Analysis of a malsegregating mouse Y chromosome: evidence that the earliest cleavage divisions of the mammalian embryo are non-disjunction-prone

Despite the clinical importance of human aneuploidy, we know little of the causes of mammalian non-disjunction. In part, this reflects the fact that, unlike lower organisms, segregation 'impaired' chromosomes are virtually non-existent in mammals. To address this issue, we have studied the mouse Y chromosome on the BALB/cWt ('Wt') inbred background, a system in which loss of the Y chromosome in gonadal tissue has been linked to hermaphroditism. Our results indicate that the Wt Y chromosome is stably transmitted during meiotic cell divisions, but non-disjoins at an extremely high frequency in mitosis. Surprisingly, the non-disjunction events are largely restricted to the earliest cleavage divisions, indicating that there is a temporal 'window' during which the Wt Y chromosome is susceptible to non-disjunction. The non-disjunction phenotype has both cis and trans components: the Wt Y chromosome malsegregates on a variety of genetic backgrounds, demonstrating an intrinsic defect; however, the incidence of non-disjunction is significantly influenced by strain background, indicating the existence of modifying loci and thus providing evidence for a genetic effect on mammalian non-disjunction. These studies suggest that the earliest cell divisions in mammals are non-disjunction-prone, an interpretation which provides an explanation for the high rate of chromosome mosaicism observed in studies of in vitro fertilization (IVF)-derived human preimplantation embryos. Further, our observations raise the possibility that the IVF setting adversely affects chromosome segregation and suggest that genetic quality be an important consideration in any attempt to improve or modify in vitro procedures for use on human eggs and embryos.     

Preliminary clinical experience with human blastocyst development in vitro without co-culture

This preliminary analysis was designed to quantify blastocyst development of supernumerary embryos without the use of feeder cells, conditioned medium or whole serum. Embryos derived from in-vitro fertilization (IVF) that were not transferred or cryopreserved were included in this study. Ova were harvested for IVF after a standard ovarian stimulation with gonadotrophin-releasing hormone agonist/ human menopausal gonadotrophin (GnRHa/HMG) or follicle-stimulating hormone (FSH). Ova were collected and culture in 150 microliters droplets of P1 medium under mineral oil, in groups at 37 degrees C under 5% CO2, 5% O2, 90% N2 (group A) or under 5% CO2 in air (group B) environment. Embryo transfer was performed 72 h post-harvest. Viable embryos not transferred or cryopreserved were placed in blastocyst medium and cultured for an additional 48 h in 5% CO2 in air. Embryos that exhibited an expanded blastocoelic cavity and well-defined inner cell mass at 120 h were counted. Of 838 supernumerary embryos cultured, 448 (53.5%) reached the expanded blastocyst stage by 120 h of culture. Patients were given the option of cryopreservation at that time. The embryos were cryopreserved using a standard protocol with serial addition of glycerol. Embryos reaching the blastocyst stage after more than 120 h of culture were not included. There was no difference in the proportions of blastocyst development between group A, 217/410 (53.5%) and group B, 231/428 (54%). To date, 16 patients have each had up to three thawed blastocysts transferred, out of whom seven became pregnant. This report demonstrates that a simple system of sequential culture generated acceptable, viable blastocyst development (54%) with supernumerary embryos, without the use of feeder cells, conditioned medium or whole serum. Recognizing the differential metabolic requirements of early and late cleavage stage embryos has enabled the application of a glucose/phosphate-free simple culture medium (P1) for up to 72 h of culture and a complex, glucose-containing medium (blastocyst medium) for subsequent blastocyst development.     

Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage

BACKGROUND: A correlation between morphology, developmental competence and chromosome abnormalities is established. However, since absolute correlations are rare, embryo selection remains one of the most arduous tasks in assisted reproduction. This study was undertaken in order to determine which chromosomal abnormalities are compatible with development to the blastocyst stage. METHODS: Embryos diagnosed by preimplantation genetic diagnosis (PGD) as chromosomally abnormal or unsuitable for transfer were cultured to day 5 or 6. Morphology and development were observed daily. After extended culture, embryos were fixed and analysed by two rounds of FISH with the same probes used for PGD. RESULTS: Some types of numerical chromosome abnormalities do not preclude full differentiation in vitro. For instance, extensive mosaicism was detected in blastocysts and trisomic embryos reached the blastocyst stage with a frequency of 37%. Interestingly, only those monosomies compatible with first trimester development (monosomy X and 21) were detected at blastocyst stage. CONCLUSION: Even though there is a strong selection against chromosomally abnormal embryos, extended culture to day 5 or 6 cannot be used as a reliable tool to select against clinically relevant chromosome abnormalities such as trisomies.     

In-vitro co-culture of early stage caprine embryos with oviduct and uterine epithelial cells.

Early stage caprine embryos were incubated with goat oviduct and uterine cells to evaluate whether these cells could be used as a somatic cell culture system to enhance development through the developmental block at the 8- to 16-cell stage during in-vitro culture. Following gonadotrophin treatment and natural mating, 2- to 4-cell embryos were surgically recovered from donor females for in-vitro culture studies. In Experiment 1, embryos were equally and randomly allotted to culture treatments of either culture medium plus caprine oviduct cells or culture medium alone. In both treatment groups, embryos were incubated in Medium-199 with 10% fetal bovine serum, 0.25% lactalbumin and 1% antibiotic-antimycotic at 37 degrees C in a humidified atmosphere of 5% CO2 in air. In Experiment 2, similar embryos were cultured in the same medium with either caprine oviduct cells, caprine uterine cells or sequentially incubated with oviduct cells and then uterine cells during a corresponding incubation interval. The culture conditions in Experiment 2 were the same as in Experiment 1. Following 72 h in culture, (Experiment 1), significantly more embryos developed through the in-vitro developmental block into blastocysts and hatched blastocysts when cultured with oviduct cells compared with no embryos developing through the in-vitro block when incubated with medium alone. In Experiment 2, caprine embryos co-cultured with oviduct cells alone resulted in more embryos developing into blastocysts and hatched blastocysts compared with those co-cultured with uterine cells alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation between fluorescence in-situ hybridization analyses and in-vitro development to blastocyst stage of embryos from Robertsonian translocation (13;14) carriers

BACKGROUND: Little is known about the extent and timing of selection against the embryos that are carriers of unbalanced translocations. METHODS: Fluorescence in-situ hybridization (FISH) with probes for chromosomes 13, 14 and 18 was performed, mostly on day 3, on 69 human embryos which were then allowed to develop further in culture to day 5, from five carriers of Robertsonian translocation (RT) t(13;14). RESULTS: Twelve normal/balanced blastocysts were replaced in seven consecutive cycles (day 5). Three cycles resulted in clinical pregnancies. The proportion of blastocysts displaying a normal/balanced karyotype was 56%, while only the 20% of blocked embryos were normal/balanced (chi(2): P < 0.05). All the embryos analysed on day 5, except one, displayed mosaicism. The percentages of diploid cells for chromosomes 13 and 14 were significantly lower than for chromosome 18 (chromosome 13: 49.0 +/- 28.0; chromosome 14: 53.0 +/- 31.8; chromosome 18: 75.7 +/- 20.4; Mann-Whitney test: P < 0.01). The embryos displaying vertical line 62% of diploid cells for at least two of the three chromosomes analysed, more frequently reached the blastocyst stage (blocked embryos: blastocysts chromosome 13: 43.1 +/- 30.3, 64.9 +/- 29.0; chromosome 18: 64.9 +/- 29.0, 83.0 +/- 12.9; Mann-Whitney test: P < 0.01). CONCLUSIONS: Normal/balanced embryos developed better but the proportion of abnormal blastocysts was still high. Preimplantation genetic diagnosis is recommended to select normal/balanced embryos from RT t(13;14) carriers.     

