FISH analysis of six chromosomes in unfertilized human oocytes after polar body removal.

PURPOSE: To develop an improved technique for estimating chromosomal abnormalities in human oocytes by fluorescence in situ hybridization (FISH) and to correlate the position of single chromatids with the chromosomal status of the oocytes. METHODS: Oocytes that were at metaphase II about 17-20 hr after insemination or intracytoplasmic sperm injection (ICSI) were treated with pronase to remove the zona pellucida and polar body (PB) and then spread on slides using HCl and Tween 20. Two rounds of FISH were performed using direct-labeled probes: chromosomes 1, 13, 21 (round 1); chromosomes X, 7, 18 (round 2). RESULTS: Of the 63 oocytes from 18 patients (mean age, 32 years), 48 (76%) had one DNA complement as expected, 9 (14%) had 2 DNA complements, 3 (5%) gave incomplete FISH signals, and 3 (5%) were not analyzable. Of the 48 oocytes with one set of DNA, 48% were haploid, 44% were aneuploid for one or more chromosomes, and 8% were polyploid. We also found an increased frequency of predivision of chromatid bivalents in aneuploid oocytes, especially for chromosome 21. CONCLUSIONS: This technique enables simultaneous assessment of six chromosomes in human oocytes, and therefore can be useful for accurately determining the incidence and causes of genetic imbalances in human oocytes and apparently low fertilization rates.

Hepatocyte growth factor in human semen and its association with semen parameters.

Hepatocyte growth factor (HGF) is a structurally unique growth factor with potent motogenic (motility inducing) effects. Studies in the murine male genital tract have suggested important associations between HGF and the acquisition of sperm motility during epididymal maturation. The aim of this study was, therefore, to determine the concentration of HGF in human semen and assess its correlation, if any, with sperm motility and other semen parameters. Semen samples were collected by masturbation and analysed using standard procedures. HGF concentrations were measured in duplicate using an enzyme-linked immunoassay technique. Total protein estimations were also made in a subset of samples. The 95 subjects were divided into three groups for analysis: normozoospermic, subnormal semen and azoospermic. HGF was detected in all samples (median 0.456, 25th centile 0.388, 75th centile 0.556 ng/ml). No significant correlations were found between semen HGF concentrations and sperm concentration, motility, total sperm count or total motile count. There were no significant differences in mean HGF concentrations between the three subgroups. In conclusion HGF is present in human semen in significant quantities. The data do not suggest HGF concentrations are correlated with parameters of sperm motility.

Fertilization failures and abnormal fertilization after intracytoplasmic sperm injection.

This study addresses the incidence of failed (0%) and suboptimal (<50%) fertilization after intracytoplasmic sperm injection (ICSI), variation in the ICSI fertilization rate for specific couples, and the causes of fertilization failure and abnormal fertilization after ICSI. Failed fertilization occurred in only 37 of 1343 cycles (3%). The risk of failure was highest (37%) when only one oocyte was injected, and was lowest (0.8%) when five or more oocytes were collected. The incidence of suboptimal fertilization and the variation in the fertilization rate were studied in 87 couples who each had three cycles of ICSI in which four oocytes were injected with ejaculated spermatozoa. Approximately 74% of these couples achieved >50% fertilization in every cycle. Only 26% of the couples had <50% fertilization in one or more cycles, and most of these (17%) had only a single cycle with suboptimal fertilization. Only four of the 87 couples (5%) had suboptimal fertilization in all three cycles. The difference between the maximum and minimum fertilization rate for a couple was used as an index of variation of the fertilization rate. It was found that 47 couples (54%) had 0-25% variation, 33 couples (38%) had 26-50% fertilization and only seven couples (8%) had >50% variation. The causes of failed and abnormal fertilization were studied in unfertilized and abnormally fertilized oocytes after staining with Hoechst 33342. In total, 1005 unfertilized oocytes were studied, of which 828 (82%) were still at metaphase II and 177 (18%) were activated. Most of the oocytes (83%) contained a spermatozoon and, in the majority of these oocytes, the sperm head was partially or completely decondensed. Hence, failure of oocyte activation was the principal cause of fertilization failure. A similar pattern was observed in activated, unfertilized oocytes, although there was a higher incidence of intact spermatozoa in these oocytes compared with metaphase II, unfertilized oocytes. Interestingly, 56% of the activated oocytes contained a decondensed sperm head which was not processed into a male pronucleus. A total of 169 abnormally fertilized oocytes was also studied. Two anomalies were found: digyny due to retention of the second polar body and its subsequent transformation into a third pronucleus, and abnormal pronuclear size and number.

