Semen quality of young adult ICSI offspring: the first results.

STUDY QUESTION: What is the semen quality of young adult men who were conceived 18-22 years ago by ICSI for male infertility? SUMMARY ANSWER: In this cohort of 54 young adult ICSI men, median sperm concentration, total sperm count and total motile sperm count were significantly lower than in spontaneously conceived peers. WHAT IS KNOWN ALREADY: The oldest ICSI offspring cohort worldwide has recently reached adulthood. Hence, their reproductive health can now be investigated. Since these children were conceived by ICSI because of severe male-factor infertility, there is reasonable concern that male offspring have inherited the deficient spermatogenesis from their fathers. Previously normal pubertal development and adequate Sertoli and Leydig cell function have been described in pubertal ICSI boys; however, no information on their sperm quality is currently available. STUDY DESIGN, SIZE, DURATION: This study was conducted at UZ Brussel between March 2013 and April 2016 and is part of a large follow-up project focussing on reproductive and metabolic health of young adults, between 18 and 22 years and conceived after ICSI with ejaculated sperm. Results of both a physical examination and semen analysis were compared between young ICSI men being part of a longitudinally followed cohort and spontaneously conceived controls who were recruited cross-sectionally. PARTICIPANTS/MATERIALS, SETTING, METHOD: Results of a single semen sample in 54 young adult ICSI men and 57 spontaneously conceived men are reported. All young adults were individually assessed, and the results of their physical examination were completed by questionnaires. Data were analysed by multiple linear and logistic regression, adjusted for covariates. In addition, semen parameters of the ICSI fathers dating back from their ICSI treatment application were analysed for correlations. MAIN RESULTS AND THE ROLE OF CHANCE: Young ICSI adults had a lower median sperm concentration (17.7 million/ml), lower median total sperm count (31.9 million) and lower median total motile sperm count (12.7 million) in comparison to spontaneously conceived peers (37.0 million/ml; 86.8 million; 38.6 million, respectively). The median percentage progressive and total motility, median percentage normal morphology and median semen volume were not significantly different between these groups. After adjustment for confounders (age, BMI, genital malformations, time from ejaculation to analysis, abstinence period), the statistically significant differences between ICSI men and spontaneously conceived peers remained: an almost doubled sperm concentration in spontaneously conceived peers in comparison to ICSI men (ratio 1.9, 95% CI 1.1-3.2) and a two-fold lower total sperm count (ratio 2.3, 95% CI 1.3-4.1) and total motile count (ratio 2.1, 95% CI 1.2-3.6) in ICSI men compared to controls were found. Furthermore, compared to men born after spontaneous conception, ICSI men were nearly three times more likely to have sperm concentrations below the WHO reference value of 15 million/ml (adjusted odds ratio (AOR) 2.7; 95% CI 1.1-6.7) and four times more likely to have total sperm counts below 39 million (AOR 4.3; 95% CI 1.7-11.3). In this small group of 54 father-son pairs, a weak negative correlation between total sperm count in fathers and their sons was found. LIMITATIONS, REASONS FOR CAUTION: The main limitation is the small study population. Also, the results of this study where ICSI was performed with ejaculated sperm and for male-factor infertility cannot be generalized to all ICSI offspring because the indications for ICSI have nowadays been extended and ICSI is also being performed with non-ejaculated sperm and reported differences may thus either decrease or increase. WIDER IMPLICATIONS OF THE FINDINGS: These first results in a small group of ICSI men indicate a lower semen quantity and quality in young adults born after ICSI for male infertility in their fathers. STUDY FUNDING/COMPETING INTERESTS: This study was supported by Methusalem grants and by grants from Wetenschappelijk Fonds Willy Gepts, all issued by the Vrije Universiteit Brussel (VUB). All co-authors except M.B. and H.T. declared no conflict of interest. M.B. has received consultancy fees from MSD, Serono Symposia and Merck. The Universitair Ziekenhuis Brussel (UZ Brussel) and the Centre for Medical Genetics have received several educational grants from IBSA, Ferring, Organon, Shering-Plough and Merck for establishing the database for follow-up research and organizing the data collection. The institution of H.T. has received research grants from the Research Fund of Flanders (FWO), an unconditional grant from Ferring for research on testicular stem cells and research grants from Ferring, Merck, MSD, Roche, Besins, Goodlife and Cook for several research projects in female infertility. H.T. has received consultancy fees from Finox, Abbott and ObsEva for research projects in female infertility.

