Integrin cell adhesion molecules in endometrium of fertile and infertile women throughout menstrual cycle

Integrins (ITGs) are ubiquitous cell adhesion molecules that undergo dynamic alterations during the normal menstrual cycle in human endometrium. The distribution of four different subunits, viz. alpha 4, alpha 6, beta 3 and beta 4 in human endometrial tissue at different stages of the menstrual cycle was studied using immunohistochemical, enzyme immunoassay and SDS-PAGE/Western blot techniques. The specificity of each mAb to their respective ligands viz., laminin (Ln), fibronectin (Fn) and vitronectin (Vn) was done by cell adhesion assays. Both alpha 6 and beta 4 subunits (Ln receptors) expressed primarily on the glandular epithelium, while glandular, stromal and luminal cells expressed predominantly alpha 4 (Fn receptor) and beta 3 (Vn receptor). The appearance of alpha 4 and beta 3 ITG subunits was found to be cell and cycle specific. The levels of both alpha 6 and beta 4 increased throughout the menstrual cycle, while beta 3 subunit appeared abruptly on cycle day 19/20. The immunostaining for alpha 4 and beta 3 was absent in 90% of infertile women. The timing of expression of alpha 4 and beta 3, the two cycle--dependent ITGs framed the putative window of implantation and suggests a role in the diagnosis of infertility. In conclusion, the absence of alpha 4 and beta 3 ITG expression in the endometrium of infertility subjects during mid luteal phase may be associated with defects in uterine function. The defective uterine receptivity may be an unrecognised cause of infertility in these group of women.     

2D-DiGE analysis of the human endometrial secretome reveals differences between receptive and nonreceptive states in fertile and infertile women

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.     

Embryo transfer in fertile and infertile cows

Pregnancy rates were compared between fertile and infertile donors in an embryo transfer program. Non-surgical embryo transfer techniques were utilized for examination of uterine contents and collection of ova. Recovered embryos were transferred surgically into suitable recipients. Three groups of donors were included in this investigation: a) fertile; b) infertile, due to known causes (diagnosed); c) infertile, due to unknown causes (undiagnosed). There were 11.3, 6.6 and 8.0 corpora lutea; 6.8, 1.2 and 1.0 fertilized ova; 3.6, 0.8 and 0.4 pregnancies per superovulation for the fertile, diagnosed and undiagnosed groups, respectively. In 23 months, unsuperovulated, fertile, diagnosed and undiagnosed donors yielded 17, 15 and 9 fertilized ova which resulted in 15, 13, and 3 pregnancies, respectively. Donors treated for uterine infections, adhesions or cystic ovaries prior to superovulation responded with 10.1, 7.6 and 4.1 corpora lutea; 1.9, 4.0 and 0.3 fertilized ova; 1.1, 3.3 and 0.2 pregnancies per superovulation, respectively. Relatively few viable embryos were recovered from donors with chronic cystic ovaries or from infertile cows of unknown etiology. Infertile donors, when compared to fertile donors, were unproductive when used for embryo transfer.     

Morphological abnormalities in the spermatozoa of fertile and infertile men

The morphological analysis of the spermatozoa from fertile and infertile men was performed using light and electron microscopy to clarify the relationship between sperm morphology and fertility. Semen samples obtained from 22 partners of pregnant women were prepared according to the protocol standardized in an international collaborative study. Semen samples from 17 patients with asthenozoospermia or varicocele were collected in a hospital. Abnormalities in the spermatozoa were classified into three types for the tails, two for the midpieces, and six for the heads according to the criteria adapted from WHO guidelines (World Health Organization, 1999: WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction (4th edition)). Approximately 14% of the spermatozoa from the fertile men had abnormal tails at the light microscopic level while approximately 44% had abnormal heads. Most types of abnormalities found in the spermatozoa from the asthenozoospermic and varicocele patients were encountered in those from the fertile men, although the semen from the fertile men contained a higher percentage of normal spermatozoa than that from the patients. These results were also confirmed at the ultrastructural level. Most abnormal cell types are encountered in semen from fertile men, although the incidence of abnormalities is low.     

