Jacqueline Mandelbaum

In Vitro Fertilization May Increase the Risk of Beckwith-Wiedemann Syndrome Related to the Abnormal Imprinting of the KCNQ1OT Gene

To the Editor: “Parental imprinting” refers to an epigenetic marking of genes that results in monoallelic expression. This phenomenon plays a critical role in embryogenesis and development. The epigenetic modification of the genome involves methylation changes and the remodeling of chromatin-associated proteins (Li 2002). Imprints are established during the development of germ cells, and the reprogramming of imprinting occurs within the first days after fertilization (Reik and Walter 2001). The alteration of normal imprinting patterns is implicated in a number of human genetic diseases. Among them, the Beckwith-Wiedemann syndrome (BWS [MIM 130650]) is an overgrowth syndrome secondary to the dysregulation of the imprinted 11p15 region (Maher and Reik 2000). Numerous mechanisms are involved in BWS, and ∼70% of cases of BWS are related to epigenetic abnormalities at the 11p15 locus, mostly demethylation of the KvDMR1 region of the KCNQ1OT (previously called “LIT1”) gene (MIM 604115) (Engel et al. 2000; Bliek et al. 2001; Gaston et al. 2001; Weksberg et al. 2001; DeBaun et al. 2002). KCNQ1OT encodes a noncoding antisense transcript within intron 10 of the KCNQ1 gene (MIM 192500) (Lee et al. 1999; Mitsuya et al. 1999; Smilinich et al. 1999) and might be involved in the regulation of parental imprinting of the centromeric domain of the 11p15 region (Fitzpatrick et al. 2002). In sheep and cattle, epigenetic abnormalities have been shown to be involved in large offspring syndrome (LOS) (Young et al. 1998). Affected animals exhibit various phenotypes, including large size at birth. In both species, the syndrome is caused by the in vitro exposure of embryos, between fertilization and the blastocyst stage, to various unusual environments. LOS is related to the loss of imprinting of the IGF2 receptor gene (MIM 147280), which ensures internalization and degradation of IGF2 and displays an antiproliferative function (Young et al. 2001). In vitro preimplantation procedures in mice are also responsible for overgrowth, owing to the abnormal expression of various imprinted genes, particularly the genes located at distal chromosome 7 (h19 [MIM 103280] and igf2 [MIM 147470] genes), orthologous to the human 11p15 region (Humpherys et al. 2001; Rideout et al. 2001). In humans, a case of BWS was recently described after in vitro fertilization (IVF) (Olivennes et al. 2001). Moreover, two recent papers (DeBaun et al. 2003; Maher et al. 2003) described an increase in prevalence of assisted reproductive technologies (ART) in patients with BWS. De Baun et al. (2003) reported a sixfold increase (4.6% vs. 0.76% in the general population) and showed that four of the six patients for whom DNA was available exhibited an isolated demethylation of KvDMR1 in the KCNQ1OT gene. Maher et al. (2003) reported a threefold increase (4% vs. 0.997% in the general population) and demonstrated that two of the six patients on whom molecular analysis could be done also exhibited an isolated demethylation of KvDMR1. Our department is a reference center in France for molecular diagnosis of BWS, and patients are referred from various medical departments (neonatology, pediatrics, genetics, and fetopathology). We studied a series of 149 patients referred for overgrowth syndromes and diagnosed as BWS, since all of them exhibited genetic or epigenetic defects at the 11p15 locus. According to the inclusion criteria described elsewhere (Gaston et al. 2001), 102 patients exhibited a complete form of BWS, and 47 exhibited an incomplete form of BWS. The techniques used to analyze the 11p15 region have been described elsewhere (Gaston et al. 2000, 2001). Epigenetic changes concerned 104 (70%) patients, most of whom (n=90) exhibited a loss of KvDMR1 methylation. Fourteen patients (9.4%) exhibited isolated hypermethylation of the H19 gene. Forty-two patients exhibited a genetic defect: 11p15 uniparental disomy (n=35; 23.5%) and germline CDKN1C (MIM 600856) mutation (n=7; 4.7%). Three patients (2%) had a chromosomal abnormality. Six of the 149 patients were born following ART. Of note, these six patients exhibited the same epigenetic abnormality (isolated demethylation of KvDMR1 with a demethylation index varying 72%–100%) (fig. 1). All of them were sporadic cases, and one was a DZ twin. The clinical features of these patients and the procedures of ART used for their conception are summarized in table 1. As shown in table 1, the phenotypes of patients born after ART were not different from phenotypes of the other patients (n=84) with isolated demethylation of KVDMR1, with the exception that only one patient born after ART exhibited ear abnormalities. These children were issued from various ART procedures: classical IVF, intracytoplasmic sperm injection (ICSI), embryo freezing, and transfer on day 2, day 3, and day 5. More recent procedures, like ICSI (two of six patients) or blastocyst transfer (one of six patients), did not prevail over other techniques. The representation of ART (4%) in our series is three times higher than that in the general population (1.3%), according to the national report of the French Ministry of Health (9,930 of 770,000 live births resulting in 1,999 from IVF, ICSI, or frozen embryo transfer). On the basis of this report, we would have expected 1.94 of the 149 patients with BWS to be born as a result of ART. To test the significance of this difference of frequencies, we used the Fisher’s exact test (P=.01) as well as the Poisson approximation (two-tailed P=.018; 95% CI 1.5–8.7). Strength of the association between exposure to ART and risk of BWS is expressed by an odds ratio of 3.2 (95% CI 1.4–7.3). This rate is the same as that described by Maher et al. (2003) but lower than that described by DeBaun et al. (2003), which addressed a prospective study. In our series and in Maher’s series, this rate is probably underestimated, as specific questions regarding ART have only been asked systematically in the past year. Figure 1 Methylation analysis of KvDMR1 in liver tissue (patient 131) and leukocytes (patients 15, 94, 98, 115, and 137) from the six patients with BWS born after ART and in leukocytes from a normal control (C). Genomic DNA was digested with BamHI and the methylation-sensitive ... Table 1 Clinical Characteristics of the Six Patients with BWS Born Following ART Although the analysis of the imprinting status at chromosome 15q11-13 in children born after ICSI did not reveal an imprinting defect (Manning et al. 2000), two recent papers reported three patients with Angelman syndrome (MIM 105830) born after ICSI (Cox et al. 2002; Orstavik et al. 2002). All three patients exhibited an imprinting defect, which is a rare cause of Angelman syndrome. As in the previous two reports (DeBaun et al. 2003; Maher et al. 2003), our data suggest that ART may favor imprinting alterations at the centromeric imprinted 11p15 locus and, consequently, the incidence of BWS. These data highlight the need to carefully follow up children born after ART to test for BWS and other diseases related to imprinted regions. Although no specific procedures of ART appear to be associated with a risk of BWS in our series, these data lend support to the importance of precisely recording these different procedures of ART, particularly the stimulation protocol, the biological technique, the stage of maturation of the gametes, the culture media used at each step, and the timing of embryo transfer.