[Genetics and male infertility]. / Genetica van de mannelijke onvruchtbaarheid.

Infertility is a problem affecting many couples with a child wish. In about half of these couples a male factor is (co-) responsible for the fertility concern. For part of these patients a genetic factor will be the underlying cause of the problems. This paper gives an overview of the studies performed in the Department of Embryology and Genetics of the Vrije Universiteit Brussel and the Centre for Medical Genetics of UZ Brussel in order to gain more insight into the genetic causes of male infertility. The studies, focusing on men with fertility problems, can be subdivided into three groups: studies on deletions on the long arm of the Y chromosome, studies on X-linked genes and studies on autosomal genes. It is obvious that Yq microdeletions should be considered as a cause of male infertility. Only for patients with a complete AZFc deletion, a small number of spermatozoa can be retrieved. However, even for these patients assisted reproductive technologies are necessary. Complete AZF deletions are found in 4.6% of the patients visiting the centres for Reproductive Medicine and Medical Genetics of the UZ Brussel and for whom no other cause of the fertility problems have been detected. Taken into consideration this low prevalence of Yq microdeletions, it is obvious that also other factors, including genetic factors, must be causing fertility problems. Potentially, gr/gr deletions (partial deletions of the AZFc region) might influence the fertility status of the patients. It remains, however, unclear which of the genes located in the deleted regions are important for the progression of spermatogenesis, in case of partial or complete AZF deletions. In our studies we have also investigated mutations in genes located on the X chromosome. In analogy to the Y chromosome, the X chromosome is interesting in view of studying male infertility since men only have a single copy of the sex chromosomes. As a consequence, mutations in genes crucial for spermatogenesis will have an immediate impact on the sperm production. The genes NXF2, USP26 and TAF7L were investigated for the presence of mutations. All observed single nucleotide changes were also present in control samples, questioning their relationship with male infertility. We also studied five autosomal genes: SYCP3, MSH4, DNMT3L, STRA8 and ETV5. Only for the genes STRA8 and ETV5, changes were detected that were absent in a control population existing of men with normozoospermia. Functional analysis of the changes in ETV5 and the localization of the change observed in STRA8 showed that also these alterations were probably not the cause of the fertility problems in these men. It can be concluded that mutations are rarely detected in men with fertility problems. This low frequency of mutations has also been confirmed in several published studies. Therefore, further research is necessary to determine the impact of genetic causes on male infertility.

CTG repeat instability in a human embryonic stem cell line carrying the myotonic dystrophy type 1 mutation.

Human embryonic stem cells (hESC) are considered to be an indefinite source of self-renewing cells that can differentiate into all types of cells of the human body and could be used in regenerative medicine, drug discovery and as a model for studying early developmental biology. hESC carrying disease-causing mutations hold promise as a tool to investigate mechanisms involved in the pathogenesis of the disease. In this report, we describe the behaviour of an expanded CTG repeat in the 3' untranslated region of the DMPK gene in VUB03_DM1, a hESC line carrying the myotonic dystrophy type 1 (DM1) mutation compared with the normal CTG repeat in two hESC lines VUB01 and VUB04_CF. Expanded CTG repeats were detected by small amount PCR, small pool PCR and Southern blot analysis in consecutive passages of VUB03_DM1. An important instability of the CTG repeat was detected during prolonged in vitro culture, showing stepwise increases of the repeat number in consecutive passages as well as a higher range of variability. This variability was present in cells of different colonies of the same passage and even within single colonies. The high repeat instability is in contrast to the previously observed stability of the repeat in preimplantation embryos and in fetuses during the first trimester of pregnancy. This in vitro culture of affected hESC represents a valuable model for studying the biology of repeat instability.

Triggering of final oocyte maturation with gonadotropin-releasing hormone agonist or human chorionic gonadotropin. Live birth after frozen-thawed embryo replacement cycles.

