Benjamin R. Emery

In Vitro Growth, Maturation, Fertilization, and Embryonic Development of Oocytes from Porcine Preantral Follicles

Abstract This study was conducted to identify an in vitro culture system that would support intact porcine follicle growth from preantral follicle to antral stages, oocyte maturation, fertilization, and embryonic development; and to evaluate factors that influence porcine preantral follicle growth in vitro. Preantral follicles isolated from prepubertal porcine ovaries were cultured for 4 days in the presence of different concentrations of porcine serum and FSH, and with different numbers of follicles per well. A series of experiments showed that porcine antral follicles can be grown at a high frequency in vitro from healthy preantral follicles with intact theca when cultured in North Carolina State University 23 medium supplemented with 1.5 ng/ml FSH, 7.5% serum, and when cultured with three follicles per well. After 4 days of culture, 68% healthy cumulus-enclosed oocytes from these follicles were obtained, and 51% of the oocytes completed meiotic maturation to the metaphase II stage. Fifty-three percent of the mature oocytes underwent fertilization, 43% of the fertilized oocytes cleaved, and 13% developed to the blastocyst stage. The results show 1) that porcine preantral follicles can grow efficiently to the antral stage using these culture conditions, and 2) that oocytes from in vitro-matured porcine preantral follicles can acquire meiotic competence and undergo fertilization and embryonic development.

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.

Body mass index is inversely related to intra-follicular HCG concentrations, embryo quality and IVF outcome

Decreased periovulatory human chorionic gonadotrophin (HCG) concentrations have been shown to be associated with diminished fertilization rates. This study evaluated if intra-follicular HCG concentration may be related to body mass in 247 IVF patients using their own oocytes and 58 patients receiving donor oocytes, and evaluated if such a relationship might affect IVF outcome. A significant inverse correlation (r = -0.353, P < 0.001) was observed between the body mass index (BMI) and intra-follicular HCG concentration. The mean HCG concentrations were significantly decreased (P < 0.001) in patients with a BMI >30 kg/m(2) compared with patients with a BMI of 20-30 kg/m(2) or BMI <20 kg/m(2) (17.6 versus 45.1 and 52.5%, respectively). The clinical pregnancy rates (P < 0.001) and embryo quality (P < 0.05) were significantly different for the three groups. In donor oocyte recipients, the pregnancy rate was significantly decreased (P < 0.0001) for recipients with a BMI >25 kg/m(2) compared with those with a BMI from 21-25 kg/m(2) and BMI <21 kg/m(2) (43.8 versus 72 and 76.5%, respectively). These data indicate that intra-follicular HCG concentration is inversely related to BMI, and may be related to a concurrent decrease in embryo quality and pregnancy rates.

Identification of human sperm transcripts as candidate markers of male fertility

To date, there has been little progress towards identifying markers of normal male fertility. The need to supplement current subjective methods that rely on variable semen parameters to assess fertility status continues to be acknowledged. Several studies have shown that spermatozoal RNAs can describe characteristic failures of the spermatogenic pathway among infertile males. In spite of the inherent heterogeneity of semen that describe the “normal” fertile male, this holds the promise of developing markers that could help identify the ever elusive idiopathic infertile male. Through the analyses of the spermatozoal transcriptome from 24 donors of proven fertility, we identified a series of transcripts that were consistently present among all individuals. The heterogeneous nature of the samples, reflected by their semen parameters, was mirrored by the variability of the observed array signal. Nevertheless, clusters of invariable transcript pairs were identified. These were founded by a single central member that was linked in constant proportion even though the absolute level of each member of the transcript pair often varied among individuals. The presence of pairs of stable transcripts suggests that among the heterogeneity observed in the sperm transcriptome, a distinct set is strictly regulated.

The aetiology of sperm protamine abnormalities and their potential impact on the sperm epigenome

During the elongating spermatid stage of spermatogenesis, there is a step-wise replacement of nuclear histones with protamines 1 and 2. In fertile men, the ratio of protamine 1/protamine 2 (P1/P2) is within the narrow range of 0.8-1.2. Ratios above or below that range are associated with infertility, exhibiting a wide range of defects including decreased sperm counts, morphology, fertilization ability, and embryo implantation capacity. In this review, we highlight studies evaluating potential causes of abnormal protamine expression, including the sequencing of genes relevant to protamine expression in both affected patients and controls. While the variants of the protamine genes themselves do not appear to be responsible for most observed defects, variants of the Contrin gene, a transcription factor and translation repressor, appear to be contributory to some cases of abnormal expression. Additionally, we explore the potential effects of abnormal protamine replacement on the epigenome of human sperm. Ongoing studies are evaluating the role of retained histones and DNA methylation in sperm, which may be affected in sperm with aberrant protamine replacement. This important area of epigenetic research has profound clinical implications.

Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment

STUDY QUESTION Is there an association between sperm DNA damage, measured by three different assays, sperm nuclear protein content and clinical outcomes in assisted reproduction treatment (ART)? SUMMARY ANSWER Sperm DNA damage measured by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and the Comet assay were significantly associated with ART outcomes in our single institution study. WHAT IS KNOWN ALREADY Abnormal protamine expression is known to be associated with sperm DNA damage and male infertility. A number of studies have shown a significant relationship between sperm DNA damage and ART outcomes. To date, there are no large studies providing direct comparisons of DNA damage tests within the same study population. Thus, the prognostic value for each method remains unknown. STUDY DESIGN, SIZE, DURATION Cross-sectional study of 238 men from infertile couples undergoing ART at the University Center for Reproductive Medicine, Utah, USA, between April 2011 and March 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS Sperm from men undergoing ART were tested for DNA damage using the alkaline Comet assay, TUNEL and flow cytometric chromatin evaluation (FCCE) assays. Histone retention was analysed using the aniline blue staining method, whereas protamine content (proteins P1 and P2) and ratio were analysed using acid urea gel electrophoresis. The prognostic value of each sperm DNA test to predict clinical pregnancy was calculated. MAIN RESULTS AND THE ROLE OF CHANCE Histone retention was associated with sperm DNA damage (P < 0.001), reduced embryo quality (P = 0.005) and clinical pregnancies (P < 0.001). The mean percentage of sperm with DNA damage was significantly higher in sperm from non-pregnant couples compared with that from pregnant couples, as measured by TUNEL assay (15.04 ± 1.16% versus 8.79 ± 0.56%; P < 0.001) and alkaline Comet assay (72.79 ± 2.49% versus 55.86 ± 2.29%; P < 0.001). There was no association between clinical pregnancies and DNA fragmentation index measured by FCCE (12.97 ± 1.46 versus 14.93 ± 1.65; P = 0.379). Of the protamine parameters analysed, only the P1/P2 ratio was associated with sperm count (P = 0.013), mens age (P = 0.037), maturity (P = 0.049) and blastocyst quality (P = 0.012). Histone retention and sperm DNA damage measured by Comet and TUNEL assays were associated with fertilization rate (P < 0.05), embryo quality (P < 0.05) and implantation rate (P < 0.05). LIMITATIONS, REASONS FOR CAUTION A potential drawback of this study is that it is cross-sectional. Generally in such studies there is more than one variable that could cause the effect. Analysing sperm is one part of the equation; there are also a number of female factors that have the potential to influence ART outcomes. Therefore, given the large and well-established role of female factors in infertility, normal sperm DNA integrity and protamination do not necessarily ensure clinical pregnancy in ART. Thus, female factors can reduce the prognostic value of sperm DNA tests. Further, our use of native semen instead of prepared sperm may have iatrogenically increased the DNA damage. WIDER IMPLICATIONS OF THE FINDINGS Alteration in sperm nuclear protein affects sperm DNA integrity. Further, with the current dataset, TUNEL and Comet assays appeared more predictive of ART success than FCCE. STUDY FUNDING/COMPETING INTEREST(S) No personal or direct financial support has been received for any of this work. The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

H1FOO Is Coupled to the Initiation of Oocytic Growth

Abstract We previously reported the discovery of a novel mammalian H1 linker histone termed H1FOO (formerly H1OO), a replacement H1, the expression of which is restricted to the growing/ maturing oocyte and to the zygote. The significance of this pre-embryonic H1 draws on its substantial orthologous conservation, singular structural attributes, selectivity for the germ cell lineage, prolonged nucleosomal residence, and apparent predominance among germ cell H1s. Herein, we report that the intronic, single-copy, five-exon (≥5301 base pair) H1foo gene maps to chromosome 6 and that the corresponding primary H1foo transcript gives rise to two distinct, alternatively spliced mRNA species (H1fooα and H1fooβ). The expression of the oocytic H1FOO transcript and protein proved temporally coupled to the recruitment of resting primordial follicles into a developing primary follicular cohort and thus to the critical transition marking the onset of oocytic growth. The corresponding potential protein isoforms (H1FOOα and H1FOOβ), both nuclear localization sequence-endowed but export consensus sequence-free and possessing a significant net positive charge, localized primarily to perinucleolar heterochromatin in the oocytic germinal vesicle. Further investigation will be required to define the functional role of the H1FOO protein in the ordering of the chromatin of early mammalian development as well as its potential role in defining the primordial-to-primary follicle transition.

