No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Predictive Roles of Proteomic Profiles in Assisted Reproduction-An Update
Abstract
The prevalence of infertility is 12-15% in sexually active couples. When, a subgroup analysis is done, 50% of
infertility can be ascribed to male factor or a combination of male and female factor. With the growing trend of male factor infertility and the desire for infertile couples to reproduce, assisted reproductive technology provides a means to overcome these challenges. Several techniques exist depending on the extent of the infertility. For cases of severe oligospermia or even azoospermia, a combination of in vitro fertilization (IVF) and intracytoplasmic injection is used. There has been birth of more than 4 million babies worldwide using the assisted reproductive techniques. In the United States alone 1% of the total babies born each year are conceived through ART However, there are very low rates of successful implantation and pregnancy per transferred embryo in in vitro fertilization. According to literature reports 70% of transferred embryos fail to implant. Also, several ART clinics perform multiple embryo transfer, rather than a single embryo transfer, which exposes patients to several complications. Proteomics has the potential to aid in selection of the best embryo. In this review, we present an appraisal; of existing scientific literature regarding the proteomic profiles of the key players impacting ART outcomes. We elaborate on the potential proteomic biomarkers that are a key to establishing a non-invasive, reliable, reproducible, and specific means of assessing endometrial receptivity, embryonic viability, aneuploidy, and fertilizing potential of the sperm. These biomarkers include a variety of both structural and functional proteins. The application of these potential biomarkers in the future will help in enhancing the ART outcomes by offering a personalized treatment for patients based upon their individualized signature proteomics profiles.
... Another compounding problem of ART is multiple pregnancy that occurs when multiple embryos, rather than a single one, are transferred, thus exposing the patient to various complications. These include an increased risk of miscarriage(s), premature labor and birth; gestational hypertension, pre-eclampsia and diabetes in women pregnant with more than one fetus including ectopic pregnancy or a combination of normal and ectopic pregnancy (Gupta et al. 2015). ...
... Assisted reproductive techniques are becoming an indispensable part of fertility treatment in patients with acute reproductive failure. The establishment of non-invasive, reliable means of assessing endometrial receptivity, embryonic viability, aneuploidy and fertilizing ability of the spermatozoa is the need of the hour (Gupta et al. 2015). Proteins form the core of every reproductive process and their altered behavior have been implicated in a myriad of pathological conditions and thus by a major class of drug targets. ...
... It is therefore becoming increasingly clear that in order to truly understand a protein, we need to identify the entire protein complement, detect covalent modifications and allow for quantitative comparisons between samples. In contrast with some biochemical methods and allied technologies that are restricted to the analysis of only a single protein in a cell or tissue (Wilkins et al. 1996), proteomics deals with the analysis of the entire population of expressed proteins, based on their expression, localization, functions, post-translational modifications and interactions at a specific condition and time (Gupta et al. 2015). ...
This Brief explores the use of proteomics as a tool for biomarker discovery in human reproduction and summarizes current findings and trends of proteomic studies in both male and female infertility. This simplifies this important but complex topic and equips the novice reader with sufficient background information on the use of proteomics in human reproduction. The up-to-date scenario on proteomic investigations will also appeal to researchers and post graduate students looking to keep abreast with the latest developments in reproductive research. This review summarizes current findings of contemporary proteomic studies on infertility in both males and females with various reproductive pathologies, and its use in predicting the outcome of assisted reproduction. In human reproduction, the search for biomarkers via proteomics is a fast-evolving approach that involves the analysis of proteins in the reproductive tissues and fluids, such as the male gametes, seminal plasma, ovarian and endometrial tissue, and follicular and uterine fluid. By comparing the protein profile of a healthy, fertile individual against that of an infertile individual, the differentially expressed proteins may give an indication to certain proteins that could serve as useful biomarkers that are related to infertility. As proteomic studies continue to unravel the dynamic proteome behind various infertility conditions, there is potential for the discovery of prognostic markers that could ultimately help in both natural and assisted human reproduction.
... A key group of proteins with altered abundance in the endometrium of PCOS women is related to apoptosis. Among these, annexin A5, a marker of apoptosis, was increased, whereas annexin A4, which is involved in the anti-apoptosis process (Gupta et al., 2015), was decreased in endometrial samples of PCOS women. In line with the results of this study, the proteomic analysis of granulosa cells, and the ovarian and visceral adipose tissue of PCOS patients has also shown up-regulation of annexin A5 (Choi et al., 2010;Ma et al., 2007;Misiti et al., 2010). ...
... To generate a reference normal endometrial proteome map and indicate whether the cause of infertility in PCOS is disturbance to this normal endometrial map, a comparative analysis of proliferative and luteal endometria in the healthy state was also carried out, which enabled identification of 21 up-regulated and 23 down-regulated proteins in the proliferative phase as compared with the luteal phase. Some of these proteins have been described previously by proteomic studies conducted on endometrium in a healthy state such as α-1antitrypsin, complement C3, fibrinogen-β, 14-3-3 protein, annexin A4, glutathione S-transferase, haptoglobin, cofilin and transthyretin (Gupta et al., 2015). However, others like apolipophorin-III, crotonase/enoylcoenzyme A (CoA), epididymis secretory sperm binding and slipin-2 are novel and their potential effects on the endometrium remain to be determined. ...
Research question
What is the molecular basis of infertility related to uterine dysfunction in women with polycystic ovary syndrome (PCOS)?
Design
In this study, differences in protein expression between PCOS and normal endometrium were identified using a proteomic approach based on two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS). The proteome of endometrium were analysed during the proliferative (on day 2 or 3 before ovulation, n = 6) and luteal phases (on day 3–5 after ovulation, n = 6) from healthy women and PCOS patients (12–14 days after spontaneous bleeding, n = 12). The differentially expressed proteins were categorized based on the biological process using the DAVID bioinformatics resources.
Results
Over 803 reproducible protein spots were detected on gels, and 150 protein spots showed different intensities between PCOS and normal women during the proliferative and luteal phases. MS analysis detected 70 proteins out of 150 spots. For four of the 70 proteins, 14-3-3 protein, annexin A5, SERPINA1 and cathepsin D, 2-DE results were validated and localized by Western blot and immunohistochemistry, respectively, and their gene expression profiles were confirmed by real-time quantitative PCR. The obtained results corresponded to the proteomic analysis. The differentially expressed proteins identified are known to be involved in apoptosis, oxidative stress, inflammation and the cytoskeleton.
Conclusions
The processes related to the differentially expressed proteins play important roles in fecundity and fecundability. The present study may reveal the cause of various endometrial aberrations as a limiting factor for achieving pregnancy in PCOS women.
... The relationship of FSH with spermatogenesis is not straightforward in men with NOA. This is manifest by the range of FSH levels seen in the present study; a preoperative FSH level of R15 IU/L resulted in greater odds of finding sperm with micro-TESE compared with normal FSH values (13). ...
... +Model Shivaji et al. (1990), Speroff et al. (1999), Suarez et al. (1999 o Yao et al. (1999), describen la importancia del plasma seminal en el aislamiento de proteínas activadoras e inhibidoras de la reacción acrosómica, que precede al desencadenamiento del mecanismo de ZP3 en la dicha reacción. La bibliografía más reciente presenta el estudio proteómico del plasma seminal en individuos normales ( Gupta et al., 2015) y con varicoceles ( Agarwal et al., 2015), como una línea de investigación que puede establecer una nueva interpretación de los estudios de infertilidad y reproducción asistida de la pareja ( Cooper, 1986). Recientemente, Bergamo et al. (2017), ha descrito la actualización de la determinación de los componentes bioquímicos clásicos (proteínas totales, glucosa, fructosa, diversos iones...), con las nuevas metodologías analíticas realizadas con los autoanalizadores actuales. ...