OBJECTIVE: To report the outcome of frozen-thawed embryo replacement cycles after GnRH-agonist triggering of final oocyte maturation in the collecting cycle with GnRH-antagonist. DESIGN: Prospective, observational, multicentric clinical study. SETTING: Tertiary university-affiliated IVF centers. PATIENT(S): Patients under observation previously had been recruited into two concurrently performed, independent, randomized controlled trials (comparing hCG with GnRH-agonist for triggering final oocyte maturation in GnRH-antagonist multiple-dose protocols in normal responder patients) encompassing a total of 228 participants. Surplus embryos or oocytes at the pronuclear stage were cryopreserved in 53 patients after hCG administration and 32 patients after GnRH-agonist administration on the basis of patient choice, pronuclear/embryo availability, and local laws. INTERVENTION(S): Transfer of frozen-thawed embryos. MAIN OUTCOME MEASURE(S): Live birth rate. RESULT(S): Thirty-one and 23 patients after administration of hCG and GnRH-agonist, respectively, started a frozen-embryo replacement cycle by September 2005, with 25 and 16 patients eventually undergoing at least one frozen-thawed ET. Live birth rate per ET was 18.5% (95% confidence interval [CI], 8.2-36.7) and 30.0% (95% CI, 14.5-51.9) after hCG and GnRH-agonist triggering, respectively. Cumulative live birth rate per patient starting a frozen-embryo replacement cycle was 16.1% (95% CI, 7.1-32.6) and 26.1% (95% CI, 12.5-46.5) for hCG and GnRH-agonist, respectively. CONCLUSION(S): The likelihood of live birth in frozen-embryo replacement cycles after GnRH-agonist triggering of final oocyte maturation does not appear to be impaired.

Preimplantation genetic diagnosis for cancer predisposition syndromes.

OBJECTIVES: Mutations in the APC, NF2 and BRCA1 genes cause adult-onset cancer predisposition syndromes. Prenatal diagnosis (PND) and selective pregnancy termination for adult-onset disorders is emotionally difficult and, in some cases, socially not well accepted. Preimplantation genetic diagnosis (PGD) appears as an attractive alternative to PND, as it ensures the establishment of a pregnancy free of the mutation from the onset, circumventing the potentially difficult decision of termination of pregnancy. METHODS: Development of single-cell PCRs using Epstein-Barr virus transformed lymphoblasts as single-cell model, followed by clinical application in PGD. RESULTS: A total of five duplex-PCRs were developed, three for adenomatous polyposis of the colon (APC), one for neurofibromatosis type 2 (NF2) and one for inherited breast and ovarian cancer caused by BRCA1 mutations. Eleven clinical cycles were performed, resulting in the birth of an unaffected girl. For one of the couples undergoing PGD for NF2, a spontaneous pregnancy ensued after five unsuccessful PGD cycles. The couple underwent chorionic villus sampling (CVS) and the application of the same protocol as used during PGD showed an unaffected fetus. CONCLUSION: In this work, we present the development and clinical application of PGD for three cancer predisposition syndromes.

Medical outcome of 8-year-old singleton ICSI children (born >or=32 weeks' gestation) and a spontaneously conceived comparison group.

BACKGROUND: There is little information about the long-term outcome of children born after ICSI. In this study, the eldest cohort of ICSI children worldwide, reaching the age of 8 years, was investigated at the prepubertal stage to monitor subsequent puberty and future fertility. To investigate possible health problems, a thorough medical and neurological examination was performed. METHODS: Medical outcome of 8-year-old singletons (n=150) born through ICSI (>or=32 weeks) was compared with that of 147 singletons of the same age born after spontaneous conception (SC). Information about their general health was obtained from the parents by means of a questionnaire. RESULTS: Fifteen of 150 ICSI children experienced a major congenital malformation compared with 5/147 SC children (P < 0.05). Pubertal staging was similar in both groups. Neurological examination did not show important differences between ICSI and SC children. ICSI children did not require more remedial therapy or surgery or hospitalization than SC children. CONCLUSION: Physical examination including a thorough neurological examination did not reveal important differences between the two groups. Major congenital malformations were significantly more frequent in the ICSI group. However, most of them were corrected by minor surgery. Further monitoring of these children at an older age is recommended.

Epithelial-mesenchymal transition process in human embryonic stem cells cultured in feeder-free conditions.

