Methods of sperm DNA extraction for genetic and epigenetic studies.

High quality DNA extractions developed for mammalian somatic cells are ineffective for sperm, due mainly to the high degree of nuclear compaction in sperm. The highly specialized nuclear proteins in sperm create a chromatin structure that is at least six times denser than histone bound DNA. Unlike somatic cells, sperm DNA is highly compacted by the replacement of histones with sperm-specific low molecular weight proteins called protamines. Both the protamines and the disulfide bridges formed within and between protamines inhibit the extraction of sperm DNA by standard techniques used for somatic cells. Here we describe the guanidine thiocyanate method reported by Hossain with additional modifications resulting in high molecular weight DNA of high quality with an A260/280 ratio ranging between 1.8 and 2.0 and an A260/230 ratio of 2.0 and greater. The DNA is efficiently digested with restriction enzymes and amplified by PCR.

The clinical utility of the protamine 1/protamine 2 ratio in sperm.

During spermiogenesis, human sperm undergo a dramatic reorganization of the chromatin in which canonical histones are replaced by two types of protamines, protamine 1 (P1) and protamine (P2). P1 and P2 are expressed approximately at a 1:1 ratio in healthy men. Alteration of this ratio is associated with male infertility. Patients with an abnormal P1/P2 ratio generally exhibit diminished semen quality, lower fertilization ability, and lower pregnancy rates when undergoing in vitro fertilization. Many studies have reported an elevated incidence of abnormal P1/P2 ratios in infertile men compared to fertile controls, and have evaluated the relationship between infertility and abnormal protamination; however, no prospective study has investigated the normal range of the P1/P2 ratio in men from the general population. Here, we report a P1/P2 reference range of 0.54 to 1.43 in a fertile, normozoospermic population. This rather wide normal range of P1/P2 led us to the conclusion that abnormal protamination is more likely indicative of other perturbations during spermatogenesis than the underlying mechanism to cause infertility. Alternatively, protamine expression may act as a checkpoint mechanism and thus be indirectly related to semen quality.

Association of aromatase (TTTAn) repeat polymorphism length and the relationship between obesity and decreased sperm concentration.

BACKGROUND: Obesity in men is associated with low sperm count, however, this finding is inconsistent. Here, we describe length of the short tandem repeat aromatase (CYP19A1) polymorphism and its relationship to increased weight and sperm count. METHODS: A cohort of 215 men was recruited from the community and BMI, hormone levels and sperm parameters were determined at enrollment. Men (196) were genotyped for length of the tetranucleotide TTTA repeats polymorphism (TTTA(n)), defined as short (S ≤ 7 repeats) or long (L > 7 repeats). Genotypes were categorized using allele combinations as 'low repeats' = S-S, or 'high repeats' = S-L/L-L. Weight and sperm parameters were examined in relation to size of TTTA(n) repeat. RESULTS: Mean (±SD) age was 29.8 ± 8.6 years and mean BMI was 25.6 ± 4.6 kg/m(2). Men with high repeats had higher estradiol (E(2)) levels (98.0 ± 33.36 pmol/l) than men with low repeats (85.9 ± 26.61 pmol/l; P= 0.026). Lower FSH levels tended to be present in men with high repeats versus men with low repeats (P= 0.052). After stratification by genotype, a negative correlation between BMI and sperm count (Pearson's coefficient = 0.406) was seen only among men with high repeats (P= 0.019). Only men with high repeats exhibited increased E(2) with increased weight. A decrease in testosterone: E(2) ratio with increasing BMI was more pronounced in men with high versus low, repeats (R(2) = 0.436 versus 0.281). CONCLUSIONS: Higher TTTA repeat numbers (>7 repeats) in the aromatase gene are associated with a negative relationship between obesity and sperm count. The effect of obesity on E(2) and sperm count appears to be absent in men with low (≤7) repeats.

The marmoset and cotton rat as animal models for the study of sperm chromatin packaging.