PHYSIOLOGICAL, ENDOCRINOLOGICAL AND ENVIRONMENTAL
DETERMINANTS OF FEMALE INFERTILITY IN LAGOS, NIGERIA
As the mammalian spermatozoa transcends from the testis to the end of the epididymal tubule, the functionally incompetent spermatozoa acquires its fertilizing capability. Molecular changes in the spermatozoa at the posttesticular level concern qualitative and quantitative modifications of proteins along with their sugar moieties and membranous lipids mostly associated with motility, egg binding, and penetration processes. Proteomic studies have identified numerous sperm-specific proteins, and recent reports have provided a further understanding of their function with respect to male fertility. High-throughput techniques such as mass spectrometry have shown drastic potential for the identification and study of sperm proteins. In fact, compelling evidence has provided that proteins are critically important in cellular remodeling event and that aberrant expression is associated with pronounced defects in sperm function. This review highlights the posttesticular functional transformation in the epididymis and female reproductive tract with due emphasis on proteomics.
Most biochemical reactions in a cell are regulated by highly specialized proteins, which are the prime mediators of the cellular phenotype. Therefore the identification, quantitation and characterization of all proteins in a cell are of utmost importance to understand the molecular processes that mediate cellular physiology. With the advent of robust and reliable mass spectrometers that are able to analyze complex protein mixtures within a reasonable timeframe, the systematic analysis of all proteins in a cell becomes feasible. Besides the ongoing improvements of analytical hardware, standardized methods to analyze and study all proteins have to be developed that allow the generation of testable new hypothesis based on the enormous pre-existing amount of biological information. Here we discuss current strategies on how to gather, filter and analyze proteomic data sates using available software packages.
We performed a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in liquid chromatography-mass spectrometry-based proteomics. We distributed an equimolar test sample, comprising 20 highly purified recombinant human proteins, to 27 laboratories. Each protein contained one or more unique tryptic peptides of 1,250 Da to test for ion selection and sampling in the mass spectrometer. Of the 27 labs, members of only 7 labs initially reported all 20 proteins correctly, and members of only 1 lab reported all tryptic peptides of 1,250 Da. Centralized analysis of the raw data, however, revealed that all 20 proteins and most of the 1,250 Da peptides had been detected in all 27 labs. Our centralized analysis determined missed identifications (false negatives), environmental contamination, database matching and curation of protein identifications as sources of problems. Improved search engines and databases are needed for mass spectrometry-based proteomics.
Infertility is an important aspect of human and animal reproduction and still presents with much etiological ambiguity. As fifty percent of infertility is related to the male partner, molecular investigations on sperm and seminal plasma can lead to new knowledge on male infertility. Several comparisons between fertile and infertile human and other species sperm proteome have shown the existence of potential fertility markers. These proteins have been categorized into energy related, structural and other functional proteins which play a major role in sperm motility, capacitation and sperm-oocyte binding. The data from these studies show the impact of sperm proteome studies on identifying different valuable markers for fertility screening. In this article, we review recent development in unraveling sperm fertility related proteins.
Approximately 1% of all men in the general population suffer from azoospermia, and azoospermic men constitute approximately 10 to 15% of all infertile men. Thus, this group of patients represents a significant population in the field of male infertility. A thorough medical history, physical examination and hormonal profile are essential in the evaluation of azoospermic males. Imaging studies, a genetic workup and a testicular biopsy (with cryopreservation) may augment the workup and evaluation. Men with nonobstructive azoospermia should be offered genetic counseling before their spermatozoa are used for assisted reproductive techniques. This article provides a contemporary review of the evaluation of the azoospermic male.
Background
Oxidative stress plays a key role in the etiology of male infertility. Significant alterations in the sperm proteome are associated with poor semen quality. The aim of the present study was to examine if elevated levels of reactive oxygen species cause an alteration in the proteomic profile of spermatozoa.
Methods
This prospective study consisted of 52 subjects: 32 infertile men and 20 normal donors. Seminal ejaculates were classified as ROS+ or ROS- and evaluated for their proteomic profile. Samples were pooled and subjected to LC-MS/MS analysis through in-solution digestion of proteins for peptide characterization. The expression profile of proteins present in human spermatozoa was examined using proteomic and bioinformatic analysis to elucidate the regulatory pathways of oxidative stress.
Results
Of the 74 proteins identified, 10 proteins with a 2-fold difference were overexpressed and 5 were underexpressed in the ROS+ group; energy metabolism and regulation, carbohydrate metabolic processes such as gluconeogenesis and glycolysis, protein modifications and oxidative stress regulation were some of the metabolic processes affected in ROS+ group.
Conclusions
We have identified proteins involved in a variety of functions associated with response and management of oxidative stress. In the present study we focused on proteins that showed a high degree of differential expression and thus, have a greater impact on the fertilizing potential of the spermatozoa. While proteomic analyses identified the potential biomarkers, further studies through Western Blot are necessary to validate the biomarker status of the proteins in pathological conditions.
Background
Alterations at the molecular level in spermatozoa and seminal plasma can affect male fertility. The objective of this study was to determine if analysis of differential expression of proteins in varying semen parameters can serve as potential biomarkers for male infertility.
Methods
The differential expression of proteins in the seminal plasma of men based on sperm count and morphology were examined utilizing proteomic tools. Subjects were categorized based on sperm concentration and morphology into 4 groups: 1) normal sperm count and normal morphology (NN); 2) normal sperm count and abnormal morphology (NA); 3) oligozoospermia and normal morphology (ON); and 4) oligozoospermia and abnormal morphology (OA). Proteomic analysis was performed by LC-MS/MS followed by functional bioinformatics analysis. Protein distribution in the NA, ON and OA groups was compared with that of the NN group.
Results
Twenty proteins were differentially expressed among the 4 groups. Among the unique proteins identified, 3 were downregulated in the NA group, 1 in the ON group and 1 in the OA group while 2 were upregulated in the ON and OA groups. The functional analysis 1) identified biological regulation as the major processes affected and 2) determined that most of the identified proteins were of extracellular origin.
Conclusions
We have identified proteins that are over-or underexpressed in the seminal plasma of men with poor sperm quality. The distinct presence of some of the proteins may serve as potential biomarkers and provide insight into the mechanistic role played by these proteins in male infertility. Further studies using Western Blot analysis are required to validate these findings.
The accurate quantitation of proteins and peptides in complex biological systems is one of the most challenging areas of proteomics. Mass spectrometry-based approaches have forged significant in-roads allowing accurate and sensitive quantitation and the ability to multiplex vastly complex samples through the application of robust bioinformatic tools. These relative and absolute quantitative measures using label-free, tags, or stable isotope labelling have their own strengths and limitations. The continuous development of these methods is vital for increasing reproducibility in the rapidly expanding application of quantitative proteomics in biomarker discovery and validation. This paper provides a critical overview of the primary mass spectrometry-based quantitative approaches and the current status of quantitative proteomics in biomedical research.
The objective of these studies was to identify differentially expressed peptides/proteins in the culture media of embryos grown during in vitro fertilization (IVF) treatment to establish their value as biomarkers predictive of implantation potential and live birth. Micro-droplets of embryo culture media from IVF patients (conditioned) and control media maintained under identical culture conditions, were collected and frozen at -80° C on days 2-3 of in vitro development prior to analysis. Embryos were transferred on day 3. Peptides were affinity purified based on their physico-chemical properties and profiled by mass spectrometry for differential expression. Identified proteins were further characterized by Western blot and ELISA, and absolute quantification was achieved by multiple reaction monitoring (MRM). We identified up to 14 differentially regulated peptides after capture using paramagnetic beads with different affinities. These differentially expressed peptides were used to generate genetic algorithms with a recognition capability of 71-84% for embryo transfer cycles resulting in pregnancy, and 75-89% for those with failed implantation. Several peptides were further identified as fragments of Apolipoprotein A-1, which showed consistent and significantly reduced expression in the embryo media samples from embryo transfer cycles resulting in viable pregnancies. Western blot and ELISA, as well as quantitative MRM results, were confirmatory. These results demonstrated that peptide/protein profiles from the culture medium during early human in vitro development can discriminate embryos with highest and lowest implantation competence following uterine transfer. Further prospective studies are needed to establish validated thresholds for clinical application.
A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm-egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm-zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility.