Feeder-free human embryonic stem cell (hESC) culture is associated with the presence of mesenchymal-like cells appearing at the periphery of the colonies. The aim of this study was to identify this early differentiation process. Long-term feeder-free hESC cultures using matrigel and conditioned medium from mouse and from human origin revealed that the appearance of mesenchymal-like cells was similar regardless of the conditioned medium used. Standard characterization confirmed the preservation of hESC properties, but the feeder-free cultures could not be maintained longer than 37 passages. The early differentiation process was characterized in the short term after switching hESCs cultured on feeders to feeder-free conditions. Transmission electron microscopy showed an epithelium-like structure inside the hESC colonies, whereas the peripheral cells revealed the acquisition of a rather mesenchymal-like phenotype. Immunochemistry analysis showed that cells at the periphery of the colonies had a negative E-cadherin expression and a positive Vimentin expression, suggesting an epithelial-mesenchymal transition (EMT). Nuclear staining of beta-catenin, positive N-cadherin and negative Connexin 43 expression were also found in the mesenchymal-like cell population. After RT-PCR analysis, Slug and Snail, both EMT-related transcription factors, were detected as up-regulated in the mesenchymal-like cell population. Taken together, our data suggest that culturing hESCs in feeder-free conditions enhances an early differentiation process identified as an EMT.

The efficiency of magnetic-activated cell sorting and fluorescence-activated cell sorting in the decontamination of testicular cell suspensions in cancer patients.

BACKGROUND: Before clinical application, the feasibility and safety of autologous testicular stem cell transplantation should be explored. Apart from limitations in their numbers, spermatogonial stem cells may also be contaminated by malignant cells. Therefore, both enrichment and decontamination before transplantation may be necessary. This study aimed at evaluating the decontaminating potential of magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) for both murine and human testicular cell suspensions. In the mouse, the effectiveness of the transplantation technique after cell sorting was also assessed. METHODS: Murine testicular cells were contaminated with 5% EL4 cells. Fresh and frozen-thawed suspensions were sorted using MACS (CD49f (+)) and FACS (CD49f(+), H-2Kb(-)) and evaluated by FACS, cell culture and transplantation into W/W(v) mice. Human testicular cells were contaminated with 5 or 0.05% CCRF-SB (SB) cells. Frozen-thawed suspensions were sorted using FACS (HLA class I(-)) and evaluated by FACS, cell culture and PCR for the B-cell receptor. RESULTS: In the mouse, the sorted fractions contained 0.39% H-2K(b)-positive and 76.55% CD49f-positive cells. After transplantation, 1 in 20 recipient mice developed a malignancy. In the human experiments, an average of 0.58% SB cells was detected after sorting. In only 1 of 11 samples, there were no SB cells observed. CONCLUSION: MACS and/or FACS are insufficient for completely depleting testicular tissue of malignant cells. Although more research on alternative decontamination techniques is necessary, developing a reliable method to screen a priori testicular tissue for malignant cells may be equally important.

New Belgian embryo transfer policy leads to sharp decrease in multiple pregnancy rate.

Since 1 July 2003, a new transfer policy aiming to reduce multiple pregnancies was brought into law in Belgium. The policy restricts the number of embryos transferred, depending on the patient's age and treatment cycle. This study aimed to evaluate the effect of this policy. Two 15-month periods before and after the start of the new law were compared for the following parameters: positive human chorionic gonadotrophin (HCG), clinical pregnancy rate and multiple pregnancy rate according to the age categories defined by the policy: <36, 36-39 and 40-42 years. HCG rates (34.2 and 32.8%) and clinical pregnancy rates (26.2 and 24.0%) per cycle were similar for the two periods. Overall, the multiple pregnancy rate was reduced from 29.1 to 9.5% (all patients) and from 28.9 to 6.2% in women <36 years. Most twins were observed in the third cycle of patients <36 years and in the first three cycles in women of 36-39 years. It can be concluded that a significant decline (P < 0.001) in multiple pregnancies was mainly observed in patients <36 years of age. Clinical pregnancy rates were not compromised by the new law. Elective single embryo transfer should be considered more seriously for women 36-39 years of age.

Does PGD for aneuploidy screening change the selection of embryos derived from testicular sperm extraction in obstructive and non-obstructive azoospermic men?