In the human, canonical histones are largely replaced by protamine 1 (P1) and protamine 2 (P2) during late spermatogenesis. Recent studies have demonstrated that abnormal replacement of the histones is associated with severe male infertility and has profound implications for early embryogenesis. In this review the hispid cotton rat and the common marmoset are evaluated as animal models for the study of chromatin packaging in sperm. The DNA sequences of protamine 1 and protamine 2 in the cotton rat are also reported. We have found that the P1 and P2 genes share a 64% and 56% amino acid identity with human, respectively. The hispid cotton rat expresses both protamines, has a similar P1:P2 ratio, and a gene sequence homologous to human, thus making it a possible model organism for chromatin studies. The common marmoset also expresses both protamine genes and has a very similar spermatogenetic characteristic to man. This review considers both animals as possible models to study the mechanisms of protamine regulation and the effects of altered expression caused by environmental or physiological perturbations.

Analysis of the meiotic recombination gene REC8 for sequence variations in a population with severe male factor infertility.

Proper regulation of meiosis is essential for normal spermatogenesis and abnormalities may be associated with infertility, as shown in both animal knockout studies and studies identifying anomalies in key proteins, such as SCP3 and MLH1. Disruptions of meiosis are associated with azoospermia or severe oligozoospermia, and may increase the incidence of sperm aneuploidy in some men. Based on its function and animal studies, REC8, a key component of the meiotic cohesion complex, has been identified as a candidate male infertility gene. In this study, we have evaluated sequence variation in the REC8 gene of severely infertile men of European descent with azoospermia or severe oligozoospermia compared to a fertile control population. The direct sequencing of these populations revealed nine polymorphic sites, four within intron/exon boarders, four within coding exons and one in the three prime untranslated region. These sites did not show significantly different allelic frequencies in the study populations compared to fertile controls. This indicates that polymorphisms of the Rec 8 gene are not a common cause of infertility in this population. Additional studies are warranted in patients with defined meiotic disruption.

Sequence analysis of the X-linked USP26 gene in severe male factor infertility patients and fertile controls.

The 1090C>T,L364F variant of the ubiquitin protease 26 (USP26) gene does not appear to be related to male infertility. Mutations of the USP26 gene do not appear to be a common cause of idiopathic azoospermia or severe oligozoospermia.

Novel UBE2B-associated polymorphisms in an azoospermic/oligozoospermic population.

AIM: To assess whether abnormalities exist in the UBE2B gene in a population of infertile human males, and to establish biologic plausibility of any discovered mutations. METHODS: We carried out polymerase chain reaction (PCR) amplification and sequence analysis of the 5'-untranslated region and six exons of the UBE2B gene, including flanking intronic regions, in a group of fertile and infertile men. Following the identification of a putative promoter region that contained single or dual triplet deletions within a 10-CGG repeat island, we evaluated the binding affinity of these identified polymorphisms as compared to the wild-type sequence to transcription factor SP1 using a DNA-protein gel shift assay. RESULTS: There was a novel exonic single nucleotide polymorphism (SNP) noted in exon 4 in 5% of infertile men. In silico 3D modeling of the altered protein showed an innocuous isoleucine for valine substitution. There were no mutations noted within any of the other exons. Three novel intronic SNPs were identified within the fertile group, and seven novel intronic SNPs identified in the infertile group. The DNA-protein gel shift assay noted that both single CGG deletion and double CGG deletion bands had approximately twice the binding affinity compared to the wild-type for SP1. The negative control confirmed no non-specific protein binding. CONCLUSION: By themselves, a single or double CGG deletion is unlikely to pose biologic significance. However, such deletions in this suspected promoter region are associated with increased binding affinity for SP1, and might represent one of several factors required for alteration of UBE2B gene expression.

Comparative analysis of follicle morphology and oocyte diameter in four mammalian species (mouse, hamster, pig, and human).

