Flow cytometric sorting of human sperm: MicroSort clinical trial update.

This report provides a summary of MicroSort efficacy in separation of X- from Y-chromosome bearing human sperm (XSort and YSort, respectively), clinical outcomes, and the sex of the resultant babies when sorted sperm were used for intrauterine insemination (IUI), in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). Clinical trial participants were married couples seeking reduced X-linked genetic disorder risk or family balancing. Sperm were stained with Hoechst 33342, sorted by flow cytometry, then used or cryopreserved for subsequent use. Fluorescence in situ hybridization (FISH) analysis determined the post-sort enrichment (purity) for X- and Y-bearing sperm. Birth and pediatric records were evaluated for incidence of congenital malformations. Between June 1994 and January 2007, patients underwent 3629 IUI cycles, 1642 IVF/ICSI cycles with fresh embryo transfer (ET) and 99 frozen embryo transfer (FET) cycles after MicroSort. Of 5871 total sorts, 74.9% were XSort and 25.1% were YSort. IVF/ICSI fertilization rate was 70.7% and 93.8% of 2PN embryos cleaved. The pregnancy rates for IUI, IVF/ICSI, and FET were 15.6, 32.0, and 33.3%, respectively, while miscarriage rates were 15.7, 14.3, and 33.3%, respectively. Post-sort purity averaged 87.9% (XSort and 73.4% (YSort. A total of 1125 clinical pregnancies yielded 943 babies born and 167 ongoing pregnancies. For babies born, XSort resulted in 92.0% females and YSort yielded 81.5% males. Postnatal follow-up showed a 2.6% major congenital malformation rate, with no recurrent pattern or clustering of malformations. FISH results confirmed MicroSort enrichment of X- and Y-bearing sperm populations that closely corresponded with the sex of the resultant child. Fertilization, cleavage, spontaneous abortion, and pregnancy rates as well as incidence of major congenital malformations were comparable to those in literature reports utilizing unsorted sperm.

Presence of N-cadherin transcripts in mature spermatozoa.

The essential mechanism involved in sperm-oolemma fusion has yet to be elucidated. Recognition and binding is initiated by specific cell surface receptor engagement between gametes. Fusion between hamster oolemma and spermatozoa is prevented in the presence of trypsin in Ca(2+)-free media, as is oocyte activation, implicating a cadherin-like adhesion. Cadherins are a family of Ca(2+)-dependent adhesion molecules that bind homotypically with their target, are morphoregulatory and function eptopically to affect tissue form and function. Cadherins and cadherin-associated molecules have been identified in testes and germinal cells, as well as ejaculated spermatozoa. Moreover, cadherins are also present in oocytes and may suggest a cadherin-mediated adhesion in sperm-oocyte interaction. We have detected antigenic epitopes recognized by N-cadherin monoclonal antibodies diffusely distributed over the entire sperm head. In addition, Western blot analysis confirmed the presence of an antibody reactive peptide in spermatozoa, testis and ovary protein extracts at the expected molecular weight for authentic N-cadherin. Total RNA was isolated from mature motile spermatozoa, as well as ovary and testis tissue, and served as template for reverse transcription-polymerase chain reaction (RT-PCR) with N-cadherin specific primers. Alignment of sequences from PCR products of testis, ovary and spermatozoa with published N-cadherin sequence was identical except for occasional base changes. We intend to develop methods to analyse this transcript from small numbers of spermatozoa from a variety of donors to determine if defects in cadherin distribution or structure may predict reduced male fertility.

L-type voltage-dependent calcium channel alpha-1C subunit mRNA is present in ejaculated human spermatozoa.

The current study adds to the growing body of evidence that RNA is present in mature ejaculated human spermatozoa. We report that a sodium dodecyl sulphate (SDS)/citric acid extraction method is superior to guanidinium isothiocyanate in terms of reproducibility of RNA recovery from motile sperm populations from individual ejaculates. Using the SDS/citric acid method, RNA was recovered from both fresh and frozen-thawed motile spermatozoa. Sperm RNA were used as templates in reverse transcription-polymerase chain reaction (RT-PCR), in an attempt to identify partial RNA transcripts of a highly conserved region within the alpha-1C (pore-forming) subunit of L-type voltage-dependent calcium channels from 11 individual donors. Control reactions employed primers derived from the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequence. In nine of the 11 specimens, gene-specific PCR products were obtained with both the GAPDH and alpha-1C primer pairs. DNA sequencing analysis confirmed that the respective spliced transcripts were amplified. The two cases in which no amplification was obtained were attributed to reduced RNA yield. These data are consistent with results from in-situ RT-PCR of rat testis sections indicating that the testis-specific calcium channel of that species was expressed uniformly in all stages of the germinal epithelium, including mature spermatozoa.

