concentrations in women with type-2 diabetes. Lancet 1999; 354: 487–88. Lobo RA. The role of progestins in hormone replacement therapy. Am J Obstet Gynecol 1992; 166: 1997–2004.

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Hulley S, Grady D, Bush T, et al. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Heart and estrogen/progestin Replacement Study (HERS) Research Group. JAMA 1998; 280: 605–13. 4 Yudkin JS, Stehouwer CD, Emeis JJ, Coppack SW. C-reactive protein in healthy subjects: associations with obesity, insulin resistance, and endothelial dysfunction: a potential role for cytokines originating from adipose tissue? Arterioscler Thromb Vasc Biol 1999; 19: 972–78. 5 McMillan DE. Increased levels of acutephase serum proteins in diabetes. Metabolism 1989; 38: 1042–46.

Authors’ reply Sir—N N Chan questions our choice of a combined rather than oestrogen-only preparation and suggests that progestagens attenuate the reported beneficial cardiovascular effects of oestrogen. Chan also asks whether women with type 2 diabetes mellitus randomly assigned HRT and placebo in our study were matched for BMI and whether we examined changes in CRP concentrations with respect to the separate components of combined HRT. Since all women participating in our study had an intact uterus, the addition of a progestagen was required for 6 months to prevent endometrial hyperplasia. Indeed, this was a specific feature of study design so that our findings would be generalisable to most women with type 2 diabetes. We are less convinced that all progestagens attenuate the apparent cardiovascular benefits of oestrogen. Indeed the reverse may be true in some cases. Two studies1,2 have reported increased (rather than decreased) CRP concentrations during HRT on the basis of oral oestrogens with or without non-androgenic progestagens. The investigators of these reports argue that proinflammatory effects of HRT might explain the increased number of cardiovascular events during the first year in women with existing cardiovascular disease seen in the HERS trial. Increases in CRP concentrations are thought to indicate increased (hepatic) synthesis rather than decreased clearance. We propose that the route of delivery of oestrogen and the relative androgenicity of the progestagen administered may account for apparent discrepancies between studies in the direction of change in CRP during HRT. In our study, the oestrogen component was delivered transdermally (not orally), thus obviating any proinflammatory effects dependent on first-pass hepatic metabolism. Moreover, norethisterone (the progestagen used in our study) is a

19-nortestosterone progestagen with androgenic properties. This is noteworthy since progestagens and androgens display anti-inflammatory effects in other tissues3 and may well have similar effects on the liver. Interestingly, triglyceride concentrations are reduced by oral norethisterone and increased by oral oestrogens. 4 In support of a net progestagenic/androgenic hepatic effect in our study, triglyceride concentrations were significantly reduced. The groups assigned HRT and placebo were matched by BMI at baseline (means 31·0 vs 31·9 kg/m2). We are aware of the limitations of disease duration as a reported characteristic of people with type 2 diabetes, but it is difficult to see how this could alter our findings. We could not correlate changes in CRP with each hormonal component because both drugs were given continuously. A large multicentre trial would be required to confirm whether an HRT regimen that reduces inflammation (such as the one used in our study) would avoid the early increase in cardiovascular events observed in HERS. If so, a regimen based on continuous transdermal oestradiol and oral norethisterone might be more desirable for postmenopausal women with diabetes or existing coronary heart disease. In the meantime, our reading of the existing evidence is that oral oestrogens increase whereas oral norethisterone decreases CRP concentrations. *Naveed Sattar, John R Petrie, Mahesh Perera, Michael Small, Mary-Ann Lumsden *Department of Pathological Biochemistry, Glasgow Royal Infirmary University NHS Trust, Glasgow G31 2ER, UK; Department of Medicine and Therapeutics, Western Infirmary, Glasgow; Department of Diabetes, Gartnavel General Hospital, Glasgow; and Departments of Obstetrics and Gynaecology, Queen Mothers Hospital, Glasgow (e-mail: [email protected]) 1

Cushman M, Legault C, Barrett-Connor E, et al. Effect of postmenopausal hormones on inflammation-sensitive proteins: the Postmenopausal Estrogen/Progestin Interventions (PEPI) study. Circulation 1999; 100: 717–22. 2 Ridker PM, Hennekens CH, Rifai N, Buring JE, Manson JE. Hormone replacement therapy and increased plasma concentration of C-reactive protein. Circulation 1999; 100: 713–16. 3 Parkar M, Tabona P, Newman H, Olsen I. IL-6 expression by oral fibroblasts is regulated by androgens. Cytokine 1998; 10: 613–19. 4 Farish E, Spowart K, Barnes JF, et al. Effects of postmenopausal hormone replacement therapy on lipoproteins including lipoprotein(a) and LDL subfractions. Atherosclerosis 1996; 126: 77–84.

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