许昌地区268例男性不育患者的细胞遗传学研究

目的 分析研究少精子症、死精子症、无精子症患者的遗传缺陷与男性不育的关系，并探讨该类患者在接受辅助生殖技术治疗前进行细胞遗传学检查的重要性。方法 采用外周血淋巴细胞染色体培养技术进行染色体核型分析。结果 268例患者中共检出异常核型65例，异常率24．3％（65／268）；其中性染色体数目异常58例，占89．3％；染色体结构异常3例，占4．6％；大Y3例，占4．6％；性反转46，XX1例，占1．5％。160例正常生育男性的染色体核型分析．共检出异常核型2例，其中大Y1例，小Y1例，异常率1．25％（2／160）。结论 染色体核型异常时引起男性不育症的重要因素之一，这类不育患者在接受辅助生殖助孕技术前进行染色体核型分析，避免将各种遗传缺陷传递给下一代，对优生优育起着重要作用。     

High oxygen atmosphere improves human follicle development in organ cultures of ovarian cortical tissues in vitro

BACKGROUND: Obtaining mature human follicles from cultured ovarian tissue may be beneficial for clinical use for women who wish to preserve fertile competence. However, the methodology of culture such as culture condition and gas atmosphere has not been well established in humans. Therefore, we investigated the effect of oxygen concentration in organ culture in order to establish an ovarian tissue culture method. METHODS: Ovarian tissue was obtained from 26-35-year-old women undergoing removal of a benign tumor (n = 12) or caesarean section (n = 16). The ovarian cortical tissues were cultured on a cell culture insert for 15 days under high (100%) and low (air, 20%) oxygen concentrations and then inspected for follicle development with light and electron microscopy. Estradiol and progesterone concentrations in the medium during culture were measured. RESULTS: The ultrastructure and the function of hormone secretion in the cultured tissues were well preserved after organ culture. The follicles developing under high oxygen were larger and more matured than those developing under low oxygen (P < 0.05). CONCLUSIONS: Human ovarian tissues can be cultured for 15 days under high oxygen concentration with the organ culture system used here. This technique could make it possible to utilize ovarian tissue for preservation of reproductive competence in cancer patients.     

In vitro culture of peri-gastrulation embryos of a macropodid marsupial

Peri-gastrulation stage tammar wallaby embryos were cultured for up to 78 h in either Dulbecco's Modified Eagle's Medium or Medium 199, in air/6% CO(2) or 95% O(2)/5% CO(2), and with added fetal calf or wallaby serum. There was little difference between the two media or sera sources, but development was markedly superior for embryos cultured in 95% O(2)/5% CO(2). Many embryos survived even prolonged culture periods up to and over 70 h, and although development continued throughout the culture period, the embryos as a whole became increasingly abnormal. Embryos explanted at the primitive streak/ regressing node stages performed better in vitro than embryos explanted at earlier or later stages. The embryo that developed the furthest had a newly formed node at the initiation of culture and after 64 h in vitro it had developed forelimb ridges, fused, beating heart tubes and mesonephric ducts. Thus high oxygen appears to be the critical component of the culture system for optimal development of primitive streak stage tammar embryos. These results provide a basis for developing culture conditions for longer term development of marsupial embryos in vitro.     

Granulocyte-macrophage colony-stimulating factor promotes human blastocyst development in vitro

The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is synthesized in the female reproductive tract and has been implicated in the growth and development of the preimplantation embryo in rodent and livestock species. To examine the effect of GM-CSF on human embryo development in vitro, surplus frozen 2-4-cell embryos were cultured in media supplemented with 2 ng/ml recombinant human GM-CSF. The addition of cytokine increased the proportion of embryos that developed to the blastocyst stage from 30 to 76%. The developmental competence of these blastocysts, as assessed by hatching and attachment to extracellular matrix-coated culture dishes, was also improved by GM-CSF. The period in culture required for 50% of the total number of blastocysts to form was reduced by 14 h, and blastocysts grown in GM-CSF were found to contain approximately 35% more cells, due primarily to an increase in the size of the inner cell mass. The beneficial effect of GM-CSF was exerted in each of two sequential media systems (IVF-50/S2 and G1. 2/G2.2) and was independent of the formulation of recombinant cytokine that was used. These data indicate that GM-CSF may have a physiological role in promoting the development of the human embryo as it traverses the reproductive tract in vivo, and suggest that addition of this cytokine to embryo culture media may improve the yield of implantation-competent blastocysts in human in-vitro fertilization programmes.     

