Sean P. Flaherty

Prediction of in vitro fertilization rates from semen variables

OBJECTIVE To assess the value of semen variables for predicting fertilization rates. DESIGN Measures of the fresh semen and the motile sperm fraction used for insemination were related to the fertilization rate by multiple regression analysis. The regression model was then used to construct a two-dimensional clinical chart. SETTING University-affiliated reproductive medicine unit. PATIENTS The results of 294 IVF cycles were analyzed retrospectively. Selection criteria were: [1] first cycle of IVF; [2] tubal and/or male factor infertility; and [3] four or more oocytes inseminated. INTERVENTIONS None. MAIN OUTCOME MEASURES The fertilization rate was related to measured variables of the fresh semen and the motile sperm fraction used for insemination. Fertilization rate was categorized as poor (< 35%) or acceptable (> or = 35%). RESULTS Multiple regression analysis demonstrated a strong correlation between the fertilization rate and the combined indexes of percentage normal morphology and grade of motility in the fresh semen and percentage progressive motility in the motile sperm fraction. A two-dimensional chart that expressed these relationships was constructed. Its accuracy of prediction was 77% for poor fertilization and 95% for acceptable fertilization. CONCLUSIONS The fertilization rate is strongly correlated with percentage normal sperm morphology in the fresh semen and the percentage progressive motility in the motile sperm fraction used for insemination. The clinical chart provides a simple but powerful tool for predicting fertilization outcome.

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 eyepiece 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.

Detection of X- and Y-bearing human spermatozoa after motile sperm isolation by swim-up

OBJECTIVE To assess the ratio of X- to Y-bearing human spermatozoa in motile fractions isolated by the swim-up technique. DESIGN The proportions of X- and Y-bearing sperm were determined in neat semen samples (control) and in motile fractions isolated from the same samples by swim-up. X- and Y-bearing sperm were simultaneously identified using chromosome-specific DNA probes and double fluorescence in situ hybridization. SETTING Hospital-based university department. PARTICIPANTS Ten healthy donors with normal semen characteristics. MAIN OUTCOME MEASURES The distribution of haploid cells (X or Y), normal size cells with two sex chromosome (XX, YY, or XY), and large cells containing two (XX, YY, or XY) or four (XXYY) sex chromosomes were measured in neat semen samples and in motile fractions prepared by swim-up. RESULTS Overall, 95% of sperm in the neat semen and swim-up fractions were labeled with the probes. The ratios of X- to Y-bearing sperm were 47.3:46.9 (neat semen) and 48.4:47.1 (swim-up fractions), which were not significantly different from a 1:1 ratio. The frequencies of sperm with normal size nuclei and two sex chromosomes (XX, YY, or XY) in the swim-up fractions were not significantly different from the controls, but there was a significant reduction in the proportion of cells with large nuclei and two (XX, YY, or XY) or four (XXYY) sex chromosomes in the swim-up fractions. CONCLUSIONS The swim-up technique does not selectively enrich either X- or Y-bearing sperm. Because the isolation of motile spermatozoa is an important procedure for routine IUI, IVF-ET, and GIFT, the results of this study are important reassurance that the sex ratio is not altered by this method of sperm preparation.

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.

Cytoskeletal Assemblies of Mammalian Spermatozoaa

It is becoming increasingly evident that the different domains of the mammalian spermatozoa possess distinct cytoskeletal assemblies. In this paper we have discussed three assemblies, found in the acrosomal, postacrosomal, and midpiece segment, respectively. Each has a distinct substructure and associates with specific sperm-membrane systems. Their protein compositions are currently unidentified, and they may be comprised of sperm-specific polypeptides. Analysis of their formation and fate during sperm-egg interaction should provide valuable insight into their role in the development of cell polarity and in the membrane-mediated steps of fertilization.