BACKGROUND: An increased incidence of aneuploid embryos has been recently described from azoospermic men. The aim of this study was to assess if embryo selection on day 5, based on morphological criteria, would be different from the selection based on PGD for aneuploidy screening (AS) in couples undergoing ICSI for male azoospermia. METHODS: Sixty-two cycles of testicular sperm extraction (TESE)-ICSI with PGD-AS were included in the analysis. Two embryologists, blinded to the PGD-AS results, retrospectively reviewed the available embryology data from day 5 embryos and selected one, two or three embryos to be transferred. These results were compared with the selected embryos based on PGD-AS. RESULTS: A total of 39 cycles from non-obstructive azoospermia (NOA) and 23 cycles from obstructive azoospermia (OA) were retrospectively analysed. If single embryo transfer (SET) had been performed, in 64.8% of the NOA cycles and 54.5% of the OA cycles, no difference in embryo choice would have occurred compared to PGD-AS and in 10.8 and 36.6% of the cycles, respectively, an aneuploid embryo would have been chosen. If double ET (DET) had been performed, in 72.9% of the NOA cycles and 86.5% of the OA cycles, no difference in embryo choice would have occurred compared to PGD-AS and in 2.7 and 4.5% of the cycles, respectively, an aneuploid embryo would have been chosen. If triple ET (TET) had been performed, the outcome would have been the same as for DET. DISCUSSION: Our results suggest that under the terms of an SET policy, the performance of PGD-AS in azoospermia would result in a higher chance of success, as the possibility of selecting a euploid embryo is enhanced.

The role of the testis-specific gene hTAF7L in the aetiology of male infertility.

The X-linked TAF7L gene is homologous to the autosomal transcription factor TAF7. Together with its testis-specific expression pattern, this might point to an important function in spermatogenesis. In order to analyse the involvement of the hTAF7L gene in the aetiology of male infertility, a total of 25 patients with maturation arrest of spermatogenesis have been analysed for the presence of mutations in this gene. Four alterations of the nucleotide sequence, with concomitant changes in the amino acid sequence, have been observed in 12 patients. All sequence alterations were also found either in a control group consisting of men with proven fertility or in a control group with men with normal spermatogenesis. Therefore, these alterations are probably polymorphisms.

Optimization and evaluation of single-cell whole-genome multiple displacement amplification.

The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minute quantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research.

Which patients with recurrent implantation failure after IVF benefit from PGD for aneuploidy screening?

Patients with recurrent IVF failure are defined as patients who are younger than 37 years and who had at least three consecutive unsuccessful IVF/intracytoplasmic sperm injection (ICSI) cycles with good quality embryos. These patients might be predisposed to chromosome errors in their embryos and therefore might benefit from preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). This technique is, however, expensive and some normal embryos might be lost due to the error rate. The aim of this retrospective study was to define those patients who would benefit most from it. One hundred and twenty-one first PGD-AS cycles for recurrent IVF failure were analysed. The aneuploidy rate, 'no embryo transfer' rate, live birth rate per embryo transfer and implantation rate were respectively 48.3, 22.3, 29.7 and 19.5%. A multivariate logistic regression analysis gave us a predictive model demonstrating that to have a 90% probability of having an embryo transfer after PGD-AS, the patient should have at least 10 mature oocytes, eight normally fertilized oocytes and six embryos for biopsy. This study suggests that most patients with recurrent IVF failure may benefit from PGD-AS. Future studies, however, should more strictly define this heterogeneous group of patients, so that comparison is easier.

Blastocyst development after assisted reproduction using spermatozoa obtained after testicular stem cell transplantation in mice.

BACKGROUND: Since its introduction in 1994, testicular stem cell transplantation (TSCT) has been widely used for research. This technique may also become important for preserving fertility in pre-pubertal cancer patients. Therefore, it is necessary to investigate the safety aspects of reproduction using spermatozoa obtained after TSCT. In this study, preimplantation development of mouse embryos, using spermatozoa obtained after TSCT, was examined. METHODS: TSCT-derived spermatozoa were used for IVF and ICSI. Embryos were cultured for five days until they reached blastocyst stage and were evaluated by differential staining. RESULTS: IVF revealed significantly lower fertilization and development rates after TSCT-IVF compared to control-IVF. Blastocysts derived from TSCT-IVF had significantly lower inner cell mass numbers (ICMs) and lower ICM/trophectoderm (TE) ratios compared to control-IVF blastocysts. No differences in fertilization and development rates were observed between TSCT-ICSI and control-ICSI, and blastocyst quality in the transplanted group was similar to that of the control blastocysts. CONCLUSION: Our study showed that after TSCT-IVF, fertilization and preimplantation development were disturbed and blastocysts showed reduced ICM and ICM/TE ratio. However, after TSCT-ICSI, both fertilization and preimplantation development were normal and blastocyst formation was comparable to control-ICSI.