Live-birth rates and multiple-birth risk of assisted reproductive technology pregnancies conceived using thawed embryos,USA 1999-2000

BACKGROUND: Increasing use of assisted reproductive technology treatments has been associated with the current rise in multiple births in the USA. Embryo cryopreservation and subsequent thawed embryo transfer may favourably impact the multiple-birth risk by relieving some pressure that patients and providers may feel to transfer several embryos in a single cycle. The study objective was to examine both live-birth rates and multiple-birth risk in thawed cycles. METHODS: The authors used a population-based sample of 21 555 assisted reproductive technology procedures performed in US clinics in 1999 and 2000 that used thawed embryos derived from the patient's oocytes. RESULTS: Both patient age and the number of embryos transferred were independent predictors of live birth. Even among women aged 20-29 years, the transfer of three embryos resulted in an increase in the live-birth rate compared with cycles in which one or two embryos were transferred. This increase in success was accompanied by an increased multiple-birth risk. In all age groups up to 40 years, the transfer of just two embryos resulted in a multiple-birth risk of 16-17%. The multiple-birth risk increased with the number of embryos transferred. CONCLUSIONS: Patient age and the number of embryos transferred significantly affect live-birth and multiple-birth rates among women who use thawed embryos.     

Intracytoplasmic sperm injection (ICSI) with transmission of a ring(Y) chromosome and ovotesticular disorder of sex development in offspring

We present a newborn infant with ovotesticular disorder of sex development and sex chromosome mosaicism with a supernumerary ring(Y), and a normal female cell line (47,XXr(Y)[10]/46,XX[40]. The ring (Y) was inherited from the child's father, and was transmitted following assisted reproductive technology and intracytoplasmic sperm injection (ICSI). The father presented with infertility and oligospermia, but cytogenetic analysis had not been carried out as part of the infertility workup. The Y containing cell line had not been seen on amniocentesis, which had shown a 46,XX apparently normal female karyotype in all cells studied. Molecular analysis using polymorphic probes from the X chromosome demonstrated that the 47,XXr(Y) cell line in the child was consistent with inheritance from the father, following meiosis I paternal non-disjunction. This report underscores the need to obtain chromosome analysis in couples with infertility who undergo assisted reproduction.     

Developmental potential of human spermatogenic cells co-cultured with Sertoli cells.

BACKGROUND: Development of an in-vitro culture system capable of supporting human early germ cell differentiation would be important for treatment of azoospermic patients. METHODS: Sertoli cells, spermatogonia and spermatocytes were isolated from testicular biopsies of 61 non-obstructive azoospermic patients, and co-cultured using Vero cell conditioned medium only or supplemented with recombinant (r)FSH or rFSH plus testosterone. Germ cell purity was checked by fluorescent in-situ hybridization (FISH) analysis. RESULTS: Best results were achieved with both hormones, which elicited 6.9% of meiosis index and 22.7% of differentiation into normal late spermatids after 2-3 weeks of culture. In-vitro matured spermatids were microinjected into oocytes to study their developmental potential. Round spermatids elicited 37.5% of fertilization and 28.6% blastocyst rates. Abnormal elongating and elongated spermatids enabled 8.3 and 27.3% fertilization rates respectively, but none achieved the blastocyst stage. Normal elongating and elongated spermatids elicited 30.5% fertilization and 42.9% of blastocyst rates. FISH analysis showed sex chromosome anomalies in all embryos, except in the case of morulae from normal late spermatids. CONCLUSIONS: Results suggest that meiosis and spermiogenesis can be resumed in vitro, with normal differentiated spermatids showing a low fertilization potential but regular rates of blastocyst formation. However, most of the embryos did not reach the morula stage and showed major sex chromosome abnormalities.     

