The importance of neuronal growth factors in the ovary.

The neurotrophin family consists of nerve growth factor (NGF), neurotrophin 3 (NT3) and neurotrophin 4/5 (NT4/5), in addition to brain-derived neurotrophic factor (BDNF) and the neuronal growth factors, glial cell line-derived neurotrophic factor (GDNF) and vasointestinal peptide (VIP). Although there are a few literature reviews, mainly of animal studies, on the importance of neurotrophins in the ovary, we aimed to provide a complete review of neurotrophins as well as neuronal growth factors and their important roles in normal and pathological processes in the ovary. Follicular assembly is probably stimulated by complementary effects of NGF, NT4/5 and BDNF and their receptors. The neurotrophins, GDNF and VIP and their receptors have all been identified in preantral and antral follicles of mammalian species, including humans. Transgenic mice with mutations in the genes encoding for Ngf, Nt4/5 and Bdnf and their tropomyosin-related kinase ß receptor showed a reduction in preantral follicles and an abnormal ovarian morphology, whereas NGF, NT3, GDNF and VIP increased the in vitro activation of primordial follicles in rats and goats. Additionally, NGF, NT3 and GDNF promoted follicular cell proliferation; NGF, BDNF and VIP were shown to be involved in ovulation; VIP inhibited follicular apoptosis; NT4/5, BDNF and GDNF promoted oocyte maturation and NGF, NT3 and VIP stimulated steroidogenesis. NGF may also exert a stimulatory effect in ovarian cancer and polycystic ovarian syndrome (PCOS). Low levels of NGF and BDNF in follicular fluid may be associated with diminished ovarian reserve and high levels with endometriosis. More knowledge of the roles of neuronal growth factors in the ovary has important implications for the development of new therapeutic drugs (such as anti-NGF agents) for ovarian cancer and PCOS as well as various infertility problems, warranting further research.

Recurrent triploid and dispermic conceptions in patients with NLRP7 mutations.

To understand the mechanisms leading to hydatidiform mole formation in patients with NLRP7 mutations, we used a combination of various approaches to characterize five products of conception, from two patients, shown by flow cytometry to contain non-diploid cells. We demonstrate that four of these conceptions are triploid and two of them originated from fertilization with more than one sperm. We show that three of these triploid conceptions fulfill the histopathological criteria of partial hydatidiform mole and one fulfills the histopathological criteria of spontaneous abortion. Our data demonstrate that some oocytes from one patient with NLRP7 mutations are not able to prevent polyspermic fertilization and highlight the importance of using several approaches to characterize the genetic complexity of molar tissues and reproductive wastage. Altogether, our previous and current data show the association of NLRP7 mutations with several types of hydatidiform moles and with triploid spontaneous abortions.

Keratinocyte growth factor and its receptor in human ovaries from fetuses, girls and women.

Keratinocyte growth factor (KGF) promotes growth of rat pre-antral follicles. There is limited information regarding its presence or that of its unique receptor (KGFR) in human ovaries, specifically in pre-antral follicles. The aim of the study was to investigate the expression of KGF and KGFR in ovarian samples from human fetuses and girls/women. The samples were prepared for immunohistochemical study of the KGF protein and for in situ hybridization to localize mRNA transcripts of KGFR. Total RNA was extracted from frozen ovarian samples, and the expression of KGF mRNA transcripts was investigated by reverse transcriptase polymerase chain reaction. In both fetuses and girls/women, the protein for KGF was detected from primordial stages in oocytes, granulosa cells (GCs) and stroma cells. Its mRNA transcripts were also detected in all extracts. The mRNA transcripts for KGFR were detected mainly in stroma cells in ovarian samples from both sources; in 10% of the samples, follicular staining was noted also in oocytes and GCs. Further studies adding KGF to the culture medium are needed to elucidate its putative role in human primordial follicle activation.

Platelet-derived growth factors (PDGF-A and -B) and their receptors in human fetal and adult ovaries.

