Dutch technical specification (NTA 8070) on devices for assisted reproductive technologies.

In 2006 the Dutch Society for Clinical Embryologists (KLEM) approached the Netherlands Standardisation Institute (NEN) for advice regarding the lack of safety and quality specifications for medical devices used in assisted reproductive technology. A project plan was drafted in accordance with NEN-standardized methods for the development of norms and Dutch technical specifications (NTA) and a working group was launched consisting of all interested parties. A framework was then set up to develop an NTA that focused on the safety of gametes, embryos and the unborn offspring. The three main parts of the NTA describe the classification of medical devices, the requirements for new and existing devices and testing methods. The content of the NTA can be considered mainly as a consensus of the participants in the working group from both industry and clinical embryologists (KLEM). A final draft was sent to representatives from or allied to the government and to a notified body, and NTA 8070, entitled Devices for Assisted Reproductive Technologies (ART), was issued in March 2008.

Does the genetic and familial background of males undertaking ICSI affect the outcome?

PURPOSE: To investigate whether the success rate of ICSI is (1) related to the etiology of infertility or (2) adversely affected by a family history of potential genetic disorders. METHODS: All men with an ICSI indication in our hospital between 1994 and 2005 were included in our cohort study. Data on the ICSI process, etiology of infertility, and family history were collected. ICSI success rates of infertility subgroups and a subgroup with a positive family history were compared to a group with unknown etiology and a negative family history. RESULTS: There was no significant difference in clinical pregnancy or delivery rates between the subgroups. Couples achieving a pregnancy underwent significantly more ICSI cycles compared to couples not achieving a pregnancy. CONCLUSION: Our results suggest that the success rate of ICSI treatment is not related to the cause of infertility or a family history positive for potential genetic disorders.

[Ten years of results of in-vitro fertilisation in the Netherlands 1996-2005]. / Tien jaar resultaten van in-vitrofertilisatie in Nederland, 1996-2005.

OBJECTIVE: To present the numbers and results of Dutch IVF treatment from 1996-2005 and to describe trends and differences between centres. DESIGN: Retrospective data-collection, description and analysis. METHOD: The annual statistics from all Dutch IVF centres covering the years 1996-2005 were collected, described and analysed. RESULTS: During this period 138,217 IVF or intracytoplasmic sperm injection (ICSI) cycles were started and 14,881 transfers of frozen-thawed embryos (cryo transfers) were performed. The number of ICSI treatments, in particular, increased to more than 6000 cycles during this period. These treatments resulted in 30,488 ongoing pregnancies (22.1% per cycle started; 19.1% for IVF and 23.4% for ICSI). The ongoing pregnancy rate per cycle increased from 17.6% in 1996 to 24.4% in 2005. The increase after cryo transfers was remarkable (from 9.4% to 17.6%). It is estimated that during this period, about 1 in 52 newborns in the Netherlands was an IVF or ICSI child (1996: 1 in 77, 2005: 1 in 43). There were differences between the individual centres regarding the ongoing pregnancy rate per cycle (range: 15.0-26.4%), the percentage of ICSI (range 20-58%), the percentage of cryo transfers per cycle (range: 4-22%) and the multiple pregnancy rate (range 5-27% in 2005). CONCLUSIONS: In the Netherlands the pregnancy rate has increased over the last 10 years as has the number of IVF treatments. Cryo transfers have become increasingly important and the multiple pregnancy rate has decreased. Although thanks to the collaboration of all centres, the current registry produces important data and works well, there are a number of limitations e.g. the retrospective nature with no validation, which must be tackled over the coming years.

Variant Klinefelter syndrome 47,X,i(X)(q10),Y and normal 46,XY karyotype in monozygotic adult twins.