Human Male Infertility Associated with Mutations in NR5A1 Encoding Steroidogenic Factor 1

One in seven couples worldwide are infertile, and male factor infertility accounts for approximately 30%-50% of these cases. Although many genes are known to be essential for gametogenesis, there are surprisingly few monogenic mutations that have been conclusively demonstrated to cause human spermatogenic failure. A nuclear receptor, NR5A1 (also called steroidogenic factor 1), is a key transcriptional regulator of genes involved in the hypothalamic-pituitary-steroidogenic axis, and it is expressed in the steroidogenic tissue of the developing and adult human gonad. Mutations of NR5A1 have been reported in 46,XY disorders of sex development and in 46,XX primary ovarian insufficiency. To test the hypothesis that mutations in NR5A1 cause male infertility, we sequenced NR5A1 in 315 men with idiopathic spermatogenic failure. We identified seven men with severe spermatogenic failure who carried missense mutations in NR5A1. Functional studies indicated that these mutations impaired NR5A1 transactivational activity. We did not observe these mutations in more than 4000 control alleles, including the entire coding sequence of 359 normospermic men and 370 fertile male controls. NR5A1 mutations are found in approximately 4% of men with otherwise unexplained severe spermatogenic failure.

Influence of sickle cell disease and treatment with hydroxyurea on sperm parameters and fertility of human males