BACKGROUND: Laboratory animals are commonly used for evaluating the physiological properties of the mammalian ovarian follicle and the enclosed oocyte. The use of different species to determine the morphological relationship between the follicle and oocyte has led to a recognizable pattern of follicular stages, but differences in follicle size, oocyte diameter and granulosa cell proliferation are not consistent across the different species. In an effort to better understand how these differences are expressed across multiple species, this investigation evaluates oocyte and follicle diameters and granulosa cell proliferation in the mouse, hamster, pig, and human. METHODS: Histological sections of ovaries from the mouse, hamster, pig, and human were used to calculate the diameter of the oocyte and follicle and the number of granulosa cells present at pre-determined stages of follicular development. A statistical analysis of these data was performed to determine the relationship of follicular growth and development within and between the species tested. RESULTS: These data have revealed that the relationships of the features listed are tightly regulated within each species, but they vary between the species studied. CONCLUSION: This information may be useful for comparative studies conducted in different animal models and the human.

Comparison of chromatin assays for DNA fragmentation evaluation in human sperm.

Sperm chromatin integrity is vital for successful pregnancy and transmission of genetic material to the offspring. We evaluated chromatin integrity in sperm from 60 infertile men and 7 fertile donors comparing the sperm chromatin structure assay (SCSA), TdT-mediated-dUTP nick end labeling (TUNEL), the sperm chromatin dispersion (SCD) test, and acridine orange staining technique (AOT). The TUNEL and SCD assays showed a strong relationship with the SCSA (r > .866; P < .001) for sperm DNA fragmentation, both in infertile men and donors of known fertility. AOT did not show any relationship with SCSA. The breakdown of the DNA fragmentation index (DFI) into 3 categories (< or =15%, >15%-<30%, and > or =30%) showed that the SCSA, TUNEL, and SCD test predict the same levels of DNA fragmentation. AOT consistently showed higher levels of DNA fragmentation for each DFI category. DNA fragmentation in sperm between infertile men and donor sperm was significantly different (P < .05) under SCSA (22.0 +/- 1.6 vs 11.8 +/- 1.4), TUNEL (19.5 +/- 1.3 vs 11.1 +/- 0.9) and SCD (20.4 +/- 1.3 vs 10.8 +/- 1.1), respectively. DNA fragmentation in sperm evaluated by AOT did not differ (P > .05) between infertile men (31.3 +/- 2.4) and donors (32.7 +/- 4.8). AOT showed extreme variations for sperm DNA fragmentation in semen from both infertile men and donors. The problems of indistinct colors, rapid fading, and the heterogeneous staining were also faced. In conclusion, SCSA, TUNEL, and SCD show similar predictive values for DNA fragmentation, and AOT shows variable and increased levels of DNA fragmentation, which makes it of questionable value in clinical practice.

Primordial and pre-antral follicles are not commonly observed in IVF aspirates.

BACKGROUND: Follicular fluid recovered from IVF patients has been proposed to be a valuable source of pre-antral and primary follicles for patient therapy and research. We evaluated the recovery of immature follicles in follicular fluid from 54 patients undergoing IVF using several techniques. METHODS: Fluid from each patient underwent several methods of follicle recovery including: filtration through a cell strainer, Ficoll-Paque density gradient, isolate density gradient, histological slide preparation, and enzymatic digestion with collagenase and DNase. RESULTS: 34 primordial and primary follicles, mean 0.63 +/- 0.27/patient, and 14 pre-antral follicles, mean 0.26 +/- 0.14/patient, were found in this study. The serum estradiol level on the day of HCG injection was significantly lower (P < 0.05) in patients in which immature follicles were recovered, compared with those without immature follicles in the follicular fluid (1779.9 +/- 167.6 versus 2246.6 +/- 153.2 pg/ml). There were no women with advanced maternal age (>39 years) who had immature follicles in the follicular fluid. CONCLUSIONS: Follicular fluid cannot be considered an efficient or reliable source of immature follicles. The presence of any immature follicles appears to be associated with cause of infertility, the random placement of the aspirating needle and may be related to the age of patient.