Klinefelter syndrome (KS; 47, XXY) is characterized by increased body height, hypergonadotrophic hypogonadism, and infertility. We describe a patient with a variant KS (47,X,i(Xq),Y) who has a twin brother with a 46,XY karyotype. Molecular studies showed that the twins were monozygotic. The presence of an isochromosome Xq in one of two monozygotic twins allows precise investigation of its phenotypic effect. The patient was somewhat shorter (3.5 cm) and had a smaller volume of the testes (8 vs. 18 ml) as compared to his twin brother. Furthermore he had increased gonadotrophin levels and an extreme oligoasthenoteratozoospermia (OAT). These data support the view that genes on Xp cause increased body height and genes on Xq cause infertility in KS. To our knowledge this is the first report on a heterokaryotypic monozygotic twin with a variant KS.

Split hand/split foot, iris/choroid coloboma, hypospadias and subfertility: a new developmental malformation syndrome?

This paper presents a patient with the following malformations: split hand and split foot on the left side, a hypoplastic fifth ray of the right hand and a hypoplastic first ray of the right foot with a small cleft between the first and second ray; eye abnormalities which consist of a complete iris coloboma of the left eye in an atypical position (cranio-temporal) and a coloboma of the choroid in the right eye; a glandular hypospadias and terato-zoospermia. Since split hand/split foot can be caused by mutations in the p63 gene, mutation analysis of this gene was performed. However, sequencing analysis did not reveal a mutation. This malformation complex may represent a new syndrome.

[Results of in vitro fertilization in the Netherlands, 1996-2000]. / Resultaten van in-vitrofertilisatie in Nederland, 1996-2000.

OBJECTIVE: To describe the annual results in all 13 Dutch in vitro fertilisation (IVF) centres in the period 1996-2000, and to look for possible differences between individual centres and years. DESIGN: Retrospective data collection, description and analysis. METHOD: The results collected on the website of the Dutch Society of Obstetrics and Gynaecology (Dutch acronym: NVOG; www.nvog.nl) in the period 1996-2000 were integrated and described, with special attention to possible differences between centres and years. RESULTS: In 1996-2000 (5 years), 63,414 IVF or ICSI treatment cycles were started in the Netherlands, and 5,884 transfers of cryopreserved embryos were performed. The number of treatment cycles increased over the years, particularly the number of ICSI cycles. The total number of ongoing pregnancies was 12,991 (20.5% per started cycle; 22.5% for ICSI and 18.3% for IVF). Particularly during the first 3 years, there was an increase in these percentages (IVF: from 16.4% (1996) to 19.2% (1998); ICSI: from 18.3% (1996) to 23.9% (1998)). There were differences between the centres in both the percentage of ongoing pregnancies per started IVF/ICSI cycle (range 13.7-25.1%) and the percentage ICSI (14-61%) and cryo-transfers per total number of treatment cycles (0-26%). It was estimated that, during this 5-year period, 1 out of every 61 Dutch neonates resulted from IVF or ICSI. CONCLUSION: The pregnancy-rates after IVF and ICSI increased during the study period, and were comparable with the rates in other European countries. Some important data are still missing from the inventory, for example regarding the number of embryos per transfer, multiple pregnancies, live births, congenital malformations and complications.

Analysis of spermatozoa from seven ICSI males with constitutional sex chromosomal abnormalities by fluorescent in situ hybridization.

PURPOSE: The objective was to estimate the risk for subfertile males with a constitutional sex chromosomal abnormality of transmitting such a chromosome abnormality to their children, conceived by intracytoplasmic sperm injection (ICSI). METHODS: Semen samples were obtained from seven severely oligospermic ICSI candidates. Six of them had a numerical sex chromosomal abnormality, including mosaic 45,X/46,XY, mosaic 46,XY/47, XXY, 47,XXY (Klinefelter's syndrome), and 47,XYY. One male had a structural abnormality, namely, an inversion of the Y chromosome. The semen was studied by three-color fluorescent in situ hybridization (FISH) with probes specific for chromosomes 18,X, and Y. RESULTS: Chromosomal aneuploidy rates of any of the three chromosomes were significantly higher than the aneuploidy rates observed in three control samples but comparable to the rates observed in 10 ICSI candidates with oligoasthenoteratozoospermia (OAT) and a normal constitutional karyotype. CONCLUSIONS: Our data indicate that males with (mosaic) sex chromosomal abnormalities have no higher risk of producing offspring with a sex chromosomal abnormality by ICSI than OAT males with a normal karyotype.

