Aitken, R. J.

Cram, D.

Krausz, Csilla Gabriella

McLachlan, R. I.

O&apos

English

Krausz, CSILLA GABRIELLA

Florence Research

Thomas BrucknerInstitute of Medical Biometry and InformaticsUniversity of HeidelbergHeidelberg, GermanyVanderleyen BiolchiAmal Alia, M.D.Thomas Strowitzki, M.D.Department of Gynecological Endocrinology andReproductive MedicineWomen’s University HospitalUniversity of HeidelbergHeidelberg, GermanyMay 22, 2008REFERENCE1. Gutsche S, von Wolff M, Strowitzki T, Thaler CJ. Seminal plasma inducesmRNA expression of IL-1beta, IL-6 and LIF in endometrial epithelialcells vitro. Mol Hum Reprod 2003;9:785–91.doi:10.1016/j.fertnstert.2008.05.083Need for standardization and confirmation of STSdeletions on the Y chromosomeTo the Editor:As clinical investigators and scientists in the Y chromo-some deletion field, we wish to express our concern aboutthe study by Feng et al. (1) that purports to show a signifi-cantly increased risk of de novo Y chromosome deletionsin children conceived by assisted reproductive technology(ART). We believe it has serious technical flaws and willserve to confuse and concern the readership.First, in terms of patient selection, it is remarkable that the37 ART fathers had the same semen quality as the controlgroup with a mean sperm density of 91.5 million/mL. Suchan inclusion criterion is at variance with worldwide ARTpractice and yet passes without comment.Most critically, we question the performance of the Y dele-tion analysis. Overdetection (false positives) is a major issue inY chromosome deletion studies, and for this reason there havebeen significant international efforts to provide a quality insur-ance program (2). Their brief method section suggests that theauthors have not participated in any quality control program.These authors have published previously (3) a staggering33% incidence of Y chromosome deletions in idiopathic non-obstructive azoospermia. This is many fold higher than thatreported by others using appropriately stringent methodsfor Yq deletion detection (4) and serves to raise questionsabout the molecular methods used to define Yq deletionsand the overall validity of the data. Independent confirmationof their data should have been sought.We also note their claim that two of the four deletions werelimited to the AZFa region. The authors (and the referee) areFertility and Sterilityclearly not aware of the limits of the AZFa region because theSTS marker sY82 is erroneously indicated as being ‘‘in theAZFa region.’’ Moreover, the four deletions found by the au-thors were all single STS deletions, which require furtherconfirmation (at least with a few contiguous markers) giventhat dealing with STS plus/minus analysis there is a highprobability of generating polymerase chain reaction (PCR)artifacts. In addition, given that the RNA recognition motif1 is present several times along the Y chromosome, the likeli-hood of finding an isolated deletion for this specific marker isnegligible. The European Academy of Andrology (EAA)Guidelines (2) for the valid detection of AZF deletions con-tain all the relevant methodological and clinical information,including the need for a reliable set of primers and the needfor confirmation of results.Finally, the authors state that ‘‘[t]he incidence of microde-letion was higher in children conceived via ICSI than in thoseconceived by IVF (3:18 vs. 1:19),’’ and yet they go on to saythat this was not statistically significant. This reveals a lack ofstatistical understanding and underscores the limited powerof this small study. Several of us were involved in a previousstudy that looked in detail into this issue in a properly se-lected group of 86 severely infertile men who were thuscandidates for intracytoplasmic sperm injection (ICSI), andwe found no evidence for de novo chromosome deletions intheir 99 ICSI-conceived male offspring (5).Robert Ian McLachlan, M.D., Ph.D.Prince Henry’s Institute of Medical ResearchClayton, AustraliaR. John Aitken, Ph.D.ARC Centre of Excellence inBiotechnology and DevelopmentUniversity of NewcastleCallaghan, AustraliaDavid Cram, Ph.D.Monash Immunology and Stem Cell LaboratoriesMonash UniversityClayton, AustraliaCsilla Krausz, M.D., Ph.D.Andrology UnitDepartment of Clinical PhysiopathologyFlorence, ItalyMoira O’Bryan, Ph.D.Monash Institute of Medical ResearchMonash UniversityClayton, AustraliaMarch 25, 2008REFERENCES1. Feng C, Wang LQ, Dong MY, Huang HF. Assisted reproductive technol-ogy may increase clinical mutation detection in male offspring. FertilSteril 2008;90:92–6.463merase chain reaction (PCR) using the primers reported pre-viously; we found a section of RNA recognition motif 1(YRRM1) was deleted, but we did not mean that all the cop-ies of YRRM1 were deleted. No Y chromosome microdele-tion was found in naturally conceived offspring and theirfathers, indicating that the detected microdeletion in ARToffspring should not be a false-positive result. Of course, ifadditional confirmation had been done, the results would bemore convincing.No statistically significant difference was found in the inci-dence of microdeletion between ICSI and IVF offspring. Thismay be due to the relatively small sample size. It is true that2. Simoni M, Bakker E, Krausz C. EAA/EMQN best practice guidelines formolecular diagnosis of Y-chromosomal