There is no information regarding the presence of platelet-derived growth factors (PDGFs) and their receptors in human ovaries. The expression of PDGF-A, -B and their two receptors, PDGFR-alpha and -beta, was investigated in ovarian samples from women/girls and from human fetuses, at the protein and mRNA levels. The samples were prepared for immunohistochemical staining for PDGF-A and -B and their two receptors and in situ hybridization for the detection of the mRNA transcripts of the receptors. Total RNA was extracted from frozen ovarian samples, and the expression of PDGF-A and -B was investigated by reverse transcription-polymerase chain reaction. The proteins for PDGF-A and -B were detected in oocytes, and in granulosa cells (GC) of 50% of the follicles from women/girls. The proteins and mRNA transcripts for the two receptors were detected in oocytes (mRNA for PDGFR-beta only in 25% of the oocytes). PDGFR-alpha mRNA was expressed in GC of a minority of the samples from women/girls, whereas PDGFR-beta protein and mRNA were identified in over 50% of the GC from this source. PDGF-A and -B transcripts were identified in all the extracts. The presence of the receptors in GC suggests that PDGFs might be involved in the activation of primordial follicles.

Tyrosine kinase B receptor and its activated neurotrophins in ovaries from human fetuses and adults.

The signals initiating the growth of primordial follicles are unknown. Growth factors such as neurotrophin 4/5 (NT-4/5) and brain-derived neurotrophic factor (BDNF) may play a role in this process. To investigate the expression of NT-4/5 and BDNF and their receptor tyrosine kinase B (TrkB) in the early developing follicles, we fixed and froze 12 ovarian samples from adolescents/adults and 31 ovaries from human fetuses. The fixed samples were prepared for immunohistochemical staining for NT-4/5, BDNF and the TrkB receptor. Total RNA was extracted from the frozen ovarian samples, and the expression of NT-4/5, BDNF and the TrkB receptor (full length and two truncated isoforms) was investigated by RT-PCR. Products were resolved by 1% agarose gel electrophoresis and image analysis. Immunohistochemical staining revealed the expression of NT-4/5 and BDNF mainly in oocytes and, in a minority of samples, also in the granulosa cells (GCs); TrkB receptor was identified in oocytes and GCs. Transcripts of NT-4/5, BDNF and all forms of TrkB receptor were identified in the samples. To elucidate whether indeed NT-4/5 and BDNF are involved in growth initiation of human primordial follicles, they should be added to the culture medium.

Presence of NGF and its receptors in ovaries from human fetuses and adults.

The ability to mature human primordial follicles in vitro would assist fertility restoration. However, the signals initiating growth of primordial follicles are unknown. Growth factors such as nerve growth factor (NGF) may play a role in this process. To investigate the expression of NGF and its receptors, p75 and TrkA, in early developing follicles (mostly primordial, primary and secondary follicles), ten ovarian samples from adolescents/adults aged 13-39 and 33 ovaries from human fetuses aged 19-33 gestational weeks (GW) were obtained and immediately fixed or frozen. The fixed samples were prepared for a study of immunocytochemical staining of NGF and its two receptors. Total RNA was extracted from the frozen ovarian samples, and the expression of NGF, TrkA and p75 was investigated by RT-PCR. Products were resolved by 1% agarose gel electrophoresis and image analysis. Immunocytochemical staining revealed the expression of NGF in granulosa cells (GC) and oocytes; TrkA was mainly in oocytes and in GC in minority of the samples; and p75 was in some of the stroma cells from fetuses aged less than 22 GW. Transcripts of NGF and TrkA were identified by RT-PCR in all samples, while those for p75 were detected only in ovarian samples from fetuses aged less than 22 GW. To elucidate if NGF is indeed involved in growth initiation of human primordial follicles, it should be added to their culture medium. The immunocytochemical detection of p75 in some of the stroma cells and transcripts in ovarian samples of fetuses less than 22 GW may suggest its role in follicular assembly.

Immunocytochemical detection and RT-PCR expression of leukaemia inhibitory factor and its receptor in human fetal and adult ovaries.