Reproductive decisions of men with microdeletions of the Y chromosome: the role of genetic counselling.

Couples dealing with microdeletions of the Y chromosome have to make decisions about their reproductive future. Do they opt for intracytoplasmic sperm injection (ICSI), artificial insemination with donor insemination (AID) or no treatment? We analysed this decision in 28 couples and investigated the role of the counsellor and the counselling process on the final decision of the couple. Ten counsellors from six fertility clinics in The Netherlands and Belgium were interviewed about their genetic counselling of couples dealing with microdeletions. The answers to the questionnaire were converted to 11 dichotomous variables. Of the 1627 tested men in the six centres, 37 (2.3%) had a microdeletion in the AZFc region, a subregion of the AZF region on the Y chromosome important for normal spermatogenesis. The decisions of 28 of them could be analysed. Most couples chose ICSI (79%). The remaining couples chose donor insemination (7%) or refrained from treatment (14%). Several variables, including the counselling procedure, the counsellor and the available treatments in the fertility centre, influenced the decision of the couple. In conclusion, most couples dealing with microdeletions in the AZF region choose ICSI. Several aspects of the process of genetic counselling appear to be related to the final decision.

Comparison between intracytoplasmic sperm injection and in-vitro fertilization (IVF) with high insemination concentration after total fertilization failure in a previous IVF attempt.

The aim of this prospective study was to evaluate whether couples with total fertilization failure in a previous in-vitro fertilization (IVF) attempt should be offered an additional IVF treatment with elevated insemination concentration or should be treated with intracytoplasmic sperm injection (ICSI). In 23 cycles 228 sibling metaphase II (MII) oocytes were randomly divided: 143 and 85 oocytes were utilized for ICSI and IVF respectively. Of the 143 injected (ICSI) oocytes, 90 (62.9%) were normally fertilized (two pronuclei), whereas 21 (14.7%) oocytes were damaged by the ICSI procedure. Of the fertilized oocytes 72 (80%) developed into transferable embryos. No fertilization at all was observed in the 85 sibling MII oocytes which were inseminated (P < 0.001). In all 23 cycles at least one embryo, obtained by ICSI, could be replaced. Eight pregnancies were achieved of which six resulted in the delivery of nine healthy children. In conclusion, for couples with no or almost no fertilization of oocytes in previous IVF attempts, ICSI appeared to be far superior to an additional IVF attempt with further elevated insemination concentrations.

Sperm analysis in a subfertile male with a Y;16 translocation, using four-color FISH.

Sperm analysis was performed in a male with oligoasthenoteratozoospermia (OAT) and a reciprocal t(Y;16) (q11. 21;q24), using four-color FISH. Intracytoplasmic sperm injection (ICSI) treatment in this patient had resulted in the birth of one chromosomally balanced and two chromosomally normal children. To assess the risk of having a chromosomally unbalanced conception after ICSI, morphologically normal spermatozoa were studied with a set of probes allowing detection of all segregation variants. There were 51% normal or balanced sperm cells. The fraction of sperm products resulting from alternate and adjacent I segregation was 87%, 12% were products of 3:1 disjunction, and the other 1% had other types of aneuploidy. If morphologically abnormal cells were also included in the FISH analysis, nearly 90% of all the spermatozoa were unbalanced. We conclude that although the majority of males with a Y/autosome translocation are infertile due to azoospermia, our patient produces sufficient morphologically and chromosomally normal spermatozoa to have chromosomally normal or balanced offspring after ICSI. Assuming that ICSI with an unbalanced spermatozoon from this patient would result in a nonviable embryo in many cases, the combination of in vitro and subsequent in vivo selection probably results in a risk of unbalanced offspring of much less than 50%. Hence, FISH studies on the sperm of translocation carriers are useful for estimating the risk of having unbalanced offspring after ICSI and in understanding the mechanisms underlying infertility in such carriers.