Problem/condition:
Since the first U.S. infant conceived with Assisted Reproductive Technology (ART) was born in 1981, both the use of advanced technologies to overcome infertility and the number of fertility clinics providing ART services have increased steadily in the United States. ART includes fertility treatments in which eggs or embryos are handled in the laboratory (i.e., in vitro fertilization [IVF] and related procedures). Because more than one embryo might be transferred during a procedure, women who undergo ART procedures, compared with those who conceive naturally, are more likely to deliver multiple birth infants. Multiple births pose substantial risks to both mothers and infants, including obstetric complications, preterm delivery, and low birthweight infants. This report provides state-specific information for the United States (including Puerto Rico) on ART procedures performed in 2012 and compares infant outcomes that occurred in 2012 (resulting from ART procedures performed in 2011 and 2012) with outcomes for all infants born in the United States in 2012.
Period covered:
2012.
Description of system:
In 1996, CDC began collecting data on ART procedures performed in fertility clinics in the United States, as mandated by the Fertility Clinic Success Rate and Certification Act of 1992 (FCSRCA) (Public Law 102-493). Data are collected through the National ART Surveillance System, a web-based data collecting system developed by CDC. This report includes data from 52 reporting areas (the 50 states, the District of Columbia [DC], and Puerto Rico).
Results:
In 2012, a total of 157,635 ART procedures performed in 456 U.S. fertility clinics were reported to CDC. These procedures resulted in 51,261 live-birth deliveries and 65,151 infants. The largest numbers of ART procedures were performed among residents of six states: California (20,241), New York (19,618), Illinois (10,449), Texas (10,281), Massachusetts (9,754), and New Jersey (8,590). These six states also had the highest number of live-birth deliveries as a result of ART procedures, and together they accounted for 50.1% of all ART procedures performed, 48.3% of all infants born from ART, and 48.3% of all ART multiple live-birth deliveries. Nationally, the total number of ART procedures performed per 1 million women of reproductive age (15-44 years), which is a proxy indicator of ART use, was 2,483. This indicator of ART use exceeded the national ratio in 13 reporting areas (California, Connecticut, Delaware, Hawaii, Illinois, Maryland, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Virginia, and DC) and was more than twice the national ratio in three reporting areas (Massachusetts, New Jersey, and DC). Nationally, among ART cycles with patients using fresh embryos from their own eggs, in which at least one embryo was transferred, the average number of embryos transferred increased with the increasing age of the woman (1.9 among women aged <35 years, 2.2 among women aged 35-40 years, and 2.7 among women aged >40 years). The percentage of elective single-embryo transfer (eSET) procedures varied substantially between reporting areas for all ages. Among women aged <35 years, who are typically considered to be good candidates for eSET procedures, the national eSET rate was 15.3% (range: 2.1% in Oklahoma to 60.4% in Delaware). Overall, ART contributed to 1.5% of all infants born in the United States (range: 0.2% in Puerto Rico to 4.7% in Massachusetts) with the highest rates (≥3.0% of all infants born) observed in four reporting areas (Connecticut, Massachusetts, New Jersey, and DC). Infants conceived with ART comprised 19.6% of all multiple-birth infants (range: 5.5% in Maine to 39.3% in Massachusetts), 19.2% of all twin infants (range: 4.4% in Puerto Rico to 39.1% in Massachusetts), and 29.6% of all triplet or higher order infants (range: 0 in West Virginia to 69.7% in Idaho). Among infants conceived with ART, 43.6% were born in multiple-birth deliveries (range: 18.7% in Delaware to 56.0% in Idaho), compared with only 3.4% among all infants born in the general population (range: 2.1% in Puerto Rico to 4.5% in New Jersey). Approximately 41% of ART-conceived infants were twin infants, and 2% were triplet and higher order infants. Nationally, infants conceived with ART comprised 5.7% of all low birthweight (<2,500 grams) infants (range: 0.8% in Puerto Rico to 15.3% in Massachusetts) and 5.8% of all very low birthweight (<1,500 grams) infants (range: 0 in West Virginia to 15.1% in New Jersey). Overall, among ART-conceived infants, 30.2% were low birthweight (range: 18.8% in DC to 45.1% in New Mexico), compared with 8.0% among all infants (range: 5.6% in Alaska to 11.6% in Mississippi and Puerto Rico); 5.5% of ART infants were very low birthweight (range: 0 in West Virginia to 12.9% in Puerto Rico), compared with 1.4% among all infants (range: 0.9% in Alaska and Idaho to 2.1% in Mississippi). ART-conceived infants comprised 4.6% of all preterm (<37 weeks) infants (range: 0.7% in Puerto Rico to 13.4% in Massachusetts) and 5.2% of all very preterm (<32 weeks) infants (range: 1.0% in Puerto Rico to 14.9% in Vermont). Overall, among infants conceived with ART, 34.9% were born preterm (range: 20.8% in Delaware and DC to 49.4% in Puerto Rico), compared with 11.6% among all infants born in the general population (range: 8.7% in Vermont to 17.1% in Mississippi); 6.5% of ART infants were born very preterm (range: 3.3% in Nevada to 14.8% in South Dakota), compared with 1.9% among all infants born in the general population (range: 1.1% in Vermont to 2.9% in Mississippi). The percentage of infants conceived with ART who were low birthweight varied from 9.3% (range: 4.1% in South Carolina to 20.9% in Puerto Rico) among singletons, to 55.2% (range: 41.5% in New Hampshire to 83.3% in South Dakota) among twins, and 95.3% (range: 85.2% in Oklahoma to 100% in several reporting areas) among triplets or higher-order multiples; comparable percentages for all infants were 6.3% (range: 4.5% in Alaska to 10.3% in Puerto Rico), 55.4% (range: 46.0% in Alaska to 69.0% in Puerto Rico), and 91.6% (range: 80.6% in Missouri to 100% in several reporting areas), respectively. The percentage of ART infants who were preterm varied from 13.2% (range: 9.4% in West Virginia to 25.4% in North Dakota) among singletons, to 61.0% (range: 47.8% in DC to 80.0% in Maine and West Virginia) among twins, and 97.7% (range: 92.7% in Massachusetts to 100% in several reporting areas) among triplets or higher-order multiples; comparable percentages for all infants were 9.9% (range: 7.3% in Vermont to 15.8% in Puerto Rico), 56.8% (range: 47.2% in Connecticut to 67.2% in Puerto Rico), and 92.6% (range: 36.4% in Oregon to 96.8% in Ohio), respectively.
Interpretation:
The percentage of infants conceived with ART varied considerably by reporting area. In most reporting areas, multiples from ART comprised a substantial proportion of all twin, triplet, and higher-order infants born, and the rates of low birthweight and preterm infants were disproportionately higher among ART infants than in the birth population overall. Among women aged <35 years, eSET procedures warrant consideration because these patients might have extra embryos available for cryopreservation, which is a good predictor of embryo quality, and might have a more favorable prognosis for a live birth than older patients. However, on average, two embryos were transferred per cycle in ART procedures among women aged <35 years, influencing the overall multiple-birth rates in the United States. Compared with ART singletons, ART twins were approximately four and a half times more likely to be born preterm, and approximately six times more likely to be born with low birthweight. Singleton infants conceived with ART had slightly higher rates of preterm delivery and low birthweight than all singleton infants born in the United States. ART use per population unit was geographically varied, with 12 states showing ART use above the national rate. Of the four states (Illinois, Massachusetts, New Jersey, and Rhode Island) with comprehensive statewide-mandated health insurance coverage for ART procedures (e.g., coverage for at least four cycles of in vitro fertilization), two states (Massachusetts and New Jersey) had rates of ART use exceeding twice the national level. This type of mandated insurance has been associated with greater use of ART and might account for some of the difference in per capita ART use observed among states.
Public health actions:
Reducing the number of embryos transferred per ART procedure and increasing use of eSET, when clinically appropriate (typically age <35 years), might reduce multiple births and related adverse consequences of ART. Improved patient education and counseling on the maternal and infant health risks of having twins are needed given that twins account for the majority of ART-conceived multiple births. Although ART contributes to increasing rates of multiple births, it does not explain all of the increases. Other explanations for multiple births not investigated in this report might include age-related factors and the role of non-ART fertility treatments, and warrant further study.