The ability to mature human primordial follicles in vitro would assist fertility restoration. However, the signals initiating growth of primordial follicles are unknown. Growth factors such as leukaemia inhibitory factor (LIF) may play a role in this process. To investigate the expression of LIF and its receptor in early developing follicles, nine ovarian samples from adolescents/adults aged 13-43 years and 23 ovaries from human fetuses aged 19-33 gestational weeks were immediately fixed or frozen. The fixed samples were prepared for a study of immunocytochemical staining of LIF and its two receptor units (LIF-R and gp 130). mRNA was extracted from the frozen ovarian samples, and the expression of LIF, LIF-R and gp 130 was investigated by RT-PCR. Products were resolved by 10% polyacrylamide gel electrophoresis and image analysis. There was strong to moderate immunocytochemical staining for LIF and LIF-R in oocytes from the primordial follicular stages onwards, and very weak to moderate staining for gp 130. LIF-R was also detected in granulosa cells of primary and secondary follicles from adolescents/adults. Transcripts of LIF, LIF-R and gp 130 RNA were identified by RT-PCR in all samples. The immunocytochemical staining and mRNA expression of LIF and its receptor are consistent with the concept that LIF might be involved in growth initiation of human primordial follicles through its receptor.

Direct comparison of detection systems used for the development of single-cell genetic tests in preimplantation genetic diagnosis.

PURPOSE: Single-cell polymerase chain reaction (PCR) requires efficient amplification and accurate detection. We compare the accuracy of heteroduplex, fluorescent-fragment, and fluorescent single-strand conformation polymorphism (F-SSCP) analysis as detection systems for analysis of a PCR assay developed for preimplantation genetic diagnosis. METHODS: A single-cell, fluorescent multiplex PCR assay was developed for the cystic fibrosis delta F508 mutation and the short tandem repeat, D21S11. Detection systems were compared by analyzing blinded PCR products. RESULTS: Amplification rates for cystic fibrosis were 89% by heteroduplex and 91% by fragment analysis, while it was 72% for D21S11 by fragment analysis. No difference in allele dropout was detected for cystic fibrosis by any method (2%). Overall accuracy was high, > 97%, although SSCP was the least accurate. CONCLUSIONS: Heteroduplex and fragment analysis proved equal in the diagnosis of a single amplified locus. We determined that fragment analysis allows maximal accuracy of detection and permits analysis of a second loci, controlling for DNA contamination and allelic dropout.

The development of preimplantation genetic diagnosis for myotonic dystrophy using multiplex fluorescent polymerase chain reaction and its clinical application.

Preimplantation genetic diagnoses (PGD) for single gene defects require considerable time and resources for the standardization of polymerase chain reactions that are rapid, sensitive and reliable. Developing tests for the trinucleotide repeat diseases, where the expansion of unstable repeats produces the phenotypes, are particularly complex. One of these disorders is myotonic dystrophy where, at present, diagnosis at the single cell level relies on the detection of the normal alleles from both the affected and unaffected parent. The incorporation of short tandem repeat polymorphisms in the assay can give additional information to improve the accuracy of diagnosis. We have developed a multiplex fluorescent reaction for myotonic dystrophy and one of two closely mapped, highly heterozygous, short tandem repeats (D19S219 and D19S559) on chromosome 19 to reduce the possibility of misdiagnosis due to contamination, act as a control for allelic drop-out and maximize the number of embryos genotyped. This protocol was designed as a general diagnosis for myotonic dystrophy, using the most informative of the two polymorphisms for each couple. Subsequently this approach was used in a PGD treatment cycle.

Quantitative measurement of transcript levels throughout human preimplantation development: analysis of hypoxanthine phosphoribosyl transferase.

We have developed a competitive reverse transcription-polymerase chain reaction (RT-PCR) sensitive enough to detect and quantify as little as 2-fold differences in gene expression in individual oocytes and embryos throughout human preimplantation development. This RT-PCR assay can be tailored for the examination of any specific gene and so will give a unique insight into human preimplantation development. This technique was used to quantify the level of hypoxanthine phosphoribosyl transferase (HPRT) expression during preimplantation development and to correlate this with embryo sex. The amount of HPRT transcripts present in the unfertilized oocyte was equivalent to 7.7 fg of competitor cDNA. At the 4-cell stage there is a significant drop (P: = 0.0006) to approximately 1.2 fg. There was no detectable difference in the HPRT levels between female and male embryos following 2 days of in-vitro culture. In contrast HPRT gene expression was higher in day 3 female embryos than in males. This is the first study to quantify gene transcripts throughout each stage of human preimplantation development and it indicates that the accumulated HPRT transcripts present in the unfertilized human oocyte undergo extensive destruction following fertilization. This work also suggests that X-inactivation occurs beyond the 8-cell stage of human preimplantation development.

