Authors: Iris Martínez Rodero and Raquel Pillado González
"Multinucleated blastomeres (MNBs) present in embryos are morphological abnormalities of unclear origin, which have been extensively correlated with chromosomal defects, lower blastocyst formation and implantation rates".
Selection of high-quality embryos is an important factor for the successful outcome of assisted reproduction technologies (ART). Nowadays, criteria for selection are mainly based on morphological features such as embryo fragmentation, cell number, blastomeres uniformity, etc. (2). The parameters studied so far have been demonstrated to be useful indicators of embryo quality. Their evaluation is performed through non-invasive light microscopy-based analyses, usually carried out once a day at specific time points. This approach intends to minimise the events of taking the embryos out of the incubators and exposing them to undesired harmful conditions.
The presence of multinucleated blastomeres (MNBs) can be regarded as one of those indicators, and even though previous studies had already connected it to DNA abnormalities and low pregnancy rates as early as in the 90s (3,4,5) the origin of this phenomenon still remains unclear. Several possible factors seem to influence its genesis, but the specific cause for its occurrence is yet to be determined (6).
Since the introduction of time-lapse imaging and monitoring technology, IVF laboratories have been able to carry out more exhaustive and continuous observations on embryo development, keeping risks at a minimum (7,8). By identifying the precise timing of specific key events of blastomere cell cycle interludes and of the embryo´s overall growth it is possible to assess its quality (7). Furthermore, time-lapse imaging and monitoring systems have facilitated the study of multinucleation (MN) in relation to its incidence in time and in the population, as well as its correlation with other morphological features and clinical variables (6,7,9).
In the IVF context multinucleation is defined as the presence of two or more nuclei in one or more blastomeres. Multiples studies even differentiate between binucleated and multi-/micro-nucleated (three or more nuclei) blastomeres (Fig 1) (1,4,7). Reports on this phenomenon range from 17% to 69% of the total of cultured embryos, depending on the groups used within assays and authors (10). Factors that may influence MNB appearance are numerous and have been repeatedly studied (6,7,9,11).
To date, several explanations have been proposed for MNBs: dysfunction of the mitotic spindle or the occurrence of karyokinesis without cytokinesis (12); DNA breaks or imperfect mitosis (13); nuclear membrane alterations (14) and even other factors not directly responsible for MNBs and yet linked to its presence (1).
FACTORS THAT MAY RELATE TO THE APPEARANCE OF MULTINUCLEATION
Results from various studies have shown a difference in the percentage of multinucleated embryos between groups that had been fertilised by traditional IVF vs ICSI. Van Royen and colleagues showed 32.7% of MN embryo in the IVF group, compared to 34.5% in the ICSI group (6). Accordingly, Walmsley et al. 2003 reported 17,2% vs 18,3% of MN in embryos derived from IVF and ICSI, respectively (11).
Apparently, the type of infertility factor seems not to affect MN rates. Some studies have reported no significant differences in the percentages of MN embryos between cases of female factor-only infertility and male factor-only infertility (32.7% and 34.7%, respectively). In addition, differences were not found between these cases and those with both partners affected by some sort of infertility, either (6).
However, different studies support the relevance of oocyte culture, specially regarding certain processes that occur naturally in in vivo conditions and that are essential for the proper embryo development. Data show that both oocytes subjected to negative conditions when cultured in vivo (like stern hypoxia, for instance) and oocytes cultured in vitro derive in a higher percentage of MN embryos (15,16).
Regarding male infertility, results are more controversial; whereas some studies reflect a higher MN rate for cases in which male factor is especially severe (normally derived for ICSI rather than IVF) (11), other authors show no significant differences (6).
Records exhibit that cycles with an accelerated ovulation induction response present increased MN rates (6,17). Furthermore, several studies have reported that embryos derived from patients from whom ten or more oocytes had been collected presented a significantly higher MN rate than embryos from groups of nine or fewer oocytes (6,8,18). This is in accordance with the fact that patients who need high doses of FSH present higher MN rates (6). Very short cycles and cases where high doses of GnRH are needed trigger the development of high numbers of premature follicles that produce oocytes, which despite being able to reach metaphase II and become fertilized fail to go through proper nuclear cleavage (6,18).