Subfertile men with constitutive chromosome abnormalities do not necessarily refrain from intracytoplasmic sperm injection treatment: a follow-up study on 75 Dutch patients.

A follow-up study was performed to investigate the impact of the detection of a chromosome abnormality in infertile men who are candidates for intracytoplasmic sperm injection (ICSI) treatment. In this collaborative study between clinical genetics centres and fertility clinics in the Netherlands, 75 ICSI couples of which the male partners had a chromosome abnormality were included. All couples were extensively counselled on the risk of having a chromosomally unbalanced child. Forty-two out of 75 couples chose to proceed with the ICSI treatment. So far, treatment has resulted in a pregnancy in 11 cases. Four of them opted to have invasive prenatal diagnosis. Despite the genetic risks related to a chromosome abnormality in infertile men, a small majority (56%) of the couples did not refrain from the ICSI treatment.

Protein synthesis and phosphorylation patterns of bovine oocytes maturing in vivo.

To investigate protein synthesis and phosphorylation during bovine oocyte maturation in vivo, oocytes were collected at consecutive times after the preovulatory luteinizing hormone (LH) peak. Therefore, heifers treated for superovulation were ovariectomized between 3 and 20 h after the maximum of the LH peak. Subsequently, cumulus-enclosed oocytes, selected from nonatretic follicles greater than 10 mm, were radiolabeled with 35S-methionine or 32P-orthophosphate for 3 h and individually prepared for gel electrophoresis. Changes in the protein synthesis patterns were observed coinciding with germinal vesicle breakdown (GVBD). No changes were detected during the ensuing maturation period or coinciding with the extrusion of the first polar body. In addition, the protein phosphorylation patterns exhibited striking differences around GVBD. In particular, a phosphoprotein band of 19 kDa and the two heavily phosphorylated proteins with apparent molecular weights between 50 and 60 kDa were present in patterns of oocytes in the germinal vesicle stage. The results are discussed in relation to previous data obtained during maturation in vitro.

The effects of alpha-amanitin and cycloheximide on nuclear progression, protein synthesis, and phosphorylation during bovine oocyte maturation in vitro.

Bovine cumulus oocyte complexes were cultured for various periods and either denuded and orcein stained or radiolabeled with 35S-methionine or 32P-orthophosphate. Specific inhibitors were added to the culture medium to investigate mRNA and protein synthesis requirements for both nuclear and cytoplasmic changes during maturation in vitro. Inhibition of mRNA synthesis by alpha-amanitin during the first 2 h of culture prevented the phosphorylation of some specific proteins preceding GVBD and decreased the occurrence of GVBD from 97% to 27%. In addition, in oocytes that had undergone GVBD, only part of the changes in protein synthesis after GVBD were observed. Addition of alpha-amanitin after 3 h of culture had no effect on meiotic maturation. When cumulus oocyte complexes were cultured in the presence of cycloheximide, the phosphorylation of specific proteins was also blocked and only 5% of the oocytes underwent GVBD. Addition of cycloheximide after 4, 6, or 8 h of culture resulted in an increasing percentage of GVBD, but the oocytes became arrested in metaphase I. When cycloheximide was added from 12 h of culture onwards, nuclear progression to metaphase II was increasingly restored. It is concluded that after the onset of culture, both mRNA and protein synthesis are necessary for the phosphorylation of specific proteins and for GVBD. Furthermore, transcription during the first hours of culture is needed for the synthesis of new proteins after GVBD.

Heterologous cell contacts and metabolic coupling in bovine cumulus oocyte complexes.