Fluorescence in-situ hybridization of sex chromosomes in spermatozoa and spare preimplantation embryos of a Klinefelter 46,XY/47,XXY male.

It has been suggested recently that 47,XXY germ cells are able to progress through meiosis to produce hyperhaploid spermatozoa. We report on a 46,XY/47,XXY Klinefelter patient whose spermatozoa were recovered from the ejaculate and used for intracytoplasmic sperm injection (ICSI). Fluorescence in-situ hybridization (FISH) analysis of the patient's spermatozoa and of spare preimplantation embryos with DNA probes specific for chromosomes X, Y and 18 revealed sex chromosome hyperploidy in 3.9% of the sperm nuclei analysed (2.23% XY18, 1.12% XX18, 0.56% YY18), while only three out of 10 spare embryos analysed were normal for chromosomes tested. The abnormalities included two diploid mosaic embryos with the majority of the blastomeres normal for the chromosomes tested, and five embryos with mostly abnormal blastomeres and chaotic chromosome X, Y and 18 patterns. None of the embryos analysed showed a XXY1818 or XXX1818 chromosome complement. The frequency of sex chromosome hyperploidy in the spermatozoa of the mosaic Klinefelter patient was higher than the mean reported for karyotypically normal males, supporting the hypothesis that 47,XXY germ cells are able to complete meiosis and produce aneuploid spermatozoa. However, most of the spermatozoa analysed were normal for sex chromosomes, and ICSI of the patient's spermatozoa did not result in a spare embryo with a uniform 47,XXY or 47,XXX chromosome complement. Instead, fertilization produced a high percentage of mosaic embryos with chaotic chromosome arrangements.

Assessment of multiplex fluorescent PCR for screening single cells for trisomy 21 and single gene defects.

A great majority of patients seeking preimplantation genetic diagnosis (PGD) are women >35 years of age. In addition to being carriers for single gene defects, these women also have a higher risk of having children with Down's syndrome (trisomy 21). For these patients, it would be advantageous if a diagnostic test for trisomy 21 was developed, which could be used in conjunction with tests for single gene defects. Here, we assessed the feasibility of developing an accurate genetic test for diagnosing trisomy 21 and the mutation causing spinal muscular atrophy (SMA) in single cells using multiplex fluorescence polymerase chain reaction (PCR). Single- and two-round PCR were developed using a combination of primers for the survival motor neuron (SMN) gene exons 7 and 8 and two chromosome 21 short tandem repeats (STRs), D21S226 and D21S11. After only 36 cycles, 88 and 68% of normal single cells were screened for SMA mutations and trisomy 21 respectively. In multiplex PCR using only two primers (SMN exon 7 and D21S11) instead of four, the efficiency of SMA diagnosis was increased to 93%. In the same reactions, the D21S11 alleles were detected in 83% of the normal single cells. Clinical applications of this assay should enable detection of those embryos that have inherited three heterozygous alleles and, therefore, benefit many PGD patients who are at an increased risk of Down's syndrome.

Production of steroids from human cumulus cells treated with different concentrations of gonadotropins during culture in vitro.

OBJECTIVE: To evaluate the output of E2 and progesterone produced by cumulus cells, derived from mature and immature oocytes, in culture medium. DESIGN: Prospective randomized study. SETTING: McGill Reproductive Center, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada. PATIENT(S): Twenty-one women, <38 years of age and with normal menstrual cycles, who were undergoing intracytoplasmic sperm injection for assisted reproduction. INTERVENTION(S): Culture medium with or without fetal bovine serum (FBS) supplemented with either a physiologic (75 mIU/mL) or a supraphysiologic (7,500 mIU/mL) concentration of gonadotropins. MAIN OUTCOME MEASURE(S): Comparison of steroid levels in culture medium. RESULT(S): Estradiol secretion was significantly increased in the culture medium with FBS supplemented with both concentrations of FSH alone compared with control. However, E2 secretion was inhibited by both concentrations of FSH with LH. The level of E2 was undetectable in the medium without FBS even after supplementation with both concentrations of FSH alone, hCG alone, and FSH with LH. Progesterone production was increased in the medium with FBS supplemented with FSH alone, hCG alone, and FSH with LH compared with control. There was no difference in progesterone levels in the culture medium without FBS supplemented with both concentrations of FSH alone and hCG alone compared with control. However, progesterone secretion was increased in the medium without FBS supplemented with a physiologic concentration of FSH with LH. CONCLUSION(S): Culture medium with FBS supplemented with a physiologic and a supraphysiologic concentration of FSH stimulates E2 secretion from cumulus cells derived from mature and immature oocytes. This suggests that it may be not necessary to add E2 to the culture medium for maturation in vitro of immature human oocytes retrieved from patients undergoing stimulated cycles.