The use of hydroxyurea has considerably modified the prognosis of sickle cell disease and many more patients now reach reproductive age. This study shows alterations of semen parameters due to sickle cell disease that seem to be exacerbated by hydroxyurea treatment. The authors suggest that a pre-treatment sperm analysis be performed and sperm cryopreservation be offered to patients before hydroxyurea treatment. Background Recent progress in the treatment of sickle cell disease, in particular the use of hydroxyurea, has considerably modified the prognosis of this disease. Many more patients now reach reproductive age. The objective of this study was to assess the potential impact of hydroxyurea on the semen of patients. Design and Methods In this retrospective multicenter study, we evaluated the sperm parameters and fertility of 44 patients and analyzed the potential impact of hydroxyurea. Results We report data from the largest series so far of semen analyses in patients with sickle cell disease: 108 samples were analyzed, of which 76 were collected before treatment. We found that at least one sperm parameter was abnormal in 91% of the patients before treatment, in agreement with published literature. All sperm parameters seemed to be affected in semen samples collected during hydroxyurea treatment, and this impairment occurred in less than 6 months, later reaching a plateau. Furthermore, after hydroxyurea cessation, while global results in 30 patients were not statistically different before and after hydroxyurea treatment, in four individuals follow-up sperm parameters did not seem to recover quickly and the total number of spermatozoa per ejaculate fell below the normal range in about half the cases. Conclusions The observed alterations of semen parameters due to sickle cell disease seem to be exacerbated by hydroxyurea treatment. Until prospective studies reveal reassuring findings, we suggest that a pre-treatment sperm analysis be performed and sperm cryopreservation be offered to patients before hydroxyurea treatment.

Y-chromosome AZFc structural architecture and relationship to male fertility

OBJECTIVE To determine if there is a relationship between various forms of partial AZFc deletions and spermatogenic failure. DESIGN Case-control study. SETTING Infertility clinic (Tenon Hospital, Paris). PATIENT(S) 557 men, comprising 364 infertile men from mixed ethnic backgrounds, and 193 men with known fertility (n = 84) and/or normospermic (n = 109). INTERVENTION(S) Characterization of 32 partial AZFc deletions. MAIN OUTCOME MEASURE(S) DAZ gene cluster divided into two families (DAZ1/2 and DAZ3/4), CDY1 gene, and Y-chromosome haplogroups. RESULT(S) We observed 18 partial AZFc deletions in 364 (4.95%) infertile men compared with 14 out of 193 (7.25%) in the control normospermic/fertile group. CONCLUSION(S) The analysis of informative Y-chromosome single nucleotide variants combined with Y-chromosome haplogroup definition enabled us to infer seven deletion classes that occur on a minimum of six Y-chromosome parental architectures. We found no relationship between either the presence or the absence of DAZ1/2, DAZ3/4, CDY1a, or CDY1b with spermatogenic failure at least on one Y-chromosome lineage. The DAZ dosage and Southern blot analyses indicated that the majority of individuals tested carried two copies of the DAZ gene, indicating a partial AZFc deletion. Our data are consistent with the hypothesis that, at least in our study populations, partial AZFc deletions may have a limited impact on fertility.

Learning curve of vitrification assessed by cumulative summation test for learning curve (LC-CUSUM)

We used the cumulative summation test for learning curve (LC-CUSUM), a specifically designed statistical tool, to evaluate the first 50 procedures performed by a trainee in vitrification and to provide a usable model for monitoring the learning process of this technique. Given the lack of models to evaluate IVF technologies, the CUSUM methodology could prove useful for quality control in laboratories.

Hormonal secretions in singleton pregnancies arising from the implantation of fresh or frozen embryos after oocyte donation in women with ovarian failure

Objective To determine whether levels of human chorionic gonadotropin (hCG), 17 β -estradiol (E 2 ), and progesterone (P) are different in the peri-implantation phase of fresh versus frozen embryos. Design Hormonal secretions were measured on days 9 and 11 after implantation and at 4, 5, and 6 weeks gestation. Patients Thirty-one pregnancies were achieved in 65 patients with ovarian failure. Seventeen singleton pregnancies developed after implantation of 4 frozen and 13 fresh embryos. Results Human chorionic gonadotropin and E 2 , contrary to P, were higher in cases of fresh embryos from the 9th day after transfer to the 5th week at which time they become statistically significant (respectively, for hCG and E 2 , 5,800.3 ± 332.3 versus 2,027.3 ± 916.3 [mean ± SD] mIU/mL for hCG and 562.3 ± 215 versus 291 ± 152 pg/mL for E 2 ). Conclusions This difference might be explained by either the higher number of fresh embryo replaced or by the fact that the number of blastomeres and also their metabolic activity could be reduced after freezing and thawing.

Adverse effect of hydroxyurea on spermatogenesis in patients with sickle cell anemia after six months of treatment

To the editor: The prognosis of sickle cell anemia (SCA), initially disastrous in the severe forms of the disease, has greatly improved, leading to increased life expectancy. More than 95% to 99% of the children diagnosed with SCA in developed countries will become young adults, reaching