The integrity of the cumulus cell processes were studied in four categories of bovine cumulus oocyte complexes (COCs) selected on their morphological characteristics. Three different types of cumulus cell process endings (CCPEs) were identified, one penetrating the cortex, another not penetrating the cortex, and a third form was intermediate and more rare in appearance. The process endings that penetrated the cortex frequently made gap junctions with the oolemma. The division of the three types of CCPEs over the four different COC categories was specific for three of the four categories. The first-category COC predominantly possessed the penetrating CCPE, the fourth-category COC possessed predominantly the nonpenetrating CCPE, and the second and third categories had both types of CCPEs. The metabolic coupling of the cumulus-oocyte contacts was assessed by means of incorporation of 3H-choline into the oocyte. The majority of category 4 COCs transferred low levels of choline into the oocyte while the majority of the oocytes of the other three categories transferred high levels of choline into the oocyte. Category 4 includes a smaller proportion of oocytes capable of cleaving after fertilization than the other three categories. This reduced developmental capacity is probably due to the loss of metabolic coupling before the onset of culture.

Changes in protein synthesis and phosphorylation patterns during bovine oocyte maturation in vitro.

Sequential protein synthesis and protein phosphorylation patterns were generated by radiolabelling bovine cumulus-oocyte complexes after various periods of culture with [35S]methionine and [32P]orthophosphate respectively. The radiolabelled oocytes were assessed for their nuclear status and used individually for gel electrophoresis. Marked changes in the protein synthesis patterns were observed exclusively after germinal vesicle breakdown (GVBD), whereas oocytes which remained in the germinal vesicle stage showed a consistent protein synthesis pattern. The changes were observed after 8 and 16 h or culture, shortly after GVBD and before first polar body extrusion. From 3 h of culture, dominant phosphoprotein bands with apparent molecular weights of 24,000 and two between 50,000 and 60,000 were observed. The latter bands displayed slight molecular weight changes, which were not closely time related. After GVBD, the phosphoprotein band with Mr 19,000 was no longer observed. This study demonstrates that specific changes in protein synthesis and protein phosphorylation are programmed during bovine oocyte maturation.

Analysis of protein synthesis in morphologically classified bovine follicular oocytes before and after maturation in vitro.

Bovine cumulus oocyte complexes (COCs) were isolated from antral ovarian follicles (4-8 mm). Immature COCs were classified into four categories, based on the homogeneity and clearness of the ooplasm and the transparency and compactness of the cumulus investment. In this study, the incorporation of TCA-precipitable 35S-methionine and the protein synthesis patterns of oocytes of these four categories were examined. Before maturation in vitro, similar incorporation rates and identical protein synthesis patterns were observed between oocytes of categories 1-3. Immature oocytes of category 4 showed reduced incorporation rates and exhibited aberrant protein synthesis patterns. After maturation in vitro, the patterns of category 4 oocytes were identical with the patterns of those in categories 1-3. The incorporation of 35S-methionine into in vitro matured oocytes was lower (P less than .001) in all categories. Based on these results, it is concluded that the initial classification of oocytes into four categories can be reduced to two categories.

Sequence divergence and selection of cap sites in the rat gamma-crystallin gene family.

The transcription initiation sites of the six rat gamma-crystallin genes were mapped by combining the results of primer extension and S1 nuclease mapping experiments. To obtain more accurate results from the S1 nuclease mapping experiments, intron-deleted clones were constructed by a novel and efficient modification of existing methods involving the use of primer extension products to seal the exons. Four of the six gamma-crystallin genes have multiple transcription start sites. The major and most of the minor transcripts start with an adenosine. Analysis of the 5' flanking sequences of the gamma-crystallin genes shows that the sequence determining the position of the cap site is merely -CA- and that its optimal distance from the first T of the TATA box is 32 base pairs. Our data further suggest that an A to G transition in the first two base pairs of the Goldberg/Hogness box of one the genes does not affect the position of its major cap site. This, together with the fact that most minor transcription start sites are located upstream from the major cap sites, suggests that in the long TATA boxes of the rat gamma-crystallin genes the major RNA polymerase 'trap site' is not directly at the beginning of the TATA sequence.