Seminal plasma is a potential source of biomarkers for many disorders of the male reproductive system including male infertility. The identification and characterisation of differentially expressed proteins in seminal plasma of man with normal and impaired spermatogenesis can help in the elucidation of the molecular basis of male infertility. We compared the protein expression profiles of seminal plasma from four different groups of men as follows: normozoospermic, asthenozoospermic, oligozoospermic and azoospermic groups, using two-dimensional differential gel electrophoresis (2-D DIGE). We found eight proteins with statistically significant increased expression in azoospermia compared with at least one of the other studied groups. The differentially expressed spots were fibronectin, prostatic acid phosphatase (PAP), proteasome subunit alpha type-3, beta-2-microglobulin, galectin-3-binding protein, prolactin-inducible protein and cytosolic nonspecific dipeptidase. Notably, PAP was increased in patients with azoospermia compared with that of all other groups. We have observed no statistically significant differences in protein expression between three of the groups: normozoospermic, oligozoospermic and asthenozoospermic. We suggest that the identified panel of proteins in our study especially PAP have a strong potential to be used as azoospermia markers. However, further investigations will be necessary to validate these markers in samples of larger and independent patient cohorts and to clarify their role in the pathogenesis of male infertility.
To identify a panel of common seminal proteins in human seminal plasma by fertile men that might be involved in successful reproduction.
Experimental study.
University hospital.
Five fertile men who conceived within 3 months before the start of the study.
None.
Proteomic analysis performed by an Ultimate 3000 Nano/Micro-HPLC apparatus equipped with an FLM-3000-Flow manager module and coupled with an LTQ Orbitrap XL hybrid mass spectrometer; gene ontology analysis.
From 919 to 1,487 unique proteins were identified per individual subject sample. Among these proteins, 83 proteins were present in all samples, including some proteins that might be involved in male fertility, such as semenogelin I, semenogelin II, olfactory receptor 5R1, lactoferrin, hCAP18, spindling, and clusterin. The gene ontology annotation analysis provided further information in describing common pattern in male fertility.
The identification of common seminal plasma proteome in fertile men could provide better insight into the physiology of male fertility and might identify novel markers of male infertility.
A bottom-up label-free mass spectrometric proteomic strategy was used to analyse the protein profiles of the human embryonic secretome. Culture media samples used for embryonic culture of patients undergoing intracytoplasmic sperm injection cycles were selected as a test case for this exploratory proof-of-principle study. The media were stored after embryo transfer and then pooled into positive (n = 8) and negative (n = 8) implantation groups. The absolute quantitative bottom-up technique employed a multidimensional protein identification technology based on separation by nano-ultra-high pressure chromatography and identification via tandem nano-electrospray ionization mass spectrometry with data-independent scanning in a hydrid QqTOF mass spectrometer. By applying quantitative bottom-up proteomics, unique proteins were found exclusively in both the positive- and negative-implantation groups, which suggest that competent embryos express and secrete unique biomarker proteins into the surrounding culture medium. The selective monitoring of these possible secretome biomarkers could make viable procedures using single-embryo transfer.
Figure
A bottom-up label-free proteomic mass spectrometric analysis of the human embryo secretome is described. This approach seems to allow quantification and identification of unique proteins related to positive- and negative-implantation groups, which can be further validated as biomarkers for selection and transfer of a single embryo.
The successful outcome of intracytoplasmic sperm injection (ICSI) with round-headed spermatozoa (globozoospermia) is reported.
A couple with infertility secondary to globozoospermia received ICSI treatment. Fertilization, cleavage and pregnancy outcomes
were recorded. This couple experienced 40, 10 and 42% fertilization rates after ICSI in their first, second and third cycles
respectively. Pregnancy did not occur in the first or second cycle but was successfully achieved after the third ICSI cycle.
It is concluded that current ICSI procedures may overcome the infertility associated with globozoospermia and result in normal
healthy livebirth without assisted oocyte activation.
Mass spectrometry-based proteomics has greatly benefitted from enormous advances in high resolution instrumentation in recent years. In particular, the combination of a linear ion trap with the Orbitrap analyzer has proven to be a popular instrument configuration. Complementing this hybrid trap-trap instrument, as well as the standalone Orbitrap analyzer termed Exactive, we here present coupling of a quadrupole mass filter to an Orbitrap analyzer. This "Q Exactive" instrument features high ion currents because of an S-lens, and fast high-energy collision-induced dissociation peptide fragmentation because of parallel filling and detection modes. The image current from the detector is processed by an "enhanced Fourier Transformation" algorithm, doubling mass spectrometric resolution. Together with almost instantaneous isolation and fragmentation, the instrument achieves overall cycle times of 1 s for a top 10 higher energy collisional dissociation method. More than 2500 proteins can be identified in standard 90-min gradients of tryptic digests of mammalian cell lysate- a significant improvement over previous Orbitrap mass spectrometers. Furthermore, the quadrupole Orbitrap analyzer combination enables multiplexed operation at the MS and tandem MS levels. This is demonstrated in a multiplexed single ion monitoring mode, in which the quadrupole rapidly switches among different narrow mass ranges that are analyzed in a single composite MS spectrum. Similarly, the quadrupole allows fragmentation of different precursor masses in rapid succession, followed by joint analysis of the higher energy collisional dissociation fragment ions in the Orbitrap analyzer. High performance in a robust benchtop format together with the ability to perform complex multiplexed scan modes make the Q Exactive an exciting new instrument for the proteomics and general analytical communities.
The prerequisite of successful implantation depends on achieving the appropriate embryo development to the blastocyst stage and at the same time the development of an endometrium that is receptive to the embryo. Implantation is a very intricate process, which is controlled by a number of complex molecules like hormones, cytokines, and growth factors and their cross talk. A network of these molecules plays a crucial role in preparing receptive endometrium and blastocyst. Furthermore, timely regulation of the expression of embryonic and maternal endometrial growth factors and cytokines plays a major role in determining the fate of embryo. Most of the existing data comes from animal studies due to ethical issues. In this study, we comprehend the data from both animal models and humans for better understanding of implantation and positive outcomes of pregnancy. The purpose of this review is to describe the potential roles of embryonic and uterine factors in implantation process such as prostaglandins, cyclooxygenases, leukemia inhibitory factor, interleukin (IL) 6, IL11, transforming growth factor-β, IGF, activins, NODAL, epidermal growth factor (EGF), and heparin binding-EGF. Understanding the function of these players will help us to address the reasons of implantation failure and infertility.
Protamine content is necessary for proper sperm chromatin condensation and subsequent male fertility. The exact effect of smoking on male fertility remains controversial. The objective of this study was to evaluate the effect of smoking on protamine content of sperm in smoker and non-smoker patients.
Protamines 1 (P1) and 2 (P2) were quantified by gel electrophoresis in the sperm of 53 smokers and 63 non-smokers. Sperm DNA fragmentation was analyzed employing the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) assay and non-condensed chromatin was evaluated using chromomycin A(3) (CMA(3)). Levels of smoking and oxidative stress markers were determined in seminal plasma using an enzyme linked immunosorbant assay (ELISA) and chemical reactions.
Protamine 2 concentrations were significantly lower (P < 0.050) in smokers than in non-smokers. In contrast P1/P2 ratios were significantly higher (P < 0.010) in smokers (1.34 ± 0.46 ng/10(6) sperm) than in non-smokers (1.11 ± 0.20 ng/10(6) sperm). The oxidative stress and smoking markers, reactive oxygen species (ROS), malondialdehyde, 8-Hydroxyguanosine (8-OHdG) and cotinine were significantly higher (P < 0.010) in smokers than in non-smokers, and correlated significantly (P < 0.050) with P1/P2 ratios. P2 showed significant negative (P < 0.050) correlations with ROS, 8-OHdG and cotinine. CMA(3) and TUNEL were also significantly higher (P < 0.010) in smokers (36.4 ± 8.1 and 17.4 ± 5.3%) than in non-smokers (29.8 ± 7.1 and 11.3 ± 4.2%).
This is the first study to evaluate the effect of smoking on protamines. Abnormal elevation of the P1/P2 ratio appears to be associated with aberrant P2 expression in smokers. These results suggest that induced oxidative stress by cigarette smoking may have significant inverse effect on the protamination process by disrupting P2.
Soluble HLA-G (sHLA-G) has been suggested as a non-invasive marker for embryo selection to improve pregnancy rates after assisted reproduction technique (ART). Our study aimed at the identification of parameters influencing the detection of sHLA-G in embryo cultures (ECs) and at the prognostic relevance of sHLA-G in a multi-centre study.