microdeletions; state of the art2004. Int J Androl 2004;27:240–9.3. Wang LQ, Huang HF, Jin F, Qian YL, Cheng Q. High frequency of Y chro-mosome microdeletions in idiopathic azoospermic men with high follicle-stimulating hormone levels. Fertil Steril 2005;83:1050–2.4. Krausz C, Degl’Innocenti S. Y chromosome and male infertility: update,2006. Front Biosci 2006;11:3049–61.5. Cram DS, Ma K, Bhasin S, Arias J, Pandjaitan M, Chu B, et al. Y chromo-some analysis of infertile men and their sons conceived through intracyto-plasmic sperm injection: vertical transmission of deletions and rarity of denovo mutations. Fertil Steril 2000;74:909–15.doi:10.1016/j.fertnstert.2008.05.026Reply of the Authors:The letter to the editor by Dr. McLachlan et al. expressedtheir concern about our recent study (1), which showed a sig-nificantly increased risk of de novo Y chromosome deletionsin children conceived by assisted reproductive technology(ART). We appreciate the concern from these doctors andothers, and we would like to discuss our study with them.The participants in our study were selected as follows.Couples requesting ART treatment for tubal infertility inour center in 2004 were informed about our research, andnearly 200 couples consented to take part. They were ran-domly divided into an in vitro fertilization (IVF) group andan intracytoplasmic sperm injection (ICSI) group. Thirty-seven male offspring conceived through ART constitutedthe study group. So semen quality is not a routine inclusioncriterion for ICSI.In our previously published study (2), Y-chromosome mi-crodeletions were detected in 13.2% of patients with idiopathicazoospermia. Among those patients, there were microdele-tions in 33.3% of the men with high serum levels of follicle-stimulating hormone (FSH). The generally accepted deletionfrequency for idiopathic severe oligospermic and azoospermicmen is 10% to 18%. We do not believe that the incidence wefound was higher than that found in other studies.As we have successfully performed Y chromosome micro-deletion detection in infertile men and quite a few publishedpapers have adopted similar methods, we used the samemethod to investigate microdeletions in offspring. The qualitycontrol program was as follows. A healthy man of provenfertility and a healthy woman were included as positive andnegative controls, respectively, and were run in parallel witheach reaction. Patients were considered negative only afterthree amplification failures. Because these details had beendescribed in our previous report (2), we omitted them fromthe current report. The European Academy of Andrology(EAA) Guidelines for the valid detection of AZF deletions(3) have been recommended as being more stringent, so wewill employ those methods in our future studies.Because SY82 locates at the proximal region of the AZFaregion and it is included in the AZFa region in some studies,we described it in the AZFa region. We performed poly-the presentation is not precise enough, but we were emphasiz-ing the difference in incidence of microdeletion between ARTand naturally conceived offspring, which was statistically sig-nificant. Cram et al. (4) found no de novo chromosome dele-tions in 99 ICSI-conceived male offspring (4), but it has beenreported that ICSI may lead to vertical transmission, expan-sion, and de novo Y-chromosome microdeletion (5). Ourstudy tries to show a potential risk that the incidence of micro-deletion might increased in ART-conceived offspring. We willenlarge the sample size and adopt more specific methods inour subsequent studies to confirm our results.Chun Feng, M.D.Li-Quan Wang, M.D., Ph.D.Min-Yue Dong, M.D., Ph.D.Fan Jin, Ph.D.He-Feng Huang, M.D.Department of Reproductive EndocrinologyWomen’s Hospital, School of Medicine,Zhejiang UniversityKey Laboratory of Women’s Reproductive Healthof Zhejiang ProvinceHangzhou, Zhejiang, People’s Republic of ChinaApril 17, 2008REFERENCES1. Feng C, Wang LQ, Dong MY, Huang HF. Assisted reproductive technol-ogy may increase clinical mutation detection in male offspring. FertilSteril 2008;90:92–6.2. Wang LQ, Huang HF, Jin F, Qian YL, Cheng Q. High frequency of Y chro-mosome microdeletions in idiopathic azoospermic men with high follicle-stimulating hormone levels. Fertil Steril 2005;83:1050–2.3. Simoni M, Bakker E, Krausz C. EAA/EMQN best practice guidelines formolecular diagnosis of y-chromosomal microdeletions. State of the art2004. Int J Androl 2004;27:240–9.4. Cram DS, Ma K, Bhasin S, Arias J, Pandjaitan M, Chu B, et al. Y chromo-some analysis of infertile men and their sons conceived through intracyto-plasmic sperm injection: vertical transmission of deletions and rarity of denovo deletions. Fertil Steril 2000;74:909–15.5. Lee SH, Ahn SY, Lee KW, Kwack K, Jun HS, Cha KY. Intracytoplasmicsperm injection may lead to vertical transmission, expansion, and de novooccurrence of Y-chromosome microdeletions in male fetuses. Fertil Steril2006;85:1512–5.doi:10.1016/j.fertnstert.2008.05.025464 Letters to the Editor Vol. 90, No. 2, August 2008

Need for standardization and confirmation of STS deletions on the Y chromosome.

https://flore.unifi.it/bitstream/2158/395130/1/395130mclachlan2008.pdf

Need for standardization and confirmation of STS deletions on the Y chromosome.

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