Successful preimplantation genetic diagnosis for sex Link Lesch--Nyhan Syndrome using specific diagnosis.

Lesch-Nyhan syndrome (LN) is a severe X-linked recessive disorder caused by a deficiency of the enzyme hypoxanthine phosphoribosyl transferase (HRT). Clinical features displayed by affected boys are particularly severe and disturbing and include hyperuricaemia, Characteristic neurological features including self-mutilation, choreothetosis, spasticity and mental retardation. A couple with a boy diagnosed with LN and a history of pregnancy termination was referred to the Hammersmith Hospital. Their affected son was born in 1982 after an uncomplicated pregnancy and vaginal delivery. Eight subsequent pregnancies had been unsuccessful. There were five therapeutic terminations and three spontaneous abortions, one at least directly caused by the sampling procedure during amniocentesis. From 1989 to 1991 two unsuccessful preimplantation genetic diagnosis (PGD) cycles by sexing were performed by DNA amplification. The mutation was characterized and a nested PCR protocol was designed which allowed the efficient amplification of the affected loci followed by the detection of the mutant allele by restriction digestion. Three PGD cycles were performed using this specific diagnostic test before a successful pregnancy was achieved resulting in the birth of a healthy unaffected baby girl.

Preimplantation genetic diagnosis of inherited cancer: familial adenomatous polyposis coli.

PURPOSE: Our purpose was to achieve preimplantation genetic diagnosis (PGD) of the dominant cancer predisposition syndrome, familial adenomatous polyposis coli (FAPC), as an alternative to prenatal diagnosis. METHODS: The affected patient was superovulated and oocytes were retrieved and fertilized by intracytoplasmic sperm injection (ICSI). Two cells were biopsied from each embryo and the whole genome was amplified by primer extension preamplification (PEP). Nested PCR was then used to amplify two APC fragments: one including the APC mutation site and the other an informative intragenic polymorphism. Both were detected by simultaneous single-strand conformation polymorphism and heteroduplex analysis. RESULTS: Four normally fertilized embryos were biopsied on day 3 post ICSI, and two cells were successfully removed from each embryo. Following PEP the APC mutation was successfully amplified in 7 of 8 cells, and the polymorphism in 6 of 8 cells. The APC mutation was detected in three embryos. This result was confirmed by identification of the mutation associated polymorphism in two cases. A single embryo was diagnosed as homozygous normal for the mutation and the polymorphism in both cells sampled. This unaffected embryo was transferred to the mother, but no pregnancy resulted. CONCLUSIONS: We report here the first diagnosis of a cancer predisposition syndrome in human preimplantation embryos. Our results indicate that difficulties associated with single-cell PCR, allele-specific amplification failure in particular, need not prevent preimplantation diagnosis of diseases with a dominant mode of inheritance, provided appropriate strategies are applied.

Assessment of the reliability of single blastomere analysis for preimplantation diagnosis of the delta F508 deletion causing cystic fibrosis in clinical practice.