Development of in-vitro-derived bovine embryos cultured in 5% CO2 in air or in 5% O2, 5% CO2 and 90% N2

To evaluate the effects of a three gas mixture of 5% O2, 5% CO2 and 90% N2 (OCN) on preimplantation embryo development, bovine in-vitro fertilization (IVF) oocytes were cultured in a defined medium (mBECM) with various supplements either under 5% CO2 in air or under OCN. When cultured in mBECM alone, embryo development was significantly stimulated in OCN compared to 5% CO2 in air (experiment 1). In the OCN atmosphere, blastocyst formation was further increased after addition of fetal bovine serum (FBS; 10%) or FBS + cumulus granulosa cells (CGC) to mBECM. The ratio of blastocysts to 8-cell embryos, number of hatched blastocysts and embryo diameter were markedly increased, and zona thickness was decreased after FBS addition. However, development up to the morula stage was fully supported by mBECM alone. There was no significant effect of beta-mercaptoethanol (ME; 10 microM) in OCN. In the 5% CO2 atmosphere, embryo development was significantly (P < 0.05) enhanced after addition of FBS + CGC + ME. In experiment 2, in OCN, FBS added at 60 h post-insemination was effective in stimulating blastocyst formation, but changes in medium volume per oocyte from 13.6 to 1.36 microliters had only a marginal effect. In conclusion, OCN gas mixture provides a suitable atmosphere for early embryo growth in vitro and mBECM + FBS in the optimal culture medium under this atmosphere.     

Block to development in cultured rat 1-cell embryos is overcome using medium HECM-1.

Rat pronuclear embryos were cultured in hamster embryo culture medium-1 (HECM-1) or a modified Krebs-Ringer bicarbonate solution (mKRB). Embryo cultures in HECM-1 were also challenged with low oxygen concentrations. In HECM-1, 57.9% (70/121) of the pronuclear embryos developed into the 4-cell stage after 48 h of culture. The rates of 8-cell, morula and blastocyst formation were 32.2% (39/121), 17.4% (21/121) and 9.9% (12/121), respectively. On the other hand, in mKRB, rat pronuclear embryos showed developmental blockages at the 2-cell and 4-cell stages, and never developed beyond the 4-cell stage. The rate of blastocyst formation under a low oxygen concentration was 20.1% (43/214), showing a significant difference from the value of 5.5% (11/201) obtained under a standard oxygen concentration (P less than 0.005). This is the first report of successful culture of rat pronuclear embryos to the blastocyst stage. Furthermore, it is suggested that protection from oxidation stress is a prerequisite for rat embryo development in vitro.     

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.     

Assisted reproduction with in-vitro-cultured testicular spermatozoa in cases of severe germ cell apoptosis: a pilot study.

BACKGROUND: Apoptosis-related cell damage is known to compromise success rates of assisted reproduction with ejaculated spermatozoa. This study was undertaken to determine whether the frequency of apoptosis-related cell damage and reproductive performance of testicular spermatozoa from men with non-obstructive azoospermia can be improved by in-vitro culture. METHODS: Testicular tissue samples were cultured for 2 days in the presence of 50 IU/l FSH and 1 micromol/l testosterone. The frequency of spermatozoa showing DNA strand breakage and plasma membrane phosphatidylserine externalization was compared in before-culture and after-culture samples. The after-culture samples were used in assisted reproduction attempts. RESULTS: In a group of 11 azoospermic patients with at least two previous intracytoplasmic sperm injection (ICSI) failures, the incidence of DNA strand breakage was high in living testicular spermatozoa from before-culture samples, but significantly lower in after-culture samples (96 versus 30%, P < 0.001). The same applied to the incidence of phosphatidylserine externalization in the motile sperm subpopulation from the before-culture and after-culture samples (83 versus 6%, P < 0.001). Seven ongoing clinical pregnancies (six with fresh embryos and one with cryopreserved embryos) were established. CONCLUSIONS: Severe testicular sperm apoptosis may become a new indication for testicular tissue in-vitro culture before ICSI.     