Analysis of unfertilized oocytes subjected to intracytoplasmic sperm injection using two rounds of fluorescence in-situ hybridization and probes to five chromosomes.

Chromosomal aberrations are the major cause of pre- and post-implantation embryo wastage and some studies suggest that half of all human conception have a chromosomal abnormality. Analysis of gametes provides information on the origin of these chromosomal aberrations. The purpose of this study was to develop a reliable multi-probe fluorescence in-situ hybridization (FISH) procedure that would enable us to investigate aneuploidy in unfertilized oocytes subjected to intracytoplasmic sperm injection (ICSI). Oocytes were spread with HCl and Tween 20 solution, and then two rounds of triple-probe FISH were performed on each oocyte using directly-labelled centromeric probes: chromosomes 1, 7, 15 (overnight hybridization); chromosomes 1, X, Y (2 h hybridization). After the first round, the slides were counterstained and evaluated, and the positions of FISH signals were recorded. For the second round, the counterstain was removed and the second probe cocktail was applied. The chromosome 1 probe was an internal control for the two hybridization procedures, while the Y chromosome probe was used to detect sperm DNA. To evaluate the method, a total of 79 oocytes from 27 patients were studied. Of these, 67 (84.8%) were successfully spread and 97% of these oocytes exhibited discernible FISH signals. Upon lysis, oocytes exhibited one or more DNA fragments (mean 1.9, range 1-3). Of the 65 analysable oocytes, 17 (26.2%) displayed a normal haploid chromosome constitution with paired spots for the two chromatids. A further 23 oocytes (35.4%) showed an ambiguous chromosome complement due to an abnormal number of DNA fragments which may have resulted from loss of DNA during spreading or to an abnormal oocyte, while 25 oocytes (38.4%) displayed aneuploidy for one or more of the chromosomes studied. In conclusion, this new approach is a quick and efficient method with which numerical chromosomal abnormalities in human oocytes can be studied; interpretation of the patterns of DNA fragments and FISH signals requires further clarification.

Estimation of aneuploidy for chromosomes 3, 7, 16, X and Y in spermatozoa from 10 normospermic men using fluorescence in-situ hybridization.

Fluorescence in-situ hybridization (FISH) is a fast and efficient method of estimating aneuploidy in human spermatozoa. In this study, we have estimated baseline disomy frequencies in spermatozoa from a group of 10 normospermic men, using stringent scoring criteria. A triple-probe FISH procedure was used for chromosomes 3, X and Y, while a double-probe FISH method was used for chromosomes 7 and 16. A total of 101273 spermatozoa were scored for chromosomes 3, X and Y, resulting in 97.83% haploidy (3X or 3Y), 0.39% disomy (33X, 33Y, 3XX, 3YY or 3XY) and 0.35% diploidy (33XX, 33YY or 33XY). A total of 100760 spermatozoa were scored for chromosomes 7 and 16, giving 98.9% haploidy (716), 0.11% disomy (7716 or 71616) and 0.27% diploidy (771616). Disomy frequencies for individual chromosomes differed (chromosome 3, 0.20%; chromosome 7, 0.05%, chromosome 16, 0.06%; X + Y, 0.19%). The frequency of disomy 3 was significantly higher than disomy 7 (P = 0.019) and disomy 16 (P = 0.022), while the frequency of sex chromosome disomy was significantly higher than disomy 7 (P = 0.0058) and disomy 16 (P = 0.0067), but not disomy 3 (P = 0.73). The disomy and diploidy (0.27-0.35%) estimates obtained for this normospermic population were generally low and were similar to other recent reports.