How successful is repeat testicular sperm extraction in patients with azoospermia?

BACKGROUND: Little is known about the extraction rate in repeated sperm retrieval procedures in azoospermic patients. This study aimed to assess the feasibility of repeated sperm recovery in these patients. METHODS: A total of 1066 azoospermic men had their first sperm recovery between 1 January 1995 and 31 December 2003. A total of 381 men had obstructive azoospermia (OA), 628 nonobstructive azoospermia (NOA) and 57 showed hypospermatogenesis. RESULTS: Overall, sperm could be retrieved in all procedures in the 598 cycles performed in OA men (100%). A total of 117, 57, 24, 11, 7 and 1 men underwent, respectively, two, three, four, five, six and seven sperm retrievals; all were successful. Of the 784 procedures performed on the 628 men with NOA, sperm could be retrieved in 384 procedures (49%). During the first testicular sperm extraction (TESE) procedure, sperm could be extracted in 261 men with NOA (41.6%). A total of 103 men had a second attempt, 34 had a third attempt, 11 had a fourth attempt, 6 had a fifth attempt and 2 had a sixth attempt. In these cycles, sperm could be extracted in, respectively, 77 (74.7%), 28 (82.3%), 11 (100%), 5 (83.3%) and 2 (100%) men. CONCLUSION: Repeated TESE ensures a high sperm recovery rate even in patients with NOA. In NOA patients, studies reporting on TESE may therefore overestimate the retrieval rate by reallocating successful patients. These data also show that when no spermatozoa can be obtained after thawing cryopreserved testicular sperm for ICSI in NOA patients, a repeat TESE procedure can be planned.

Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders.

BACKGROUND: Human embryonic stem (hES) cells are pluripotent cells usually derived from the inner cell mass (ICM) of blastocysts. Because of their ability to differentiate into all three embryonic germ layers, hES cells represent an important material for studying developmental biology and cell replacement therapy. hES cell lines derived from blastocysts diagnosed as carrying a genetic disorder after PGD represent in vitro disease models. METHODS: ICMs isolated by immunosurgery from human blastocysts donated for research after IVF cycles and after PGD were plated in serum-free medium (except VUB01) on mouse feeder layers. RESULTS: Five hES cell lines were isolated, two from IVF embryos and three from PGD embryos. All lines behave similarly in culture and present a normal karyotype. The lines express all the markers considered characteristic of undifferentiated hES cells and were proven to be pluripotent both in vitro and in vivo (ongoing for VUB05_HD). CONCLUSIONS: We report here on the derivation of two hES cell lines presumed to be genetically normal (VUB01 and VUB02) and three hES cell lines carrying mutations for myotonic dystrophy type 1 (VUB03_DM1), cystic fibrosis (VUB04_CF) and Huntington disease (VUB05_HD).

Study of two markers of apoptosis and meiotic segregation in ejaculated sperm of chromosomal translocation carrier patients.

BACKGROUND: To try to explain the infertility of chromosomal translocation carrier patients, we compared the expression of two markers of apoptosis in the sperm of patients and of fertile donors, and we studied the meiotic segregation in the ejaculated sperm of these translocation carriers. METHODS: Twenty semen samples of translocation carriers, [reciprocal (n=14) and Robertsonian translocations (n=6)], were compared with the semen samples of donors (n=20). Different tests were applied: annexin V binding assay; terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL); and fluorescence in situ hybridization (FISH). RESULTS: The annexin V binding assay in sperm of patients with chromosomal translocation (n=17) showed a significantly increased proportion of sperm with externalized phosphatidylserine (PS) than in the control group (n=20, P

A lower ongoing pregnancy rate can be expected when GnRH agonist is used for triggering final oocyte maturation instead of HCG in patients undergoing IVF with GnRH antagonists.