The expression pattern of MUC1 glycoforms and other biomarkers of endometrial receptivity in fertile and infertile women

Changes in the surface epithelium of the endometrium, characterized in part by alterations in cell-surface molecules, sex steroid receptors and the appearance of pinopodes, coincide with the window of endometrial receptivity in the menstrual cycle. This study was performed to evaluate the usefulness of hematoxylin and eosin staining, scanning and transmission microscopy, and MUC1 glycoform, sex steroid receptor, and interleukin receptor (type 1) expression as biomarkers of endometrial receptivity using carefully characterized clinical fertile and infertile groups of women. Using a combination of immunohistochemistry and scanning electron microscopy (SEM) called scanning immunoelectron microscopy (SIM), we confirmed that MUC1 mucin was not associated with the endometrial pinopodes, which have been linked with embryo adhesion. We also showed that failure of embryo implantation was associated with an abnormal endometrial expression of MUC1 mucin, and retention of nuclear progesterone receptor (PR) particularly in epithelial cells. Hematoxylin and eosin staining, transmission electron microscopy (TEM), SEM in isolation and immunohistochemistry for interleukin receptor were not shown to be useful markers. Progesterone-dependent regulation of MUC1 appears to be an important factor in determining endometrial receptivity.     

Frequency of detection of Ureaplasma urealyticum and Mycoplasma hominis in cervical canal and the Douglas pouch of infertile and fertile women

The group of organisms commonly referred to as genital mycoplasmas comprise species most often found in genitourinary tract of sexually active adults as common commensal inhabitants, or pathogens which can possibly cause many different pathologies like: non-gonococcal urethritis, bacterial vaginosis, cervicitis, endometritis or pelvic inflammatory disease. The problem of their morbidity and the possible influence they have on human fertility is still not clear. The aim of this study was to find out whether two investigated species- Ureaplasma urealyticum and Mycoplasma hominis can be detect more often in a group of infertile women. 74 women participated in the study and were assigned to one of 2 groups of patients: infertile women and fertile women without any sign of genital tract infection. Swabs from the cervical canal of the uterus and the fluid from the Douglas pouch were taken during the gynecological examination and laparoscopic procedure. Two diagnostic methods were used: biochemical method- commercial diagnostic kit- Mycoplasma IST 2 and PCR method. The results showed that Ureaplasma urealyticum and Mycoplasma hominis were detected among both fertile and infertile women with nearly the same frequency, much more often in cervical canal than in the Douglas pouch. Ureaplasma urealyticum was more common pathogen than Mycoplasma hominis in both groups and locations. The achieved results point out that the role of genital mycoplasmas in human infertility is still unclear and require further investigations.     

Proteomic analysis of human follicular fluid from fertile women

Background

Follicular fluid is a unique biological fluid in which the critical events of oocyte and follicular maturation and somatic cell-germ cell communication occur. Because of the intimate proximity of follicular fluid to the maturing oocyte, this fluid provides a unique window into the processes occurring during follicular maturation. A thorough identification of the specific components within follicular fluid may provide a better understanding of intrafollicular signaling, as well as reveal potential biomarkers of oocyte health for women undergoing assisted reproductive treatment. In this study, we used high and low pH HPLC peptide separations followed by mass spectrometry to perform a comprehensive proteomic analysis of human follicular fluid from healthy ovum donors. Next, using samples from a second set of patients, an isobaric mass tagging strategy for quantitative analysis was used to identify proteins with altered abundances after hCG treatment.

Results

A total of 742 follicular fluid proteins were identified in healthy ovum donors, including 413 that have not been previously reported. The proteins belong to diverse functional groups including insulin growth factor and insulin growth factor binding protein families, growth factor and related proteins, receptor signaling, defense/immunity, anti-apoptotic proteins, matrix metalloprotease related proteins, and complement activity. In a quantitative analysis, follicular fluid samples from age-matched women undergoing in vitro fertilization oocyte retrieval were compared and 17 follicular fluid proteins were found at significantly altered levels (p < 0.05) between pre-hCG and post-hCG samples. These proteins belong to a variety of functional processes, including protease inhibition, inflammation, and cell adhesion.

Conclusions

This database of FF proteins significantly extends the known protein components present during the peri-ovulatory period and provides a useful basis for future studies comparing follicular fluid proteomes in various fertility, disease, and environmental exposure conditions. We identified 17 differentially expressed proteins after hCG treatment and together these data showed the feasibility for defining biomarkers that illuminate how the ovarian follicle microenvironment is altered in various infertility-related conditions.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9077-6) contains supplementary material, which is available to authorized users.     