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization.

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi-probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.

Failure of multitube sperm swim-up for sex preselection.

OBJECTIVE: To use double-label fluorescence in situ hybridization to evaluate a modified swim-up procedure that is purported to be effective for preconceptual sex selection. DESIGN: Controlled, blinded study. SETTING: University hospital laboratories. PATIENT(S): Donor males reporting for routine semen analysis. MAIN OUTCOME MEASURE(S): Percentages of X- and Y-bearing spermatozoa in neat semen and in two swim-up fractions, determined using double-label fluorescence in situ hybridization. RESULT(S): No clinically significant change from a 1:1 ratio was found in the distribution of X- or Y-bearing spermatozoa after double-label fluorescence in situ hybridization following a modified swim-up procedure and irrespective of the time (15, 30, 45, and 60 minutes) allowed for swim-up. CONCLUSION(S): Using fluorescence in situ hybridization, a modified swim-up procedure was evaluated for its purported ability to skew the relative percentages of X- and Y-bearing spermatozoa. No clinically significant change in the ratio of X- to Y-bearing spermatozoa was detected independent of time. Therefore, clinical application of this procedure should be strongly discouraged.

Albumin gradients do not enrich Y-bearing human spermatozoa.

The aim of this study was to evaluate objectively whether or not discontinuous albumin gradients enrich the proportion of Y-bearing human sperm. A blinded, collaborative trial design was employed whereby a licensed centre prepared the sperm fractions using licensed procedures, coded the sperm slides and then sent them to an independent laboratory for determination of the X:Y ratio in each sperm fraction using X and Y chromosome-specific probes and double label fluorescence in-situ hybridization (FISH). The identification codes and FISH results were collated by an independent third observer. Two albumin gradient methods which are currently used by licensed centres for male sex pre-selection, protocol 3 and modified protocol 3, were tested. Essentially the same results were obtained for the two methods. Highly motile sperm fractions were recovered from the albumin gradients, and the recoveries of motile spermatozoa (1.3-8.5%) were within the optimal range reported to produce maximal enrichment of Y-bearing spermatozoa. FISH analysis, however, revealed no enrichment for Y-bearing spermatozoa with either method, and the overall X:Y ratios were not significantly different from 1.0. Some samples showed marginal enrichment of Y-bearing spermaotozoa, whereas others showed marginal enrichment of X-bearing spermaotozoa. In conclusion, this collaborative study has demonstrated that the protocol 3 and modified protocol 3 albumin gradient procedures do not enrich Y-bearing spermatozoa. The clinical use of albumin gradients for male sex preselection should be reconsidered in the light of this and other evidence.

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography.

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.

Application of modern molecular techniques to evaluate sperm sex selection methods.

The aim of this paper is to review modern approaches which have been used to evaluate sex pre-selection procedures. Two approaches can be used, polymerase chain reaction (PCR) and fluorescence in-situ hybridization (FISH). FISH is currently the method of choice for evaluating sex selection procedures because: (i) FISH accurately identifies the sex chromosome of individual spermatozoa using specific probes for the X and Y chromosomes and a two-colour detection system; and (ii) large numbers of spermatozoa can be screened in a short period of time. Of the published sex pre-selection methods tested using FISH, only flow cytometry has been shown to produce a clinically significant enrichment of X- and/or Y-bearing human spermatozoa. Studies have shown that 12-step Percoll gradients produce a slight but clinically insignificant enrichment of X-bearing spermatozoa, swim-up techniques do not appear to enrich either X- or Y-bearing spermatozoa, and discontinuous albumin gradients do not enrich Y-bearing spermatozoa. Despite this evidence, some of these methods continue to be used clinically, so it is vital that sex selection methods are properly evaluated using reliable methods such as double-label FISH before they are introduced for clinical use.

The influence of sperm morphology and the number of motile sperm inseminated on the outcome of intrauterine insemination combined with mild ovarian stimulation.