Can spermatozoa with abnormal heads gain access to the ovum in artificially inseminated super- and single-ovulating cattle?

The collective efficiency of barriers in the female tract against spermatozoa with abnormal heads was studied. In Experiment 1, Day 6 ova/embryos were recovered nonsurgically from superovulated (n = 24) and single-ovulating (n = 44) cows following artificial insemination with semen of bulls selected for normal spermatozoal motility (> or = 50%) and high content (> 30%) of spermatozoa with misshapen heads, random nuclear vacuoles or the diadem defect. To assess characteristics of spermatozoa capable of traversing barriers in the female tract, accessory spermatozoa were classified morphologically (x 1250) and compared with those of the inseminate. Superovulated cows proved inadequate for assessment of accessory spermatozoa due to evidence of poor sperm retention in the zona pellucida; thus, only single-ovulating cows were used. Accessory spermatozoa (n = 479) from 31 ova/embryos recovered from 44 cows were more normal in head shape than those in the inseminate (76 vs 62%; P < 0.05). Spermatozoa with normal head shape, but with nuclear vacuoles appeared as accessory spermatozoa at the same frequency as they were found in the inseminate (20 vs 17%, respectively). Only sperm cells with subtly misshapen heads appeared as accessory spermatozoa. In Experiment 2, semen pooled from 4 bulls having large numbers of spermatozoa exhibiting a gradation from severely asymmetrically misshapen heads to subtly misshapen heads was evaluated. Again, the accessory sperm population (960 sperm cells recovered from 64 ova/embryos) was enriched with spermatozoa of normal head shape relative to the inseminate (53 vs 26%, respectively; P < 0.05). Sperm cells with only nuclear vacuoles and those with subtly misshapen heads were not different between the accessory and inseminate populations (11 vs 8%, and 20 vs 25%, respectively). We conclude that morphologically abnormal spermatozoa are excluded from the accessory sperm population based upon severity of head shape distortion.

Chromatin structural changes in sperm after scrotal insulation of Holstein bulls.

The reported effects on semen quality ascribed to testicular heat stress generally relate to traits impacting sperm transport and fertilizing ability but not to the genetic material contained by the sperm. To characterize the effects of testicular heat stress on sperm chromatin, susceptibility of DNA in sperm nuclear chromatin to in situ acid denaturation was measured by flow cytometry after staining with acridine orange using the sperm chromatin structure assay (SCSA). Semen was collected from Holstein bulls at 3-day intervals, before and after 48-hour scrotal insulation, until the morphologically abnormal sperm content in raw semen exceeded 50%. After cryopreservation in egg yolk-citrate extender, semen was thawed and sampled during incubation in vitro at 38.5 degrees C. Overall, SCSA results showed that chromatin susceptibility to denaturation was increased for sperm collected post- vs. preinsulation and was more pronounced for sperm presumably in the testes during insulation than for those sperm presumably in the epididymides. Increased susceptibility was detected as early as the first collection postinsulation; however, chromatin of sperm presumably in the proximal epididymis during insulation did not appear to have been detrimentally affected. Chromatin susceptibility to denaturation increased with increased incubation time in vitro, but the rate of change in susceptibility during incubation did not differ among pre- vs. postinsulation specimens. We conclude that elevated scrotal temperatures adversely affect both epididymal and testicular sperm by reducing sperm chromatin stability. The effects of heat stress on the chromatin of epididymal sperm were more subtle than those exhibited by testicular sperm but detectable within close proximity to the heat stress event.

The impact of sperm morphology evaluated by strict criteria on intrauterine insemination success.