Following the birth of a baby girl confirmed to be homozygous normal for the delta F508 deletion causing cystic fibrosis (CF), many single-gene defects have been diagnosed by polymerase chain reaction (PCR) for preimplantation genetic diagnosis (PGD). A few misdiagnoses have been reported but no large-scale studies have been performed to assess the accuracy of diagnosis in a clinical setting. Here we focus on a series of 15 delta F508 PGD cycles performed at the Hammersmith hospital in an 18 month period. All the spare embryos that had not been selected for transfer after clinical diagnosis were disaggregated and the blastomeres were analysed individually to confirm the clinical results and assess the reliability of single blastomere analysis by the nested PCR method. A total of 484 blastomeres from 112 embryos of different delta F508 genotypes were analysed. The amplification rate for nucleated blastomeres was 95 per cent and the overall accuracy of diagnosis was 89 per cent. Using these figures, we calculate that the chance of selecting an affected embryo instead of a homozygous unaffected or heterozygous carrier is 1.3 per cent, and 0.3 per cent of selecting an affected embryo as unaffected when heterozygotes were not considered for transfer. Misdiagnoses risks were negligible when embryos were considered for transfer after obtaining two concordant results from the same embryo. This study highlights the fact that heterozygous carrier embryos are more often associated with misdiagnoses, due to the failure of amplification of one of the two alleles in heterozygous cells (allele dropout (ADO)) and undetected contamination. In a recessive condition such as CF, ADO cannot result in a serious error. Misdiagnoses due to contamination are potentially more dangerous, they, however, can be limited by only selecting homozygous unaffected embryos for transfer as the risks are quadrupled when heterozygotes are also considered for transfer. For diagnoses of dominant conditions we strongly recommend the systematic analysis of two blastomeres per embryo and the transfer of only embryos with two independent concordant results.

Paternal transcripts for glucose-6-phosphate dehydrogenase and adenosine deaminase are first detectable in the human preimplantation embryo at the three- to four-cell stage.

The transition between dependence on maternal transcripts and proteins inherited in the oocyte and embryonic gene expression in the human preimplantation embryo occurs at the four- to eight-cell stage. Recently, studies using reverse transcriptase polymerase chain reaction (RT-PCR) have detected paternal transcripts for the Y-linked genes, ZFY and SRY, and the myotonic dystrophy associated protein kinase gene, DK, as early as the late pronucleate one-cell stage. However, expression at the protein level has not been demonstrated and its function at these early stages is unknown. Using coding sequence polymorphisms to distinguish maternal and paternal transcripts, we have examined the transcription of two ubiquitously expressed genes: X-linked glucose-6-phosphate dehydrogenase (G6PD) and adenosine deaminase (ADA). Both G6PD and ADA are housekeeping genes with TATA-less promoters which, because of their roles in metabolism and ubiquitous expression, may provide a more reliable indication of the timing of activation of the embryonic genome. They also each have biallelic polymorphisms with a high heterozygosity ratio which can be detected by restriction digestion. Couples undergoing in vitro fertilization (IVF) were screened for these polymorphisms. Individual spare oocytes and embryos at different stages of preimplantation development were analyzed by RT-PCR and appropriate restriction digestion in those cases in which the male partner carried a different allele to the female partner. In addition, since only female embryos inherit the paternal allele of X-linked G6PD, cDNA was also analyzed for ZFX/ZFY transcripts to identify the sex of each embryo. One hundred and twenty three individual oocytes and embryos were analyzed by RT-PCR and restriction digestion to detect the paternal transcripts from the polymorphic alleles. Maternal transcripts for G6PD, ADA, and ZFX were detected in all oocytes and embryos and at all stages. Following restriction digestion, paternal G6PD and ZFY transcripts were first detected at the four-cell stage and paternal ADA transcripts in an embryo at the three-cell stage coinciding with the onset of dependency on transcription from the embryonic genome. This approach should be widely applicable to other genes since similar polymorphisms exist in the coding regions of many genes.

Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients.

We have used multicolour fluorescent in situ hybridisation (FISH) with DNA probes for chromosomes X, Y and l to analyse spare untransferred cleavage-stage embryos after preimplantation diagnosis to avoid X-linked disease. In total, 93 morphologically normal embryos were available from seven patients (six of proven fertility) who had undergone fourteen in vitro fertilisation (IVF) cycles. The chromosome patterns observed were classified into four groups; normal, abnormal (non-mosaic), mosaic and chaotic (uncontrolled division). Approximately half of the embryos were normal for the chromosomes tested. Two embryos only were aneuploid (non-mosaic) throughout but, after excluding those showing chaotic division, 30% were considered to be chromosomal mosaics. Of these, a minority had arisen because of mitotic non-disjunction or chromosome loss or gain, whereas the majority were ploidy mosaics, with haploidy being the most common. The occurrence of chaotically dividing embryos was strongly patient-related, i.e. some patients had 'chaotic' embryos in repeated cycles, whereas other patients were completely free of this type of anomaly. 'Chaotic' embryos are unlikely to progress beyond implantation. These findings have important implications both for routine IVF and preimplantation genetic diagnosis.