OBJECTIVE: To determine the influence of sperm morphology and the number of motile sperm inseminated on the outcome of IUI in hMG-stimulated cycles and to establish lower limits for these variables below which the expectation of pregnancy is limited. DESIGN: Retrospective study of data from 1990 to 1992. SETTING: Tertiary referral Reproductive Medicine Unit. PATIENTS: Couples with bilaterally patent fallopian tubes, and > or = 200,000 motile sperm recovered in a trial preparation before treatment. No other semen criteria were used to exclude couples. Women were stimulated with hMG irrespective of whether they were ovulatory or anovulatory. The study comprised 163 couples who underwent 330 cycles. MAIN OUTCOME MEASURES: Pregnancy rate (PR) per cycle was related to the percentage normal sperm morphology in the fresh semen sample and the number of motile sperm inseminated after sperm preparation by swim-up or Percoll gradients. RESULTS: The overall PR was 16.1% per cycle. The PR was highest in the first cycle of treatment (21.4%) and declined in the second and third cycles. The miscarriage rate was 10.4% and the incidence of multiple pregnancies was 13.9%. Two groups of patients were defined on the basis of sperm morphology: a "poor outcome" group ( < or = 10% normal) and a "good outcome" group ( > 10% normal). The PRs in these two groups were 4.3% and 18.2%, respectively, and the cumulative PRs after three cycles were 8.3% and 40.1%, respectively. The number of motile sperm inseminated did not significantly affect the PR. CONCLUSIONS: The degree of teratozoospermia affected the PR in hMG-stimulated IUI cycles and a normal morphology value of 10% in the fresh semen distinguished couples with good and poor outcomes. In contrast, the number of motile sperm inseminated did not significantly influence IUI outcome.

Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection.

The aim of this study was to determine why oocytes remain unfertilized or develop three pronuclei after intracytoplasmic sperm injection (ICSI). Unfertilized and abnormally fertilized oocytes were fixed in glutaraldehyde, stained with Hoechst 33342 and examined by fluorescence microscopy to identify oocyte, sperm and polar body DNA. One-pronuclear oocytes were considered to be unfertilized. A total of 285 unfertilized oocytes were examined (104 ICSI cycles). Overall, 83% of these oocytes were not activated (still at metaphase II) while 17% had activated and formed a single (female) pronucleus. About 66% of the unfertilized, metaphase II oocytes contained a swollen sperm head, indicating that the oocyte was correctly injected but had failed to activate and complete the second meiotic division. Premature chromosome condensation of the sperm DNA was evident in 6% of these metaphase II oocytes (4% of the unfertilized oocytes). The swollen sperm head was located among the oocyte chromosomes in 5% of the metaphase II oocytes. Other causes of failed fertilization in the metaphase II oocytes were the failure of sperm head decondensation (11%) and ejection of the spermatozoon from the oocyte (23%). A similar pattern was observed in one-pronuclear oocytes (52%, swollen sperm head; 28%, intact, undecondensed sperm head; 20%, ejection of the spermatozoon), which indicates that asynchronous pronuclear development does not explain the presence of one-pronuclear oocytes. A total of 41 three-pronuclear oocytes were examined and all had a single polar body, which indicates that the retention of the second polar body leads to the formation of the third pronucleus. In conclusion, this study demonstrates that: (i) the major cause of fertilization failure after ICSI is failure of oocyte activation; (ii) ejection of the spermatozoon into the perivitelline space is not a major cause of fertilization failure; and (iii) sperm head decondensation and oocyte activation after ICSI can occur independently.

Assessment of fertilization failure and abnormal fertilization after intracytoplasmic sperm injection (ICSI).