OBJECTIVE: To determine the relationship between IUI success and the level of morphologically normal sperm, evaluated using strict criteria, in the raw semen. DESIGN: Evaluation of semen quality characteristics and pregnancy results for 538 stimulated IUI cycles. SETTING: University medical center infertility clinic. PATIENT(S): Women undergoing IUI with their partner's semen as treatment for infertility (n = 193). INTERVENTION(S): Ovulation induction using clomiphene citrate, hMG, or both; preparation of raw semen using wash and swim-up or Percoll; deposition of prepared semen at the uterine fundus. MAIN OUTCOME MEASURE(S): Pregnancy status after IUI. Percentage morphologically normal sperm in raw semen, evaluated using strict criteria. Sperm concentration and percentage motile sperm in raw and prepared semen. RESULT(S): Pregnancy rates (PRs) per cycle were not different when the percentage of morphologically normal sperm in raw semen was < 5%, 5% to 9%, 10% to 19%, 20% to 29%, and > or = 30% (6.5% +/- 3.9%, 13.6% +/- 3.2%, 8.8% +/- 2.4%, 7.1% +/- 2.5%, and 9.7% +/- 3.3%, respectively). Pregnancy rates did not differ among age groups, infertility diagnoses, ovarian stimulation protocols, or semen preparation methods. CONCLUSION(S): The percentage of morphologically normal sperm in the raw semen, as judged by strict criteria, did not affect IUI PR. Intrauterine insemination appears to be a successful treatment modality for male factor infertility, even when the percentage of morphologically normal sperm in raw semen is very low.

Dithiothreitol effects on human sperm quality.

Human semen normally coagulates immediately after ejaculation and then undergoes liquefaction during the next 15 to 60 minutes. Incomplete seminal liquefaction can result in impaired sperm motility and make clinical evaluation and manipulation difficult. Dithiothreitol, a mucolytic agent that reduces the mucoprotein disulfide bonds in sputum, has been found to induce liquefaction of incompletely liquefied semen in vitro. We studied the effects of dithiothreitol on sperm motility, viability, acrosomal integrity and morphology. A semen sample was provided by 45 healthy, young men at the University of Arizona. Of the specimens 10 (22%) demonstrated incomplete liquefaction. Sperm motility and motion characteristics of untreated (control) semen and semen treated with dithiothreitol were objectively evaluated using computer assisted semen analysis. Sperm cell membrane integrity and mitochondrial integrity were measured by fluorescence microscopy using the deoxyribonucleic acid specific fluorochrome propidium iodide and the mitochondria specific fluorochrome rhodamine-123, respectively. Acrosomal integrity was determined using the fluorescent stain chlortetracycline. Sperm morphology was evaluated using bright field microscopy. For completely liquefied semen (35 cases) dithiothreitol reduced sperm motility (59.1 +/- 1.2% untreated versus 53.2 +/- 1.2% treated, p < 0.01) and motion characteristics. However, dithiothreitol had no statistically significant effect on motility on sperm in the group with incompletely liquefied semen (10 cases). Sperm cell membrane, mitochondrial and acrosomal integrity was unaffected by dithiothreitol regardless of liquefaction status. Dithiothreitol caused a significant increase in abnormally large sperm head morphology in the group with completely liquefied semen. The minimal effects of dithiothreitol on sperm motility traits and viability support its use as a possible aid in the evaluation and manipulation of incompletely liquefied semen.

Effects of egg yolk-citrate and milk extenders on chromatin structure and viability of cryopreserved bull sperm.

Semen from four Holstein bulls was evaluated to compare effects of four extender treatments on postthaw semen quality. Extender fractions A and B, either heated whole milk or 20% egg yolk-citrate, were combined to yield the extender treatments 1) milk and milk, 2) milk and egg yolk-citrate, 3) egg yolk-citrate and milk, and 4) egg yolk-citrate and egg yolk-citrate. Semen was evaluated at thawing and after 30, 60, 120, and 180 min of incubation at 38.5 degrees C. Flow cytometry showed that acridine orange-stained sperm were most susceptible to in situ DNA denaturation when fraction A was milk. For sperm stained with rhodamine 123, flow cytometry showed that the proportion with intact mitochondrial membrane potential was lowest of all treatments at thawing but greatest at 180-min incubation with milk and milk extender. Flow cytometry of propidium iodine-stained sperm showed greatest proportion of cell membrane intact sperm when fraction A was egg yolk-citrate. Light microscopy showed the lowest proportion of cell membrane intact sperm with milk and milk extender after eosin-aniline blue vital staining. Postthaw motility scores tended to be reduced when both extender fractions were egg yolk-citrate. Results demonstrate differential extender effects on postthaw semen quality and indicate that altering extender composition or sequence of adding extender components may improve postthaw quality of cryopreserved sperm.

Comparison of semen quality in young and mature Holstein bulls measured by light microscopy and flow cytometry.