Comparison of abdominal skin temperature between fertile and infertile women by infrared thermography: A diagnostic approach

This retrospective study aimed to evaluate the differences in abdominal temperature (AT) between fertile (n=206; age) and infertile (n=250) women between the ages of 30 and 39 years. We evaluated the differences in two distinctive skin temperatures by thermography: ΔT₁ (CV8 index) – difference in temperature between the mid-abdomen (CV8 acupuncture area) and ventral upper arm (VUA) and ΔT₂ (CV4 index) – difference in temperature between the lower abdomen (CV4 acupuncture area) and VUA. The results indicated that the ΔT₁ and ΔT₂ of infertile women were significantly lower (by 1.05 °C and 0.79 °C, respectively; p<0.001, both) compared to those of fertile women. Additionally, the area under the curve of ΔT₁ (0.78) was greater compared to that of ΔT₂ (0.736), and its threshold was set at 0.675 °C, by which, the sensitivity and specificity of ΔT₁ for determination of fertility were found to be 80.8% and 68.4%, respectively. In conclusion, infertility is associated with lower AT. The decrease in AT in infertile women might be due to poor blood perfusion to the core muscles and tissues of the body. These findings provide a basis for further research for evaluation of clinical feasibility of thermography for analysis of infertility in women. Further evaluation of the influence of AT on fertility outcomes is required to determine the causal relationship between AT and infertility.     

Removal of vertebrate-dispersed fruits in vegetation on fertile and infertile soils

Summary The hypothesis that more plant species with vertebrate-dispersed fruits occur on fertile soils because there is a greater probability of fruit removal from the parent plant was tested at 16 sites around Sydney, Australia. Removal rates from artificial fruit spikes were two and a half times greater on fertile than infertile soil sites, although this was not quite statistically significant. High variability in removal rate between sites was evident irrespective of fertility. Most removal occurred during the day indicating that birds were important consumers, rather than nocturnal mammals. Bird abundance and diversity did not differ between soil types. More frugivorous species were found in plant communities growing on fertile soil. Two models could explain the patterns observed. Firstly, plants with vertebrate-dispersed fruits could be favoured on fertile soils because of a high abundance of frugivorous birds accomplishing seed dispersal. Alternatively, plants with vertebrate-dispersed fruits could be favoured on fertile soil sites for some other reason and frugivorous birds could be attracted to these areas of abundant food. The correlation between soil fertility and the percentage of vertebrate-dispersed fruits was stronger than the correlation between soil fertility and removal rates and suggests that the second model is more likely to be true. Frugivorous birds are unlikely to be responsible for the high percentage of species with vertebrate-dispersed fruits in fertile soil environments.     

Free amino acid concentration in hydatid cyst fluids from fertile and infertile human and animal Echinococcus granulosus.

The aim of this study was to search and compare free amino acid composition of fertile and infertile cyst fluids obtained from humans and animals infected naturally with Echinococcus granulosus, by using automated analysis based on cation-exchange chromatography with post-column ninhydrin derivatization system. 11 free amino acids from fertile (sheep origin), nine from infertile (cattle origin), 13 from infertile (human origin) hydatid cyst fluids and 19 amino acids from sera of patients with hydatid infection were detected. The levels of glycine, alanine, valine and lyrosine in fertile and infertile hydatid cysts fluids were significantly higher than in sera from patients with hydatid cysts. Glycine level in the fertile hydatid cyst fluids (sheep origin) was significantly higher than those of infertile cysts fluids (cattle and human origin) and sera with hydatid patients. Glycine level in fertile hydatid cyst fluids was about two times more concentrated in infertile cattle cyst fluids, 10 times more concentrated in infertile human hydatid cyst fluids and 13 times more concentrated in sera with hydatid patients. On the other hand, alanine and valine concentration in the fertile and infertile cyst fluids were at similar level with the exception that valine level in fertile cyst fluids was 12 times more concentrated in infertile human cyst fluids. The levels of tyrosine, citrulline, leucine, isoleucine and lysine amino acids in fertile and infertile hydatid cyst fluids were similar. Our findings with respect to fertile and infertile cysts fluids showed that free amino acids concentrations in cyst fluids were significantly, higher in sera from patients with hydatid cyst. Total amount of free amino acids content in fertile and infertile cyst fluids was three to eight times higher from that of human sera with hydatid patients.     