In total 4212 EC from 2364 cycles were randomly collected from 29 German ART centres and analysed for sHLA-G by Luminex-based technology.
Among test and culture conditions, only the cleavage stage of the embryo was identified as an independent factor for sHLA-G detection (P < 0.001). Overall, sHLA-G was significantly associated with pregnancy after ART [P < 0.001; odds ratio: 2.0 (95% CI: 1.7-2.4)], suggesting that sHLA-G testing might improve the pregnancy rate from 30 to 40%. Importantly, the sHLA-G status of embryos could be associated with pregnancy after single embryo transfer [P = 0.002; odds ratio: 3.3 (95% CI: 1.5-6.8)] doubling the probability of pregnancy rate to 26% after sHLA-G testing. The patient's age, number of transferred embryos, morphological grading [EXP(B): 4.3 (95% CI: 2.1-8.9)] of embryos and sHLA-G status [EXP(B): 2.3 (95% CI: 1.8-3.1)] were independent predictors of pregnancy, with the latter two being most powerful.
This study provides significant evidence that the morphological scoring system is still the best strategy for the selection of embryos but that sHLA-G might be considered as a second parameter if a choice has to be made between embryos of morphologically equal quality.
Glycosaminoglycans, especially heparin, are involved in various cell processes such as apoptosis, cell cycle control, platelet activation, capacitation, acrosome reaction and sperm decondensation. Heparin-binding proteins (HBPs) are essential constituents of human seminal fluid, which bind to sperm lipids containing the phosphorylcholine group and mediate the fertilization process. We utilized a proteomic set-up consisting of affinity chromatography, isoelectric focusing (IEF) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI TOF/MS) for protein analysis of human HBPs. We resolved 70 different spots on two-dimensional (2-D) gel and subsequently identifi ed these proteins. Forty different types of proteins were identified. Functional analysis revealed that 38% of the proteins belonged to the enzyme category, 20% were involved in RNA processing and transcription, 18% in structure and transport function, and 16% in cell recognition and signal transduction. We also identified 8% of proteins with unknown functions, although their expression in seminal fluid has been documented. Proteins of seminal fluid that bind heparin may be directly involved in sperm capacitation and acrosome reaction (AR), which are the two critical steps for fertilization. This information on HBPs would be useful for identifying potential biomarkers of fertility in the near future.
Male factors account for 40% of infertility cases. The identification of differentially expressed proteins on spermatozoa from fertile and infertile men can help in the elucidation of the molecular basis of male infertility. The aim of this study was to compare sperm proteomes from 3 different groups: fertile men, normozoospermic men consulting for infertility, and normozoospermic men with an impaired capacity for fertilization (IVF-failure). We used differential proteomics with isobaric tags for relative and absolute quantitation (iTRAQ) labeling, and LC-MS analysis to identify proteins that are differentially expressed. Three hundred forty eight unique proteins were identified and quantified. The analysis identified 33 proteins that were differentially expressed in the IVF-failure group vs. the fertile group. Comparison of the infertile and fertile groups revealed that 18 proteins appeared to be differentially expressed. Four proteins were similarly altered in the IVF-failure and infertile groups: semenogelin 1 (SEMG1), prolactin-induced protein (PIP), glyceraldehyde-3-phosphate dehydrogenase (GAPDHS), and phosphoglycerate kinase 2 (PGK2). These protein markers were selected for validation using multiple reactions monitoring mass spectrometry (MRM-MS) and further confirmed by western blot analysis. Overall, these results suggest that a panel of proteins may be used as biomarkers for future studies of infertility.
Mammalian sperm motility is a prerequisite for in vivo fertilization and alterations in this parameter are commonly observed in infertile males. However, we still do not have a complete understanding of the molecular mechanisms controlling it. The aim of this study was to identify proteins involved in human sperm motility deficiency, by using TMT protein labeling and LC-MS/MS. Two complementary approaches were used: comparison between sperm samples differing in motility (asthenozoospermic versus normozoospermic); and comparison between sperm subpopulations of fractionated normozoospermic samples differing in motility (non-migrated versus migrated). LC-MS/MS resulted in the identification of 1157 and 887 proteins in the first and second approach, respectively. Remarkably, similar proteomic alterations were detected in the two experiments, with 80 proteins differentially expressed in the two groups of samples and 93 differentially expressed in the two groups of subpopulations. The differential proteins were analyzed by GO, cellular pathways and clustering analyses and resulted in the identification of core deregulated proteins and pathways associated with sperm motility dysfunction. These included proteins associated with energetic metabolism, protein folding/degradation, vesicle trafficking and the cytoskeleton. Contrary to what is usually accepted, the outcomes support the hypothesis that several metabolic pathways (notably mitochondrial-related ones) contribute towards regulating sperm motility.
The human endometrium is receptive for implantation of a blastocyst, for only 4–5 days in each menstrual cycle. Failure of implantation is a major reason for infertility in women, and the inability to achieve endometrial receptivity is responsible for much of the failure of reproductive technologies. Endometrial receptivity requires alterations in the uterine luminal and glandular cells, particularly in terms of their secretory capacity and altered expression of adhesion molecules, along with decidualization of the endometrial stroma, which in women is initiated during the receptive phase, regardless of the presence of a blastocyst. Increased leukocyte numbers are also important. The microenvironments provided by the endometrium during the receptive phase and which support implantation are highly complex and constantly changing. The present review summarizes work from our laboratories and others, regarding these microenvironments, how they impact on receptivity and how they are disturbed in infertile women. Such microenvironments can also be manipulated to provide new contraceptive strategies for women.
Objective:
To identify the male molecular causes of failures of IVF (with a deficient binding of spermatozoa to the zona pellucida, without any obvious oocyte anomaly), which are undetected by classical sperm analysis.
Design:
Case-control prospective study.
Setting:
University hospital.
Patient(s):
Proteomic profiles of spermatozoa in patients with a complete failure of fertilization and no spermatozoa bound to the zona pellucida were compared with those of controls (men with normal fertilization and cleavage rates after classical IVF for tubal indication).
Intervention(s):
All samples were analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) after being divided into three fractions according to their isoelectric point.
Main outcome measure(s):
Differentially expressed proteins between infertile men and controls were identified by mass spectrometry.
Result(s):
Seventeen proteins differentially expressed between cases and controls were found. Twelve of these proteins were identified by mass spectrometry, and two may influence gametes interaction: laminin receptor LR67 and L-xylulose reductase (P34H).
Conclusion(s):
This study shows that 2D-DIGE might be useful in finding potential targets for diagnosis and prognosis of idiopathic infertility in IVF.
Oxidative stress has been established as one of the main causes of male infertility and has been implicated in many diseases associated with infertile men. It results from high concentrations of free radicals and suppressed antioxidant potential, which may alter protein expression in seminal plasma and/or spermatozoa. In recent years, proteomic analyses have been performed to characterize the protein profiles of seminal ejaculate from men with different clinical conditions, such as high oxidative stress. The aim of the present review is to summarize current findings on proteomic studies performed in men with high oxidative stress compared with those with physiological concentrations of free radicals, to better understand the aetiology of oxidative stress-induced male infertility. Each of these studies have suggested candidate biomarkers of oxidative stress, among them are DJ-1, PIP, lactotransferrin and peroxiredoxin. Changes in protein concentrations in seminal plasma samples with oxidative stress conditions were related to stress responses and to regulatory pathways, while alterations in sperm proteins were mostly associated to metabolic responses (carbohydrate metabolism) and stress responses. Future studies should include assessment of post-translational modifications in the spermatozoa as well as in seminal plasma proteomes of men diagnosed with idiopathic infertility.