Lastly, even though differences in GnRH doses have been associated to significant differences in the incidence of MN embryos, similar results have not been observed when using different hormones (like rFSH, r-hFSH, purified urinary FSH or urinary gonadotropin, for instance) (6).
Multiple studies have been conducted on patients ranging from 25 to 45 years old. Several authors divided data in 5-year interval groups in order to verify whether patient age correlates with MN. However, the only significant difference was found when comparing women of +40 with younger ones of -35, presenting higher degree of MN in the first group (6,9).
Chromosome polymorphisms consist in heterochromatin variability. These are usually located in the long arms of chromosomes 1, 9 and 16, and the short arms of chromosomes from the groups D and G (13, 15 and 21, 22 and Y) (19). Even though such polymorphisms are generally regarded as normal within karyotypes (20), studies indicate that some of them might be associated with certain clinical problems such as abnormal spermatogenesis (21), infertility (22,23), recurrent miscarriages (24,25) and higher rate of chromosome abnormalities among blastomeres at the cleavage stage (26,27). Sun and collaborators hypothesized that couples with chromosome polymorphisms might experience a higher rate of embryo multinucleation (19). Nevertheless, the authors found no association between chromosome polymorphisms and MN embryo formation in couples undergoing IVF (19).
- Cellular fragmentation
Although MN may appear regardless of the cellular fragmentation levels, several papers support the correlation between these two features (6,28,29). In particular, Van Royen et al. divided the level of fragmentation in three categories: F1 (≤10%), F2 (10-20%) and F3 (20-30%); this study presented evidence for higher MN in F2 and F3 when compared to F1, but similar to each other (6).
- Cleavage rate
When 3-cell and 5‐cell day-2 embryos were observed under the microscope, both types exhibited significantly higher multinucleation (28.2% -50%) than regular ones with the ideal 4-cell cleavage pattern (with only 16.8% MN). Similarly, day-3 embryos with the typical 8‐cell stage showed significantly lower multinucleation (15.5%) than 7‐cell and 9‐cell embryos (6).
As it has been mentioned, ideal 4-cell and 8-cell stages show similar MN percentages. However, application of time-lapse imaging has revealed a significant decrease in MN from the 2-cell to the 4-cell stage (from 43.2% to 15.0%). The analysis of MN in 2-cell embryos indicated that, after cleavage, the majority (52%) of 2-cell MN embryos became mononucleated, whereas only a lower percentage (34%) showed MNBs, and about 14% were of poor quality (with only one or no visible nucleus at all) (9).
This decrease in the MN rate suggests that 2-cell MN embryos are able to self-correct their nuclear abnormalities. But this repair mechanism has been observed in both euploid and aneuploid embryos, therefore it cannot be used as an indicator of chromosomal normality during embryo selection (8,9,30). An extended duration of both 2-cell and 4-cell stages has been proposed as a possible indicator of the occurrence of nuclear self-correction (9).
INCIDENCE OF MULTINUCLEATION IN CLINICAL IVF
As previously exposed, it is through time-lapse imaging that a far higher percentage of multinucleation (25%) has been detected compared to static observations on day 2 at 42 hours post-insemination (hpi) (<5%) (7). These observations have demonstrated that multinucleation is a frequent event that, according to Yilmaz et al., is present in at least one embryo in 41.3% of IVF cycles (31). Data provided by Desai and colleagues reported that approximately 56% of binucleated embryos and 48% of those with three or more nuclei went on to form blastocysts that met the appropriate criteria for vitrification (7). In addition, data from different studies point to binucleation being more frequent than blastomeres with 3 or more nuclei (7,31,32).
At the same time, multinucleation has provided an additional criterion for embryo selection, since it is mainly observed in those of poor quality and is associated with direct and/or reverse cleavage (7). It has been observed that, out of all embryos found showing direct and/or reverse cleavage, at least one fourth were also multinucleated (7).