Androgen receptor gene CAG repeats length in fertile and infertile Tunisian men

Several reports implicated a relation between the trinucleotide (CAG) repeat length in the androgen receptor (AR) gene and male infertility. But such result was not reproduced in others. To test this hypothesis, we investigated the number of (CAG) repeats in the AR gene among two groups of infertile (n = 129) and fertile Tunisian men (n = 98), using polymerase chain reaction (PCR) targeting the AR CAG repeat tract, followed by electrophoresis on polyacrylamide gel (6%). For statistical analysis we used Student, Kolmogorov-Smirnov (KS) and chi(2)-tests. Significance was reached when P < 0.05. No statistically significant difference in the mean length of the CAG repeat was found between infertile and control groups (P = 0.47). Moreover, using KS test, we have not found a difference in the distribution of allele frequencies between infertile and controls (D(obs) = 0.046 < D(crit) = 0.180). We also did not found a statistically significant relationship between the size of the CAG repeat and impaired sperm production in Tunisian population. Our results may be attributed to the high probability that infertile males may represent a heterogeneous group with respect to the causes of defective spermatogenesis.     

Comparison of seed removal by ants in vegetation on fertile and infertile soils

Plants which have seeds adapted for dispersal by ants (myrmecochores) are more common in some vegetation types than others. In Australia, myrmecochores are common in sclerophyll shrublands which are associated with infertile soils but not in forests on fertile soils. One hypothesis to explain this difference is that ants are less abundant or less active in seed removal on fertile soils. A test of this hypothesis was carried out on four sites in the Royal National Park, south of Sydney, by measuring the number of myrmecochorous seeds removed by ants in a 24 h period. It was found that the removal rates of seeds were slightly higher on fertile sites although this difference was not significant. The hypothesis that a lower removal rate on fertile soils could account for the lower percentage myrmecochory was therefore rejected. Apart from the gross removal rate of seeds by ants, other differences in ant behaviour might be important. It is possible that a greater proportion of ant species unfertile soils are seed predators or ineffective seed dispersers, and evidence obtained indicates that this is worthy of further research.     

Granulocytes in the endometrium of post-partum women

Endometrial samples of women at various stages of gonadal activity after parturition were examined for the presence and numbers of endometrial granulocytes. Although samples at all the stages contained significant numbers of the granulocytes (i.e. greater than 7/high-power field), the 100% values for late-proliferative and adaptation hyperplasia were significantly higher than the values for the resting (81.8%), early (82.4%) and mid- (87.9%) proliferative and secretory (83.3%) phases. We suggest that this correlates with the suggestion that the granulocytes constitute a receptor system for oestrogens.     

Roles of kisspeptin in IVF/ICSI-treated infertile women and in human granulosa cells

Kisspeptin, a crucial central regulator of reproduction, has been used as a trigger in in vitro fertilization (IVF) treatment. This study aimed to investigate the roles of kisspeptin in IVF treatment in infertile females (n = 30); and in steroidogenesis in human granulosa-like tumor cell line (KGN). In the human study, blood was collected at three time points including (1) the beginning of gonadotropin stimulation (Phase I), (2) around eight days after gonadotropin stimulation (Phase II), and (3) on the day of ovum pick-up (Phase III). Follicular fluid (FF) was collected at Phase III. Serum human chorionic gonadotropin (hCG) was measured 15 days after embryo transfer and fetal heart beats were determined around 42 days of menstrual cycle to classify the subjects into successful and unsuccessful groups. FF kisspeptin levels were higher in successful compared with unsuccessful subjects (P < 0.01). Kisspeptin levels were significantly higher in FF than in serum in successful subjects (P < 0.05) but were comparable in unsuccessful subjects. Serum kisspeptin was comparable among three phases in the successful group but its levels in Phase III were significantly lower compared with Phase I in the unsuccessful group (P < 0.01). Serum kisspeptin in Phase II/III had positive correlations with serum E2 in Phases II and III and the outcomes of IVF/intracytoplasmic sperm injection (ICSI) treatment including serum hCG levels. For the cell experiment (n = 3), kisspeptin treatment in the presence of FSH together with IGF-1 enhanced CYP19A1 (aromatase) mRNA expression compared with control. FSH alone increased aromatase concentrations in the supernatant compared with control and kisspeptin at the dose of 10^-2 mmol/L with FSH enhanced aromatase concentrations in the supernatant compared with FSH alone (P < 0.001 all). In conclusion, kisspeptin enhanced aromatase expression and secretion and was associated with positive outcomes of IVF/ICSI treatment. Further studies regarding supplementation of kisspeptin could reveal its beneficial effects on IVF/ICSI treatment.