Recurrent implantation failure refers to failure to achieve a clinical pregnancy after transfer of at least four good-quality embryos in a minimum of three fresh or frozen cycles in a woman under the age of 40years. The failure to implant may be a consequence of embryo or uterine factors. Thorough investigations should be carried out to ascertain whether there is any underlying cause of the condition. Ovarian function should be assessed by measurement of antral follicle count, FSH and anti-Müllerian hormone. Increased sperm DNA fragmentation may be a contributory cause. Various uterine pathology including fibroids, endometrial polyps, congenital anomalies and intrauterine adhesions should be excluded by ultrasonography and hysteroscopy. Hydrosalpinges are a recognized cause of implantation failure and should be excluded by hysterosalpingogram; if necessary, laparoscopy should be performed to confirm or refute the diagnosis. Treatment offered should be evidence based, aimed at improving embryo quality or endometrial receptivity. Gamete donation or surrogacy may be necessary if there is no realistic chance of success with further IVF attempts. Recurrent implantation failure is an important cause of repeated IVF failure. It is estimated that approximately 10% of women seeking IVF treatment will experience this particular problem. It is a distressing condition for patients and frustrating for clinicians and scientists. In this review, we have discussed the definition and management of the possible underlying causes of recurrent implantation failure.
Proteomic technologies have begun providing evidence that viable embryos possess unique protein profiles. Some of these potential protein biomarkers have been identified as extracellular and could be used in the development of a noninvasive quantitative method for embryo assessment. The field of assisted reproductive technologies would benefit from defining the human embryonic proteome and secretome, thereby expanding our current knowledge of embryonic cellular processes. Copyright © 2013 American Society for Reproductive Medicine, Published by Elsevier Inc.
Seminal plasma serves as a natural reservoir of antioxidants. It helps to remove excessive formation of reactive oxygen species (ROS) and consequently, reduce oxidative stress. Proteomic profiling of seminal plasma proteins is important to understand the molecular mechanisms underlying oxidative stress and sperm dysfunction in infertile men.
This prospective study consisted of 52 subjects: 32 infertile men and 20 healthy donors. Once semen and oxidative stress parameters were assessed (ROS, antioxidant concentration and DNA damage), the subjects were categorized into ROS positive (ROS+) or ROS negative (ROS-). Seminal plasma from each group was pooled and subjected to proteomics analysis. In-solution digestion and protein identification with liquid chromatography tandem mass spectrometry (LC-MS/MS), followed by bioinformatics analyses was used to identify and characterize potential biomarker proteins.
A total of 14 proteins were identified in this analysis with 7 of these common and unique proteins were identified in both the ROS+ and ROS- groups through MASCOT and SEQUEST analyses, respectively. Prolactin-induced protein was found to be more abundantly present in men with increased levels of ROS. Gene ontology annotations showed extracellular distribution of proteins with a major role in antioxidative activity and regulatory processes.
We have identified proteins that help protect against oxidative stress and are uniquely present in the seminal plasma of the ROS- men. Men exhibiting high levels of ROS in their seminal ejaculate are likely to exhibit proteins that are either downregulated or oxidatively modified, and these could potentially contribute to male infertility.
Unlabelled:
Human follicular fluid (HFF) has been proven to contain biologically active molecules and proteins that may affect follicle growth and oocyte fertilization. Based on this concept, HFF proteomic characterization is having a significant impact in the delineation of a biomarkers' profile for oocyte quality estimation and, maybe, for in vitro fertilization (IVF) success improvement. Follicular fluid is characterized by a vast protein complexity and a broad dynamic range of protein abundances that hinder its analysis. In this study we determined a proper solubilization and resolution method of HFF in 2-DE, minimizing sample manipulation, protein loss, and experimental artifacts. According to our methodology some low-abundance proteins were detected and identified by MS. Identified proteins were then functionally cross-linked by a pathway analysis. The generated path highlighted the occurrence in HFF of a tight functional-network in which effectors and inhibitors control and balance a space- and time-dependent induction/inhibition of inflammation, coagulation, and ECM degradation/remodeling. Such fine modulation of enzymatic activities exerts a fundamental role in follicle development and in oocyte competence acquiring. Alpha-1-antitrypsin resulted in the core protein of the delineated net and we interestingly detected its differential incidence in FF and serum from two small cohorts of patients who underwent IVF.
Biological significance:
Human ovarian follicular fluid (HFF) is the in vivo microenvironment for oocyte during folliculogenesis. It contains biologically active molecules that may affect oocyte quality, fertilization, and embryo development. HFF is also one of the most abundant "waste product" in assisted reproduction. This makes HFF a readily accessible source of biomolecules for competence evaluation of collected oocytes. The methodological improvement we obtained in proteomics characterization of HFF lead to a wide overview on the functional correlation existing between several fluid components and on how their aberrant occurrence/activity may affect oocyte quality and ovulation.
The analysis of endometrial secretions offers a window on human peri-implantation events that have hitherto been difficult to study noninvasively. Uterine secretomic analysis provides a sensitive means of interrogating the contents of secretions, which have been shown to play a key role in determining endometrial receptivity and embryo-endometrial signaling, and in providing a nourishing environment to the preimplantation embryo. Compared with other means of assessing the endometrium with genomics or proteomics, secretomics offers a nondisruptive approach, allowing analysis during conception cycles. It also provides information on the downstream molecular profile directly encountered by the embryo, offering an integrated review of the complete endometrial surface rather than information representative of only a discrete biopsy site. In this article, recent data derived from uterine secretomics are reviewed. In addition to in vivo studies, recent data from in vitro studies that are changing our understanding of the role of the endometrium in embryo selection are reviewed. Finally, the role of uterine secretions in nutrition and early development programming of the embryo is considered.
Although male factors account for approximately 50% of all infertility, the mechanisms underlying their origin are unknown. Currently, clinicians rely primarily on semen analyses to predict male reproductive potential and chart treatment success. Even when invasive procedures are performed, the causes of male factor infertility frequently remain elusive. Recently, the advent of new technologies has spurred the search for novel male infertility biomarkers, and the detection of genes, proteins, or metabolites unique to the infertile male holds much promise. The concept that a cost-effective, noninvasive, and accurate set of biomarkers can be identified to diagnose male factor infertility is tantalizing. This review focuses on the various methodologies used in the discovery of novel biomarkers along with their findings. Specific attention is paid to recent advances in the fields of genetics, proteomics, and metabolomics.
A noninvasive test for endometriosis would be useful for the early detection of endometriosis in symptomatic women who have pelvic pain and/or subfertility with normal ultrasound results. This would include nearly all cases of minimal-to-mild endometriosis, some cases of moderate-to-severe endometriosis without a clearly visible ovarian endometrioma, and cases with pelvic adhesions and/or other pelvic pathology that might benefit from surgery to improve pelvic pain and/or subfertility. This overview discusses the diagnostic performance of noninvasive or semi-invasive tests for endometriosis, including panels of known peripheral blood biomarkers, protein/peptide markers discovered by proteomics, miRNA, and endometrial nerve fiber density. Tests with high sensitivity and acceptable specificity have been developed; some have been validated in independent populations and are therefore promising. To make real progress, international agreement on biobank development is needed for standard operating procedures for the collection, treatment, storage, and analysis of tissue samples and for detailed clinical phenotyping of these samples. Furthermore, it is necessary to validate the diagnostic accuracy of any promising test prospectively in an independent symptomatic patient population with subfertility and/or pain without clear ultrasound evidence of endometriosis and with a clinical indication for surgery, divided into cases with laparoscopically and histologically confirmed endometriosis and controls with laparoscopically confirmed absence of endometriosis.
Objective:
To identify the relative abundance of proteins in pooled reactive oxygen species (ROS)-positive (ROS+) and ROS-negative (ROS-) semen samples with the use of two-dimensional differential in-gel electrophoresis (2D-DIGE).
Design:
Spermatozoa suspensions from ROS+ and ROS- groups by 2D-DIGE analysis.
Setting:
Tertiary hospital.
Patient(s):
20 donors and 32 infertile men.
Intervention(s):
Seminal ejaculates evaluated for semen and proteomic analysis.
Main outcome measure(s):
Semen samples from 20 donors and 32 infertile men were pooled, divided into ROS+ and ROS- groups based on the cutoff value of <20 relative light units/s/10(6) sperm and frozen. From each pooled group, spermatozoa were labeled with Cy3/Cy5 fluorescent dye. Duplicate 2D-DIGE gels were run. Image analysis was performed with the use of Decider software. Protein spots exhibiting ≥1.5-fold difference in intensity were excised from the preparatory gel and identified by liquid chromatography-mass spectrometry. Data were analyzed with the use of Sequest and Blast programs.