The assessment of fertilization is an important part of intracytoplasmic sperm injection (ICSI) and oocytes are routinely examined about 17 h after injection using Nomarski differential interference contrast optics. However, it is not possible to conclusively determine the aetiology of fertilization anomalies in this manner, so cytological studies were undertaken to determine the causes of failed and abnormal fertilization after ICSI. Oocytes which exhibited no evidence of fertilization, one pronucleus (PN) or 3 PN were fixed in glutaraldehyde, stained with Hoechst 33342 and examined by fluorescence microscopy to identify PN, metaphase chromosomes, sperm heads and polar bodies. A total of 428 unfertilized oocytes were examined from 170 ICSI cycles. Overall, 82% of these unfertilized oocytes were still at metaphase II (non-activated) while the remaining 18% were activated and had 1 PN and two polar bodies. The majority (71%) of the metaphase II oocytes contained a swollen sperm head, which indicates that the spermatozoon was correctly injected but the oocyte did not activate and complete its second meiotic division. The swollen sperm head was located among the metaphase chromosomes in 4.3% of these oocytes, while in some cases (6.6%), the sperm chromosomes had undergone premature chromosome condensation (PCC). Other aetiologies of failed fertilization in these metaphase oocytes were ejection of the spermatozoon from the oocyte (19%) and complete failure of sperm head decondensation (10%). A similar pattern of anomalies was found in 1 PN oocytes, although the ratios were different (swollen sperm head, 51%; ejection of the spermatozoon, 19%; undecondensed sperm head, 30%). Seventy abnormally fertilized oocytes were also examined, of which 63 had 3 PN and a single polar body, indicating that the unextruded second polar body developed into the third PN. In conclusion, the present study demonstrates that the principal cause of fertilization failure after ICSI is failure of oocyte activation and not ejection of the spermatozoon from the oocyte. It is also apparent that further studies are needed to elucidate the mechanisms that control oocyte activation and sperm head decondensation in injected oocytes.

Successful treatment of severe male factor infertility in 100 consecutive cycles using intracytoplasmic sperm injection.

In this report, we present the results of our first 100 consecutive cycles of intracytoplasmic sperm injection (ICSI). Overall, fertilization occurred in 98% of cycles and embryos were transferred in 94% (2.6 embryos per cycle). About 50% of patients had embryos frozen. The overall fertilization rate was 71%, of which 4% were abnormally fertilized (three pronuclei). A total of 30 clinical pregnancies were established (32% per transfer), resulting in 18 singleton, six twin and one triplet ongoing pregnancies. The implantation rate per embryo was 15%. There were no significant differences in the fertilization or pregnancy rates between patients who had only occasional motile spermatozoa in the ejaculate, semen that was too poor for routine in-vitro fertilization (IVF), or who had failed routine IVF and/or subzonal sperm injection (SUZI). A group of 18 patients were treated with both ICSI and routine IVF on their first cycle because of the high likelihood of failed fertilization due to poor sperm morphology < 20% normal). In this group, ICSI oocytes had a fertilization rate of 76% compared to only 15% for the routine IVF (control) oocytes, and six patients conceived after transfer of ICSI embryos (33%), indicating that ICSI can be used successfully on 50% of the oocytes if fertilization failure is expected. Similarly, patients who had failed to become pregnant with SUZI achieved excellent results after ICSI. There were no significant differences between ICSI and routine IVF in the proportions of grade 1, 2 or 3 embryos on day 3 post-oocyte recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

Discontinuous Percoll gradients enrich X-bearing human spermatozoa: a study using double-label fluorescence in-situ hybridization.

The aim of this study was to evaluate the efficacy of enriching X-bearing human spermatozoa using 12-step (25-80%) discontinuous Percoll gradients. X- and Y-bearing spermatozoa were simultaneously identified in neat semen (controls) and in 80% Percoll fractions from the same samples using double-label fluorescence in-situ hybridization and chromosome-specific DNA probes. Hybridization and labelling efficiencies of 95-99% were obtained in all samples. The mean ratio of X- to Y-bearing spermatozoa in the controls was 49.0:48.2, whereas there was a significant enrichment (P < 0.0001) of X-bearing spermatozoa in the Percoll fractions (mean X:Y ratio was 55.1:41.1 or 1.35). The ratio varied from 1.1-1.5 in individual Percoll samples. There were no significant differences in the proportions of aneuploid spermatozoa (XX, YY, XY) between the control and Percoll fractions. We conclude that 12-step discontinuous Percoll gradients enrich X-bearing spermatozoa, but the degree of enrichment is insufficient for use in preconceptional sex selection.

The influence of sperm morphology and the acrosome reaction on fertilization outcome after sub-zonal injection (SZI) of human spermatozoa.