By using time-lapse, multinucleation has been repeatedly observed to be a reversible event in a high proportion of embryos (7,32). Multinucleation reversibility has been reported to be as high as 73.4% (32); this has been calculated as the proportion of embryos in which multinucleation was detected at 2-cell stage, but not visible at 4-cell stage (likely due to self-correction mechanisms, as above-mentioned). In fact, Aguilar and collaborators reported 127 multinucleated embryos at 4-cell stage out of the 479 ones initially observed to present this feature at 2-cell stage. De novo multinucleation at the 4-cell stage in turn was observed in 36 embryos (32).
IMPACT OF MULTINUCLEATION ON IVF OUTCOMES
Multinucleation has traditionally been related to both low blastocyst formation (33) and implantation rates (5,6,17,28,34), and linked to the likely presence of chromosome abnormalities, which consequently results in embryo arrest (35). Nevertheless and despite all the existing evidences, there is still much controversy regarding multinucleation; reports have been published revealing cases in which fully binucleated 4-cell stage embryos had eventually developed into euploid blastocysts and genetically normal children (31,34).
Although some preimplantation genetic testing (PGT) studies have shown that not all multinucleated embryos are chromosomally abnormal (31,32,36) multinucleation is predominantly associated to chromosomal defects and poor implantation prognosis (3,31,37). Kligman and colleagues published that 74.5% of multinucleated embryos were chromosomally abnormal, compared to 32.3% of non-multinucleated embryos (3). Years later, Ambroggio et al. revealed an increased incidence of aneuploidy of MN 4-cell stage embryos when compared to single-nucleated embryos (85% vs 78%), suggesting that multinucleated embryos should not be recommended for transfer in IVF cycles (37). These results were confirmed when, from 395 MN embryos tested for PGT, Yilmaz et al. reported that 82.5% of MN blastomeres exhibited two nuclei, whereas the remaining blastomeres presented a single or three or more nuclei (31).
Noteworthy, binucleated patterns of multinucleation may be less detrimental, since a high percentage of embryos with such feature are euploid, compared to embryos exhibiting three or more nuclei in a single blastomere (38).
Embryo morphokinetics was studied and related to the multinucleation status in a study conducted by Meseguer’s team (32). In the study, 53.4% of a total 1676 embryos included were MN. Based upon the reported data, differences in morphokinetics between multinucleated and non-multinucleated embryos at both 2-cell and 4-cell stages comprise cleavage events involving the completion of the first mitosis and the length of the S-phase. These differences affected the following parameters: t2, t3, second cell cycle (cc2=t3-t2), t4, t6, t7 and t8. These results allowed to conclude that, if multinucleation remains at 4-cell stage, it takes longer for the embryos to complete the next cell cycle (cc3=t5-t3). Should this be true, the restoration system would not be efficient if MNBs were still observed after the 2-cell stage (32).
The origin of the multinucleation phenotype has been suggested to be multiple: disruption of intracellular restructuring, remodelling or imprinting in the developing oocyte, or even alterations in DNA replication, cytokinesis or compaction during the first cell cycle (16). If multinucleation appears as a result of defects in cell function, differences in morphokinetics between MN and non-MN embryos during these early stages may be expected (32).
Opinions on the impact of the multinucleation phenotype on implantation rates diverge from each other:
On one hand, cell stage for MN appearance has been proposed to exert the highest effect on the implantation rate. Authors supporting this claim are divided into two positions: those who affirm that the presence of MN at the 2-cell stage is actually insignificant in terms of differences on implantation rates, but it is at 4-cell stage when it does have a measurable negative effect (32); and the authors who argue implantation rates to be significantly reduced when MN is already observed at the 2-cell stage (8).
On the other hand, the school of Meriano and coauthors affirm that binucleation is less harmful than any other type of multinucleation (16). However, Aguilar and colleagues explained that their differences with Meriano were found on the frequency of image acquisition and the systems used to measure multinucleation (32); whereas the former acquired one picture in seven different focal planes every 20 minutes, the latter recorded images every 2.5 minutes (32).