Result(s):
A total of 1,343 protein spots in gel 1 (ROS-) and 1,265 spots in gel 2 (ROS+) were detected. The majority of protein spots had similar expression, with 31 spots were differentially expressed. Six spots were significantly decreased and 25 increased in the ROS- sample compared with the ROS+ sample.
Conclusion(s):
Significantly different expression of protective proteins against oxidative stress was found in ROS-compared with ROS+ samples. These differences may explain the role of oxidation species in the pathology of male infertility.
Failure of the endometrium to achieve receptivity results in infertility, and it is also a rate-limiting step in in vitro fertilization (IVF) success. The microenvironments provided by the endometrium during the receptive phase and that support implantation are highly complex and constantly changing as implantation progresses. Although a number of gene array studies have defined mRNA changes across the cycle, with infertility, and in IVF cycles, these have not generally been informative due in part to the subsequent regulation of transcription and posttranslational modifications of the proteins. State-of-the-art proteomic technologies now enable analysis of changes in the endometrium and its secretome related to cycle phase and associated with infertility. These techniques include two-dimensional differential in-gel electrophoresis, isobaric tags for relative and absolute quantitation, and multiplex analyses of selected panels of markers. Subsequent definition of cellular location, timing of production of identified proteins, and their regulation by steroid hormones and blastocyst-derived factors provide indications of their functions and their relationship to the establishment of pregnancy. Proteins discovered by proteomic analyses and fully evaluated will provide the differentiative profiles necessary to inform clinical practice and serve as an end point for optimizing stimulation cycles in IVF clinics as well as more clearly defining the molecular mechanisms underlying successful implantation.
Proteomic technologies represent new strategies towards high-throughput, simultaneous analysis of thousands of biological molecules leading to the discovery of biomarkers for early diagnosis, prognosis and prediction of pregnancy outcome. Proteomics have additional relevance in understanding pathophysiology and the development of molecularly targeted therapeutics. Comparison of normal human amniotic fluid proteome with that coming from pregnancies carrying fetuses with chromosomal abnormalities facilitated the detection of panels of potential biomarkers for prenatal detection of fetal aneuploidies. Candidate biomarkers for the early prediction of preeclampsis are also available, while four biomarkers (defensins-2 and -1, calgranulin-C, and calgranulin-A), which were called the «MR score», can quickly and accurately detect potentially dangerous infections and predict premature birth.Researchers remain hopeful that proteomic studies will allow for the identification of either one protein marker or of a panel of markers for prenatal detection of fetal aneuploidies and pregnancy complications that could be usefully employed for diagnostic purposes or improvement of the current screening methods. For maximum predictive power however, biomarkers should be selected for further comparative analysis of expression and structural modifications in large numbers of samples from chromosomally normal and abnormal pregnancies obtained from different populations.
Prolactin inducible protein (PIP) is a 17 kDa glycoprotein. It binds to many proteins including fibrinogen, actin, keratin, myosin, immunoglobulin G, CD4, and human zinc-alpha-2 glycoprotein. Its ability to bind a large array of proteins indicates its multifaceted role in various biological processes, such as fertility, immunoregulation, antimicrobial activity, apoptosis, and tumor progression. Here, we present the first report of native human serum albumin (HSA)-PIP complex formation in seminal plasma. The complex was purified by chromatographic separation techniques, analyzed by gel electrophoresis, identified by MALDI-TOF mass spectrometry and validated by co-immunoprecipitation coupled with western blotting experiments. Moreover, the behavior of complex in solution was analyzed by dynamic light scattering and interacting residues were identified by in silico protein-protein docking. The purified protein complex shows two bands (67 kDa and 17 kDa) on SDS-PAGE gel and a single band (~85 kDa) on native PAGE gel. The predicted complex structure has 13 intermolecular hydrogen bonds, which may contribute to the overall stability of the complex. As HSA has been known to preserve the motility of sperm, native HSA-PIP complex formation may point towards an important role of PIP, which can directly be correlated with male fertility/infertility.
Infertility affects approximately 15% of couples with equivalent male and female contribution. Absence of sperm in semen, referred to as azoospermia, accounts for 5-20% of male infertility cases and can result from pretesticular azoospermia, non-obstructive azoospermia (NOA), and obstructive azoospermia (OA). The current clinical methods of differentiating NOA cases from OA ones are indeterminate and often require surgical intervention for a conclusive diagnosis. We catalogued 2048 proteins in seminal plasma from men presented with NOA. Using spectral-counting, we compared the NOA proteome to our previously published proteomes of fertile control men and postvasectomy (PV) men and identified proteins at differential abundance levels among these clinical groups. To verify spectral counting ratios for candidate proteins, extracted ion current (XIC) intensities were also used to calculate abundance ratios. The Pearson correlation coefficient between spectral counting and XIC ratios for the Control-NOA and NOA-PV data sets is 0.83 and 0.80, respectively. Proteins that showed inconsistent spectral counting and XIC ratios were removed from analysis. There are 34 proteins elevated in Control relative to NOA, 18 decreased in Control relative to NOA, 59 elevated in NOA relative to PV, and 16 decreased in NOA relative to PV. Many of these proteins have expression in the testis and the epididymis and are linked to fertility. Some of these proteins may be useful as noninvasive biomarkers in discriminating NOA cases from OA.
To investigate the expression of Spindlin 1 (Spin 1) isoform2 and assess its function in mouse testis.
First, reverse-transcription polymerase chain reaction (RT-PCR) was used to determine whether Spin1 isoform2 is present in mouse testis. Then the expression patterns of the isoform between newborn and adult mice testes were compared by immunoblot analysis. Finally, the diversity of its localization in mice testes at different ages (days 0, 7, 14, 21, 28 and 60) was observed by immunohistochemistry. The localization of the protein in mouse sperm was also investigated by immunofluorescence.
The RT-PCR results show that Spin1 isoform2 is present in mouse testis. As shown by immunoblot analysis, the isoform was more highly expressed in adult testes compared with newborn testes. Interestingly, Spin1 isoform2 did not show up in the cytoplasm of primary spermatocytes until day 14. Also, the protein exists at the tail of the mouse sperm.
Spin1 isoform2 is a protein expressed highly in adult testis, which might be involved in spermatogenesis and could be necessary for normal sperm motility.
The receptive endometrium represents a physiologic state of the uterus when embryo implantation is possible. It occurs at a discreet stage of the menstrual cycle referred to as window of implantation, outside of which the uterus is refractory to the initiation of pregnancy. In modern society, assisted reproductive technologies (ART) are an ever-growing demand to counter infertility; however, pregnancy rates remain below expectations, not least because current diagnostic tools fail to provide accurate assessment of endometrial receptivity. In the last decade, widespread arrival of large-scale analytical techniques has brought a stream of studies seeking to identify specific biomarkers of endometrial receptivity by extracting global molecular information from endometrial biopsies. The latter are an undesired requirement for dating the endometrium, which has prompted development of alternative strategies whereby large-scale analyses and non-invasive methods can converge. In this context, secretomics represents an attractive possibility to assess endometrial maturation and receptivity. Endometrial-cell secretions poured into the uterine cavity are suitable for collection and analysis without the need of biopsying, and might provide important additional molecular information reflective of endometrial physiology and day of cycle. If properly validated, the outgoing results would represent a step forward in the development of diagnostic tools to assess endometrial receptivity.
To identify factors secreted by the human embryo and correlate levels with embryo morphology and pregnancy outcome.
A laboratory-based study of human embryo protein synthesis and secretion and a retrospective analysis of spent embryo culture media as it relates to pregnancy outcome.
University-based academic IVF program.
IVF patients who had donated cryopreserved human pronuclear-stage embryos. Patients undergoing fresh IVF cycles resulting in a blastocyst transfer who donated spent media drops.
In vitro embryo culture and collection of spent media.
Protein analysis and identification by two-dimensional gel electrophoresis and mass spectrometry, ApoA1 quantification by ELISA, and mRNA analysis by quantitative reverse transcriptase-polymerase chain reaction.