The usefulness of sub-zonal injection (SZI) for the treatment of severe male factor infertility has been restricted by low and unpredictable fertilization rates and the high risk of polyspermy after the injection of multiple spermatozoa. In this prospective study, we have evaluated whether sperm morphology and the percentage of acrosome-reacted spermatozoa at the time of injection can be used to predict SZI fertilization outcomes. Populations of motile spermatozoa equivalent to those injected were collected from the medium/oil interface immediately after SZI of each cohort of oocytes. Morphology was assessed using the World Health Organization 1987 criteria and the acrosomal status of spermatozoa was determined after staining with rhodamine-conjugated Pisum sativum agglutinin. A fertilization index (FI) was calculated to express the actual fertilizing potential of the spermatozoa injected. In all, 67 patients underwent 72 SZI cycles. The overall fertilization and polyspermy rates were 36 and 47% respectively, and a clinical pregnancy rate per transfer of 22% was achieved. Linear regression analysis demonstrated a statistically significant relationship between morphology and the FI (r = 0.506, P < 0.0001). Patients with < or = 10% normal morphology always had a FI < or = 10%, and this was reflected by low fertilization and polyspermy rates and the high number (32%) of cycles with complete failure of fertilization in this group. In patients with > 10% normal morphology, there were two patterns: low (< or = 10% FI) or high (> or = 10% FI) fertility. This was evident in the fertilization (23 and 85%, respectively), and polyspermy (25 and 68%, respectively) rates of these two patient sub-groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of the separation of X- and Y-bearing sperm on albumin gradients using double-label fluorescence in situ hybridization.

OBJECTIVE: To determine the ratio of X- to Y-bearing human spermatozoa in fractions isolated from discontinuous albumin gradients. DESIGN: The proportions of X- and Y-bearing sperm were determined in neat semen samples (control) and in albumin-separated fractions from the same samples. Two albumin methods were used: a two-layer method (experiment 1) and a three-layer method (experiment 2). X- and Y-bearing sperm were identified simultaneously using chromosome-specific DNA probes and fluorescence in situ hybridization. SETTING: Hospital-based university department. PARTICIPANTS: Healthy donors with normal semen characteristics. MAIN OUTCOME MEASURES: The proportions of haploid cells (X or Y) and cells with two sex chromosomes (XX, YY, or XY) were determined. RESULTS: Labeling efficiencies were > 96% in all samples. Control samples showed a 1:1 ratio of X- to Y-bearing sperm. Fractions isolated on albumin gradients showed a slight, but statistically significant enrichment of X-bearing sperm. This was evident with both albumin methods. CONCLUSIONS: Discontinuous albumin gradients do not enrich Y-bearing sperm as previously reported.

A fluorescent in situ hybridization analysis of the chromosome constitution of ejaculated sperm in a 47,XYY male.

Two semen samples from a 47,XXY male were examined using chromosome-specific DNA probes and fluorescent in situ hybridization (FISH) to determine the distribution of sex chromosomes and an autosome (chromosome 17) in the sperm. A motile population of sperm was also prepared from one sample using the swim-up technique to compare the motile and total sperm populations. Chromosomes were localized using single FISH and a biotinylated chromosome 17 probe (TR17), or double FISH using a biotinylated X chromosome probe (TRX) and a digoxigenin-labelled Y chromosome probe (HRY). Labelling efficiencies were 95-98%. Ploidy levels were estimated by measurement against a microscope eye-piece graticule. The overall ratio of X- to Y-bearing sperm was 47% to 48.4% in the neat samples, and 48.4% to 45.3% in the swim-up fraction. Neither of the ratios was significantly different from 1:1. The frequencies of monosomic and disomic (but otherwise haploid sperm) were not different from the frequencies we observed in normal donors. In contrast, the frequencies of both diploid and tetraploid cells were increased in the neat samples of the XYY male. In the swim-up fractions, however, none of these parameters differed from those of ten normal semen donors. These results support the hypothesis that the extra Y chromosome in XYY men is eliminated during spermatogenesis.