In any case, it has been demonstrated that patterns of multinucleation at 4-cell stage are correlated with low implantation rates, while any of the other cases has been reported to decrease the chances to achieve pregnancy (16,32).
Multinucleation is a common and reversible event observed in human IVF embryos, and it is specially frequent as binucleation at two-cell stage. It is associated with chromosomal defects and altered morphokinetic parameters, eventhough binucleation patterns seem to be the less severe. Regarding multinucleation impact on implantation rate, results are controverted. It seems that implantation rates are not affected when multinucleation appears as two nuclei in two-cell stage.
Although the presence of multinucleated blastomeres in human embryos has been associated with the above-mentioned undesired characteristics in IVF embryos, the reasons explaining its appearance and occurrence in time and its relationship with patient specifications have not been deeply studied until time-lapse systems became available.
Even though different causes have been suspected to lie behind MNB development, none of them have been proved actually represent the main responsible. Nevertheless, a growing number of studies provide data untangling the relationship between MN and assisted reproduction fertilization methods (IVF and ICSI), stimulation cycles, infertility factors, culture conditions and other embryonic morphological characteristics. Even though sometimes results from different studies may seem contradictory, this might be accredited to the differences in sample sizes.
All the above said, it seems reasonable to highlight the need for further research on this issue. It would be highly helpful to unveil the actual triggers of multinucleation, to develop optimal ART practices that avoid increasing MN incidence, and to unravel any other correlation with adverse embryo features during development. Deeper knowledge would help improving embryo assessment methods and, consequently, increase the rates of successful ART outcomes.
Authors: María Caballero Sastre & Lidón Carretero Vilarroig
“Due to the current lifestyle and habits, high production of ROS and oxidative stress are the most common mechanisms responsible for DNA damage”
Fig 1. Example of two techniques to detect DNA fragmentation in a sperm sample. A) TUNEL assay: sperm in green indicate DNA fragmentation. B) Sperm Chromatin Dispersion test: the halo around both central sperm nuclei indicate non-fragmented DNA, while the absence of halo in the sperm in the upper right hand corner indicates DNA fragmentation (1).
Male factor plays a role in 33% of couples experiencing infertility problems. Although it is less understood than its female counterpart, a great effort has been made in order to understand its basis. The World Health Organization (WHO) periodically updates its Laboratory Manual for the Examination and Processing of Human Semen, as well as the reference parameters (2). However, since results from conventional seminograms vary depending on temporal factors such as stress, period of abstinence or sample collection, it is difficult to diagnose male infertility using conventional semen analyses (3). This makes also necessary to understand the process behind male gamete formation (spermatogenesis) in order to achieve a better comprehension of the molecular basis of male infertility. Within spermatogenesis, meiotic problems may occur resulting in sperm cells carrying chromosome alterations, and during spermiogenesis sperm DNA becomes highly compacted by replacing histones by protamines. Humans express two protamines (P1 and P2) in a 1:1 ratio; an imbalance of this ratio results in male infertility (2). Measurement of sperm DNA integrity as a marker of sperm quality has recently gained interest for embryologists.
The study of sperm DNA fragmentation is highly indicated in cases of implantation failure or repeated miscarriages. There is a wide range of tests currently used aimed to analyze sperm DNA integrity (Fig. 1). These techniques make it possible to determine whether there is a high rate of single-strand or double-strand breakpoints (SSB or DSB) in the DNA; in such cases the fertilizing and replicative ability of the sperm cell may be jeopardised.
CONVENTIONAL SEMEN ANALYSIS
Conventional semen analyses are the first step to measure semen parameters. Using fresh ejaculated semen, andrologists evaluate sperm motility, vitality and morphology, as well as its concentration along with other seminal components, and total volume of the ejaculate. WHO gives the following results as a reference for normality (2):
- Volume of at least 1.5 mL. The total amount of fresh ejaculated semen may vary depending on the abstinence period. Thus, a period of abstinence of 3-7 days before the analysis is recommended.