Random samples of cryopreserved, milk-extended semen, collected from 20 Holstein bulls at about 14 mo of age (young) and again at about 4 yr of age (mature), were evaluated at thawing and during 3-h incubation to compare semen quality of young versus mature bulls. Evaluation by differential interference contrast microscopy showed greater proportions of cytoplasmic droplets in semen from young versus mature bulls. Mature bulls exhibited greater proportions of intact acrosomes in freshly thawed semen than did young bulls. Evaluation of sperm chromatin structure by flow cytometry after staining with acridine orange showed lower values for mature versus young bulls, indicating resistance of DNA in nuclear chromatin to acid denaturation increased with age. Correlations between ages for most sperm morphology, acrosome integrity, and flow cytometry variables were high and positive. Nonreturn rate for young bulls was positively related to morphologically normal sperm and acrosomal integrity and negatively related to flow cytometry traits. Results suggest semen quality of young bulls was related to subsequent quality as mature bulls. With flow cytometry, differences were detected between semen samples that were not evident with light microscopy.

Effect of 1,3-dinitrobenzene on prepubertal, pubertal, and adult mouse spermatogenesis.

Exposure of prepubertal, pubertal, and adult mice to 0, 8, 16, 32, 40, or 48 mg 1,3-dinitrobenzene (m-DNB)/kg body weight and measuring responses 1-25 d posttreatment (dpt) demonstrated significant effects on testicular function only at 48 mg/kg dosage. m-DNB had no effect on body or testis weights with the exception of reduced adult mouse testis weights at 22 dpt with 48 mg/kg (p less than .05). None of the exposures resulted in detectable levels of germinal epithelial cells in the ductus epididymis. Exposure of prepubertal and pubertal mice to m-DNB caused only minimal nonsignificant changes in the relative percent of testicular cell types present up to 25 dpt. The adult mice testicular cell type ratios, in particular the round and elongating spermatid populations, changed significantly at doses of 48 mg/kg. Also, a reduction in the percent tetraploid cells occurred at d 1, suggesting these cells may be a primary target of m-DNB action. Caput and caudal sperm from mice exposed to m-DNB prior to puberty did not demonstrate an increased susceptibility to DNA denaturation when analyzed by the sperm chromatin structure assay. However, in pubertal mice, m-DNB exposure further exaggerated the abnormal chromatin structure that normally characterizes sperm during the onset of sperm production. In adult mice, 48 mg/kg resulted in increased susceptibility to DNA denaturation of caput sperm chromatin at 11 dpt (p less than .05) and in caudal sperm at 22 dpt (p less than .01). The abnormal chromatin structure of cauda sperm from adult mice was highly correlated with sperm head morphology abnormalities (ABN; 0.82 to 0.95, p less than .01, 11 and 22 dpt, respectively), but showed lower correlations with dose (0.60 to 0.79, p less than .01, 11 and 22 dpt, respectively). For pubertal mice, a positive relationship was also observed between the variation of sperm chromatin structure abnormalities and ABN. The effect of m-DNB on testicular function in prepubertal and pubertal mice appear to be less pronounced than in adult mice. Furthermore, following exposure to the same dosage, the effect of m-DNB is less severe in adult mice than that observed for adult rats as reported in the companion paper.

Flow cytometric analysis of effects of 1,3-dinitrobenzene on rat spermatogenesis.

Exposure of 100-d old rats to 1,3-dinitrobenzene (m-DNB) at dosages up to 48 mg/kg resulted in disruption of spermatogenesis as measured by flow cytometry (FCM) of acridine orange-stained sperm and testis cells. One day (d 1) after a single exposure to 48 mg/kg m-DNB, FCM measurements of caput epididymal fluid cells demonstrated the presence of testicular germinal epithelial cells apparently sloughed off into the epididymis. Also, at d 1 after the same exposure, a decrease in pachytene spermatocytes was observed. By d 16 after exposure to 32 or 48 mg/kg, testicular damage was evidenced by an alteration of cell type ratios in FCM-analyzed populations of testicular cells. Extensive recovery of cell type ratios occurred by d 32. At d 16, dosages of 32 and 48 mg/kg caused alterations of sperm chromatin structure as determined by the flow cytometric sperm chromatin structure assay (SCSA); 48 mg/kg caused alterations at both d 16 and d 32. Exposure to m-DNB caused a dose response increase in percent sperm head morphology abnormalities (%ABN) assessed in cauda epididymal and vas sperm. A slightly higher correlation existed between dose and SCSA alpha t values (d 16, .78; p less than .01) than between dose and %ABN (d 16, .70; p less than .01). Also, a higher correlation existed between standard deviation of alpha t (SD alpha t) values and %ABN (.97; p less than .01) than between dose and %ABN (.70; p less than .01). This study demonstrated rapid and unique FCM procedures originally derived for reproductive toxicology studies in mice to be equally useful for studies in rats.