FISH Analysis of Six Chromosomes in Unfertilized Human Oocytes After Polar Body Removal

AbstractPurpose: To develop an improved technique for estimatingchromosomal abnormalities in human oocytes byfluorescence in situ hybridization (FISH) and to correlate theposition of single chromatids with the chromosomal status ofthe oocytes. Methods: Oocytes that were at metaphase II about17–20 hr after insemination or intracytoplasmic sperm injection(ICSI) were treated with pronase to remove the zonapellucida and polar body (PB) and then spread on slides usingHCl and Tween 20. Two rounds of FISH were performedusing direct-labeled probes: chromosomes 1, 13, 21 (round1); 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 FISHsignals, and 3 (5%) were not analyzable. Of the 48 oocyteswith one set of DNA, 48% were haploid, 44% were aneuploidfor one or more chromosomes, and 8% were polyploid. Wealso found an increased frequency of predivision of chromatidbivalents in aneuploid oocytes, especially for chromosome 21. Conclusions: This technique enables simultaneousassessment of six chromosomes in human oocytes, and thereforecan be useful for accurately determining the incidence andcauses of genetic imbalances in human oocytes andapparently low fertilization rates.

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)

What Is the Best Strategy for Using Embryos and Presenting Results in Assisted Reproductive Technology? A Strategy for Valid Comparisons of Results

Presentation of results is now an important facet of assisted reproductive technology (ART), for quality control, counseling of patients, research and development, accreditation of clinics, and establishment of national and international registers (1). Results can also have a financial impact on clinics, especially in countries such as the United Kingdom, where publication and public scrutiny of clinical results are requirements for accreditation by the Human Fertilization and Embryology Authority (HFEA), or in countries in which there is vigorous commercial competition between clinics. An enormous number of factors influence ART results: cultural, financial, ethical, clinical, and administrative practices and clinical factors such as female age at treatment, etiology of infertility, ovarian stimulation regimes, patient history, previous treatment cycles, treatment modality, number of oocytes available for insemination, number of embryos transferred, and embryo selection (2–4). One cannot, and therefore should not, make comparisons of results unless equivalent data sets are being compared. The introduction of undue variance due to uncontrolled factors can skew the results and suggest differences that do not exist, and this in turn can lead to misinformation and unwarranted changes in clinical practice. For example, one should not compare in vitro fertilization (IVF) pregnancy rates for the United States, where it is common practice to transfer three, four, or five or more embryos, with those for other countries where only two embryos are routinely transferred, because the pregnancy rate is correlated with the number of embryos transferred (4). Another perception that arises from invalid comparisons is that one form of treatment is more effective than another. A good example of this is intracytoplasmic sperm injection (ICSI). The high initial pregnancy rates achieved after the widespread introduction of ICSI (5–7) prompted some practitioners to speculate that we should inject all oocytes and abandon routine IVF insemination. This was short-sighted for several reasons, not least of which was that it completely ignored the issue of congenital abnormalities and the developmental outcome of children produced by ICSI (8) at a time

A Prospective Trial of Intrauterine Insemination of Motile Spermatozoa Versus Timed Intercourse

STUDY OBJECTIVE The efficacy of intrauterine insemination (IUI) of selected motile sperm. DESIGN Prospective randomized sequential alternating cycle trial comparing IUI with luteinizing hormone (LH)-timed intercourse. SETTING Clinical infertility service. PATIENTS Couples selected included unexplained infertility (n = 73), cervical mucus hostility (n = 24), moderate semen defect (n = 110), and severe semen defect (n = 78). Two hundred eighty-five couples undertook 600 IUI cycles and 505 LH-timed intercourse. RESULTS Overall, IUI was slightly more effective than LH-timed intercourse with a pregnancy rate of 6.2% versus 3.4% per cycle. When individual categories were considered only, IUI for severe semen defect was significantly better (5.6% versus 1.3%, P less than 0.05). The first IUI cycle was more effective when compared with both subsequent IUI cycles and the initial LH-timed cycle. Overall, 74% (27/37) of IUI pregnancies occurred in the first cycle. CONCLUSIONS Compared with LH-timed intercourse, IUI provided little or no improved expectation of pregnancy but was beneficial in couples with severe semen defect. The occurrence of pregnancy was limited per cycle and confined essentially to the initial cycle of treatment. Continued IUI is considered to be unrewarding.