By protein gel electrophoresis, apolipoprotein A1 (ApoA1) was increased in the culture media from good-quality blastocysts (n = 6 embryos) compared to either cleavage-arrested embryos (n = 6 embryos) or poor-quality blastocysts (n = 6 embryos) using spent media from culture days 4 and 5, respectively. Apolipoprotein A1 concentrations were 23.1% greater in day 5 spent culture media from good-grade blastocysts (n = 30) when compared to poor-grade embryos (n = 30). However, in a group of patients (n = 20) with transfer of two good-quality blastocysts, ApoA1 levels from day 5 spent media did not correlate with embryo implantation and pregnancy. Quantitative reverse transcriptase-polymerase chain reaction confirmed the presence of ApoA1 mRNA transcripts in human blastocysts.
Apolipoprotein A1 is produced by human preimplantation embryos, and increased levels are present in spent culture media containing blastocysts of higher morphologic grade. These results suggest a role for lipoproteins in early embryologic development.
Generating a catalogue of sperm nuclear proteins is an important first step towards the clarification of the function of the paternal chromatin transmitted to the oocyte upon fertilization. With this goal, sperm nuclei were obtained through CTAB treatment and isolated to over 99.9% purity without any tail fragments, acrosome or mitochondria as assessed by optical microscopy and transmission electron microscopy. The nuclear proteins were extracted and separated in 2-D and 1-D gels and the 2-D spots and 1-D bands were excised and analysed to identify the proteins through LC-MS/MS. With this approach, 403 different proteins have been identified from the isolated sperm nuclei. The most abundant family of proteins identified are the histones, for which several novel members had not been reported previously as present in the spermatogenic cell line or in the human mature spermatozoa. More than half (52.6%) of the proteins had not been detected in the previous human whole sperm cell proteome reports. Of relevance, several chromatin-related proteins, such as zinc fingers and transcription factors, so far not known to be associated with the sperm chromatin, have also been detected. This provides additional information about the nuclear proteins that are potentially relevant for epigenetic marking, proper fertilization and embryo development.
To analyze information on assisted reproductive technologies (ART) performed globally.
Data on access, efficacy, and safety of ART were collected for the year 2003 from 54 countries.
National and regional ART registries globally.
Patients undergoing ART globally.
Collection and analysis of international ART registry data.
Number of cycles performed in reporting countries and regions globally for different ART procedures with resulting pregnancy, live birth and multiple birth rates.
A total of 433,427 initiated cycles reported in this registry resulted in 173,424 babies born. This corresponded to a delivery rate per aspiration of 22.4% for in vitro fertilization (IVF), 23.3% for intracytoplasmic sperm injection (ICSI), and a delivery rate per transfer of 17.1% for frozen embryo transfer. Although there is wide variation among countries and regions, the overall proportion of deliveries with twins and triplets from IVF and ICSI was 24.8% and 2.0%, respectively. There were wide variations in access, and compared with the previous report (year 2002), there was a 3.9% increase in the number of reported cycles and a minor increase in the delivery rate per aspiration. There was also a marginal decline in the mean number of embryos transferred and in the rate of multiple births.
ART access, efficacy, and safety varies greatly globally. Collection and analysis of data over time will benefit ART patients, providers, and policy makers.
This study has identified the first protein, lipocalin-1, in the secretome of human blastocysts that is associated with chromosome aneuploidy. The development of a noninvasive technique to determine an embryo's developmental competence, including chromosomal constitution, by analysis of spent IVF culture medium will be a powerful tool for embryo selection in assisted reproductive technologies.
Binding of sperm to egg in the mouse has been proposed to depend primarily on interactions between lectin-like egg-binding proteins on the sperm plasma membrane and complementary carbohydrates on the specialized extracellular matrix of the egg known as the zona pellucida. An alternative model posits that initial sperm-egg binding depends on the interaction of a sperm surface protein with a supramolecular complex of the three mouse zona pellucida glycoproteins (mZP1, mZP2, mZP3); the role of carbohydrate recognition in this paradigm is thought to be minimal (Gahlay G, Gauthier L, Baibakov B, Epifano O,Dean J. 2010. Gamete recognition in mice depends on the cleavage status of an egg's zona pellucida protein. Science.329:216-219). This perspective reviews these recent findings,and considers them in light of evidence favoring a major role for lectin-like interactions. An alternative model, the domain specific model for mammalian gamete binding, could reconcile some of the conflicting observations.
Proteomics refers to the analysis of expression, localization, functions, posttranslational modifications, and interactions of proteins expressed by a genome at a specific condition and at a specific time. Mass spectrometry (MS)-based proteomic methods have emerged as a key technology for unbiased systematic and high-throughput identification and quantification of complex protein mixtures. These methods have the potential to reveal unknown and novel changes in protein interactions and assemblies that regulate cellular and physiological processes. Both gel-based (one-dimensional [1D] gel electrophoresis, two-dimensional [2D] polyacrylamide gel electrophoresis, 2D difference in-gel electrophoresis [DIGE]) and gel-free (liquid chromatography [LC], capillary electrophoresis) approaches have been developed and utilized in a variety of combinations to separate proteins prior to mass spectrometric analysis. Detailed protocols for global proteomic analysis from adipose-derived stem cells (ASCs) using two central strategies, 2D-DIGE-MS and 2D-LC-MS, are presented here.
fertile women the patterns of proteome during the prereceptive (day 2 after LH surge) and receptive (day 7 after LH surge) phases were analyzed by two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. A total of 31 differentially expressed proteins (17 up-regulated and 14 down-regulated) were identified between the two phases, which is helpful in understanding the biological mechanisms underlying human endometrial receptivity. (Fertil Steril (R) 2011;95:1161-3. (C) 2011 by American Society for Reproductive Medicine.)
Endometrial secretions in the uterine cavity contain mediators important for endometrial receptivity and embryo implantation. Unbiased analysis of uterine fluid from a receptive versus nonreceptive time of the menstrual cycle and in fertile and infertile women will provide new insights into uterine receptivity. We hypothesized that proteomic analysis of human uterine lavages would identify proteins important for the establishment of pregnancy in humans. Lavages collected from fertile (n = 7) and infertile (n = 8) women during the midsecretory (MS) phase, and from fertile women during the midproliferative (MP) (n = 7) phase, were assessed using 2D-differential in gel electrophoresis (2D-DiGE) over a pI 4-7 range. Statistical analysis revealed 7 spots that were significantly decreased in the MP compared to the MS phase, while 18 spots showed differential expression between fertile and infertile women. A number of proteins were identified by mass spectrometry, including antithrombin III and alpha-2-macroglobulin, whose production was confirmed in endometrial epithelium. Their staining pattern suggests roles during embryo implantation. Assessment of the human endometrial secretome has identified differences in the protein content of uterine fluid with respect to receptivity and fertility.
To compare pregnancy and implantation rates when embryos are selected based on a single Day 3 (D 3) morphology score vs. a GES score plus sHLA-G expression.
A prospective randomized study (n = 214) undergoing fresh ICSI cycles. Embryos were selected for transfer based on either Day 3 morphology score (Group A) or GES-scoring plus sHLA-G expression (Group B).
Clinical [35/107 (33%) vs. 52/107 (49%)] and ongoing pregnancy [20/107 (19%) vs. 52/107 (49%)] rates were significantly different between Group A and Group B (p < 0.05). Implantation rates were not significantly different between Group A [52/353 (15%)] and Group B [73/417 (18%)] (p < 0.05). The number of pregnancies lost during the first trimester was nearly 12 times higher in Group A [25/52 (48%)].
The miscarriage rate was significantly lower in Group B than Group A and the pregnancy results were superior when embryos were selected based on GES plus sHLA-G expression.
A key step in assisted reproduction is the assessment of embryo viability in order to identify the embryo(s) most likely to result in pregnancy. Currently used embryo assessment systems are largely based on morphology and cleavage rate. While these systems have been pivotal in improving implantation and pregnancy rates and reducing multiple gestations, their precision is still insufficient. The limitations of strategies based on morphology have led to the investigation of adjunctive technologies for non-invasive assessment of embryo viability in assisted reproduction. These include the measurement of glucose, pyruvate, or amino acid levels in the embryo culture media, assessment of oxygen consumption by the embryo, genomic and proteomic profiling, and most recently, analytical examination of the embryonic metabolome. As the number of ART cycles increases worldwide, improvements in the ability to quickly and non-invasively identify the best embryos for transfer become an increasingly more important goal for reproductive medicine.