Development of an siRNA-based method for repressing specific genes in renal organ culture and its use to show that the Wt1 tumour suppressor is required for nephron differentiation

Wt1 is a tumour suppressor gene, mutation of which is a cause of Wilms' tumour, a childhood renal nephroblastoma. Wt1 is expressed in a rich pattern during renal development suggesting that it acts at three stages: determination of the kidney area, the differentiation of nephrons and maturation of glomeruli. Wt1-/- mice confirm that Wt1 is essential for the inception of kidney development; cells that ought to form kidneys die by apoptosis instead. Specific human WT1 mutations cause defects of glomerular maturation (Denys-Drash and Frasier syndromes), providing circumstantial evidence for action of Wt1 during glomerular maturation. There is, however, no genetic evidence for a function during nephron differentiation because this stage is never reached in Wt1-/- mice. We have therefore developed a novel technique, based on small interfering RNA (siRNA), to repress the expression of Wt1 and other specific genes at different stages of kidney development in culture. We find that early repression of Wt1 phenocopies the Wt1-/- mouse, but later repression prevents cells differentiating into nephrons and causes them instead to proliferate abnormally, possibly mimicking aspects of Wilms' tumour. In line with established hypotheses about genetic pathways that control kidney development, we find that repressing Pax2 using siRNAs represses Wt1 expression and blocks both bud growth and nephron differentiation, but that repressing Wnt4 blocks nephron differentiation without affecting Wt1 expression. As well as illuminating previously inaccessible aspects of Wt1 biology, our results suggest that siRNA in organ culture will be a powerful method for analyzing other developmental pathways and testing the effects of stage-specific loss of tumour suppressor genes.     

Extended embryo culture in human assisted reproduction treatments

In order to evaluate the niche of extended embryo culture in an IVF programme, retrospective analysis of non-selected IVF patients, who underwent ovarian stimulation from April 1998 to June 1999 in a single private practice assisted reproductive technology centre, was performed. Embryos were cultured for 48 h in S1/G1.2 medium followed by 48 to 72 h of culture in S2/G2.2 to day 5 or day 6. Only fertilized oocytes exhibiting two pronuclei from donor and non-donor IVF and intracytoplasmic sperm injection (ICSI) cases were examined to determine the relationship between embryo cell number on day 3 and subsequent rate of blastocyst formation. Results indicated that a proportional relationship existed between the number of blastomeres present in day 3 embryos and the rate of blastocyst formation. Fifty-four per cent of embryos that had six cells on day 3 formed blastocysts, while 76% of those embryos with eight cells formed blastocysts. Blastocyst development did not increase further when embryos had more than eight cells on day 3, indicating that embryos with greater cell numbers on day 3 are not always predictive of a greater likelihood of blastocyst formation. Fertilized oocytes exhibiting two pronuclei from donors produced significantly more blastocysts (67%) than those from IVF patients (52%; P < 0.01), and had a significantly higher implantation rate (54%) compared with IVF patients (30%; P < 0.01). Furthermore, blastocyst cryopreservation resulted in significantly higher implantation rates than cryopreserved cleavage stage embryos (P < 0.001).     

Screening for microdeletions of Y chromosome genes in patients undergoing intracytoplasmic sperm injection.

The potential of assisted reproduction techniques to transmit genetic defects causing male infertility raises questions concerning the need for a systematic genetic screen and counselling. Deletions of the long arm of the Y chromosome are frequently associated with a failure of spermatogenesis. The search for Y specific sequences and for the gene families RNA binding motif (RBM) and deleted in azoospermia (DAZ) have been introduced in many laboratories. The incidence of Y microdeletions varies widely between studies, from 1-55%. These differences are mainly related to study design. The highest incidence of microdeletions has been reported in well selected idiopathic azoospermic patients. Since microdeletions have been reported also in non-idiopathic patients, it is important to define what is the deletion frequency in unselected patients. We report Y chromosome microdeletion screening in 134 unselected patients undergoing intracytoplasmic sperm injection (ICSI). In the first part of the study we tested six Y chromosome markers. We found three patients with microdeletions (2.2%). Subdivision of the study population revealed a deletion incidence of 4.7% in azoospermic/cryptozoospermic patients; an incidence of 7% in idiopathic patients and an incidence of 16% in idiopathic azoospermic/cryptozoospermic patients. The second part of the study consisted of a screen for the presence of the Y chromosome genes, DBY, CDY, XKRY, eIF-1A, DAZ and BPY2. No additional gene-specific deletions were found. Further data on gene specific screening are needed especially for selected idiopathic patients.