Sperm chromosome aneuploidy as related to male factor infertility and some ultrastructure defects.

Some men have elevated levels of sperm chromosome aneuploidy. In this study, we have evaluated and summarized sperm aneuploidy rates in male infertility patients and control groups. The mean aneuploidy rate for five chromosomes (X, Y, 13, 18, 21) was 1.2 ± 0.1 for fertile controls, 1.4 ± 0.1 for a general population control group, and 5.8 ± 1.14 for the patients. When the patients were classified by the type of male factor infertility, the total aneuploidy rate was 2.6 ± 0.3 in men with moderately diminished semen quality (n = 7), 4.0 ± 0.3 patients with severe teratoasthenooligozoospermia, and 15.9 ± 3.8 for men with rare ultrastructure defects such as round head only syndrome or severe tail agenesis. Some infertility patients have a severely elevated level of sperm chromosome aneuploidy, which may contribute to infertility or diminish the likelihood of a successful outcome from IVF/ICSI. The severity of sperm chromosome aneuploidy appears to be proportional to the severity of abnormal semen quality: in particular, abnormal morphology. The high rates of aneuploidy in patients with severe ultrastructure defects suggest that caution should be employed in counseling those patients prior to IVF/ICSI.

Review: Diagnosis and impact of sperm DNA alterations in assisted reproduction

Sperm nuclear and chromatin abnormalities are common among infertile men and are known to influence natural reproduction. These abnormalities are also considered detrimental to normal fertilization, embryo development, and successful implantation and pregnancies following assisted reproductive treatment (ART). Abnormalities in the sperm nucleus can be broadly classified into sperm chromosomal abnormalities (aneuploidies) and sperm DNA abnormalities such as abnormal packing, DNA integrity, or DNA fragmentation. For the past 30 years, numerous tests have been developed to quantify these abnormalities in sperm. In this chapter, we review the causes of sperm DNA and chromosomal abnormalities, describe the commonly used tests to evaluate these abnormalities, and finally review the impact of these abnormalities on male fertility and ART outcomes. We also performed a comprehensive meta-analysis and systematic review from the existing literature to summarize the effect of sperm DNA fragmentation on ART outcomes such as fertilization rate, embryo quality, and clinical pregnancies. A review of the literature presented in this chapter suggests that sperm nuclear and chromatin abnormalities are associated with male infertility, and they reduce the probability of a successful pregnancy following ART.

The Correlation of Sperm Chromatin Decondensation Following In Vitro Exposure to Heparin and Sperm Penetration Rates

Purpose:The aim of this study was to evaluate the possible correlation of low-dose heparin-induced decondensation of sperm chromatin with sperm concentration, motility, morphology, membrane hypoosmotic response, ejaculate volume, and the ability of sperm to penetrate zona-free hamster oocytes.Methods:Twenty-two donors of known fertility and 105 patients undergoing evaluation at an andrology laboratory were evaluated by standard World Health Organization semen analysis techniques and a modified sperm penetration assay (SPA). An aliquot was also incubated for 60 min and Hams F10 medium containing 50 USP/ml heparin. The percentage of sperm undergoing chromatin decondensation was evaluated and correlated to SPA rates and semen quality parameters.Results:No significant correlation was observed between semen parameters and decondensation rates. A nonsignificant (P = 0.11) inverse correlation (P = −0.21) wax observed between SPA rates and chromatin decondensation. Significant (P < 0.001) differences were observed in the decondensation rate of donors (3.7 ± 0.6), patients with normal SPA rates (7.8 ± 1.5), and patients with decreased SPA rates (21.7 ± 1.8). The decondensation rates were significantly different (P < 0.01) between patients with a normal SPA rate and patients with a decreased SPA rate.Conclusions:These data indicate a significant inverse relationship between the SPA rate, which has previously been shown to correlate highly with fertilization ability, and heparin-induced sperm chromatin decondensation.