BACKGROUND: Eliciting an endogenous LH surge by GnRH-agonist for the induction of final oocyte maturation may be more physiological compared with the administration of HCG. However, the efficacy of this intervention in patients treated for IVF with GnRH antagonists remains to be assessed. METHODS: 106 patients were randomized to receive either 10 000 IU urinary HCG or 0.2 mg Triptorelin for triggering final oocyte maturation. Ovarian stimulation for IVF was performed with a fixed dose of 200 IU recombinant FSH and GnRH antagonist was started on stimulation day 6. Luteal phase was supported with micronized vaginal progesterone and oral estradiol. The study was monitored continuously for safety and stopping rules were established. RESULTS: No significant differences were present in the number of cumulus-oocyte complexes retrieved, in the proportion of metaphase II oocytes, in fertilization rates or in the number and quality of the embryos transferred between the two groups. However, a significantly lower probability of ongoing pregnancy in the GnRH agonist arm prompted discontinuation of the trial, according to the stopping rules established (odds ratio 0.11; 95% confidence interval 0.02-0.52). CONCLUSIONS: Lower probability of ongoing pregnancy can be expected when GnRH agonist is used for triggering final oocyte maturation instead of HCG in patients undergoing ovarian stimulation for IVF with GnRH antagonists.

Is chromosome analysis mandatory in the initial investigation of normovulatory women seeking infertility treatment?

BACKGROUND: There is no agreement about the frequency of chromosomal abnormalities (CAs) in the female partner of an infertile couple and therefore there is no evidence base for determining whether karyotype analysis is mandatory before the initiation of infertility treatment. The aim of this prospective study was to estimate the prevalence of karyotype abnormalities in normovulatory women attending an infertility clinic and compare it to that known to be present in the newborn female population. METHODS: Cytogenetic testing was performed in 1206 women with normal ovulatory cycle seeking infertility treatment. At least 15 GTG-banded metaphases were analysed in each case. In the case of a structural abnormality, fluorescent in situ hybridization (FISH) analysis and high resolution banding (HRB) were performed on a new blood sample to elucidate the aberration. When mosaicism was suspected, the number of analysed metaphases was increased to a total of 115 and an additional analysis of 200 metaphases was done on a second blood sample. RESULTS: A chromosomal abnormality was demonstrated in 0.58% (95% CI: 0.28-1.19) of cases which did not differ significantly from that reported in female newborns (0.79%; 95% CI: 0.68-0.94). Balanced reciprocal translocation was observed in 0.4% of patients (n = 5), paracentric inversion of chromosome X in 0.08% (n = 1) and gonosomal mosaicism in 0.08% (n = 1). However, chromosomal aberrations were less common among females with primary infertility compared to those with secondary infertility (0.25 versus 1.25%, P = 0.04). CONCLUSIONS: The present study suggests that routine cytogenetic analysis cannot be advocated in normovulatory infertile women. Nevertheless, the relatively higher frequency of abnormal karyotypes in women with secondary infertility indicates that this subgroup of patients might benefit from a routine karyotype analysis.

DAZL expression in human oocytes, preimplantation embryos and embryonic stem cells.

In humans, the Deleted in Azoospermia Like (DAZL) gene is believed to function in the development of primordial germ cells and in germ cell differentiation and maturation because the expression of DAZL is only found in the germ and non-germ lineage of the reproductive system and in embryonic stem (ES) cells. The present study examined the presence of DAZL transcripts in the last stages of oocyte maturation, in ES cells, and throughout the preimplantation development; the link between gametes and ES cells. The finding of DAZL transcripts in the last stages of oogenesis and during the first two cell cycles of the preimplantation development was expected, because DAZL is a germ cell marker and the transcripts present at that time are generally encoded by the maternal genome. During the third cell cycle, DAZL showed a variable expression pattern, which may point to the maternal to embryonic transition. After the third cell cycle, transcripts were again consistently detected, suggesting embryonic DAZL transcription. In blastocysts, DAZL transcripts were only detected in those of good quality and this as well in the inner cell mass (ICM) as in the trophectoderm (TE). The presence of DAZL transcripts in the ICM and in ES cells was not surprising since both can lead to the formation of germ cells, but TE cells cannot. The quality-related expression of DAZL in blastocysts, and especially its trophectodermal expression, might imply other functions for DAZL beyond germ cell development.