- Concentration of at least 15 million sperm cells per millilitre (15 mil/mL). Samples with less than 15 mil/mL are classified as oligozoospermic, whereas samples with no sperm cells are classified as azoospermic. Samples with a concentration below 100,000 sperm cells per millilitre are considered cryptozoospermic.
- Motility and vitality. Even though sperm cells acquire their characteristic forward and rapid movement during capacitation, the way they move within a fresh sample is a quality indicator. Proper movement consists in progressive and linear displacement, while those sperm cells with circular or even missing movement are considered less capable of fertilising. Such samples are classified as asthenozoospermic. Following these WHO’s reference guidelines, sperm samples are considered normal if linear progressive movement is found in at least 32% of the cells.
- Correct morphology in at least 4% of the sperm cells. This feature refers to (4): (i) sperm cells with one head and one tail; (ii) sperm cells with a round/oval-shaped head; (iii) lack of cytoplasmic droplet. Samples with less than 4% of morphologically normal sperm cells are classified as teratozoospermic.
Fluorescent In Situ Hybridization (FISH) in spermatozoa is a complementary technique to conventional semen analyses. This test is indicated in cases of altered seminal values, miscarriages, implantation failure, non-oocyte-driven fertilization failure cause or anomalous embryonic development (5). The objective of this procedure is to verify the chromosome number in human sperm cells. As haploid cells, these must contain 23 chromosomes each. FISH assays use fluorescent probes to recognize specific regions of certain chromosomes in every test. The 5-probe FISH test analyzes chromosomes X, Y, 13, 18 and 21, while the 9-probe FISH test also includes chromosomes 15, 16, 17 and 22. Altered results can suppose a high percentage of diploid, dysomic or hyperploidic sperm cells, which may compromise fertilization and embryo development (Fig. 2). In such cases, a cycle of in vitro fertilization is indicated, followed by preimplantation genetic test (IVF-PGT) by aneuploidy screening, in order to transfer only euploid embryos. Due to all different variables that may influence the result of a conventional semen analysis (6-8) and FISH assay (7), DNA fragmentation analysis has gained attention over the last years.
Fig 2. FISH analysis of testicular human sperm. Sperm nuclei hybridized by chromosome 18 (green) and Y (red) probes. (a) Diploid sperm nucleus exhibiting signals for chromosomes 18,18 and YY.. (b) Nucleus with an absent signal for chromosome 18 (left) and nucleus displaying gonosomal dysomy for the Y chromosome (5).
SPERM DNA FRAGMENTATION
According to Sakkas et al., DNA damage in spermatozoa can be induced by six main mechanisms:  apoptosis during spermatogenesis,  DNA strand breaks produced during remodeling of sperm chromatin typical of spermiogenesis,  oxygen radicals during sperm transport through the seminiferous tubules and the epididymis,  endogenous caspases and endonucleases,  radiotherapy and chemotherapy and  environmental toxicants (8). Some of these mechanisms seem to be involved in specific cases of DNA damage due to a variety of factors such as varicocele (10), bacterial infections (11), age (15) or abstinence (13).
The main types of DNA damage include are mismatch, base adducts, crosslink, base modification, pyrimidine dimerization, abasic sites, thymidine glycol and oxidation at guanosine sites. When evaluating DNA fragmentation, two kind of breaks are considered: single strand breaks (SSBs) and double strand breaks (DSBs) (Fig. 3). Although both type of DNA lesions are present in sperm of both fertile and infertile men, the latter have been observed to present higher levels of DNA fragmentation (14).
Each kind of DNA break has a different origin. For instance, nucleases usually generate both types, whereas chromatin remodelling may cause DSBs produced by topoisomerase II and reactive oxygen species (ROS), and other radical molecules generate mainly SSBs, DNA adducts and abasic sites (16, 17).
Whereas SSBs are slightly detrimental and easy to repair, DSBs represent serious harmful lesions that can lead to chromosome aberrations (16, 18). Fortunately, cells present different pathways to face and repair DNA damage. The main mechanisms present in germline cells are direct reversal repair, nucleotide excision repair, base excision repair, mismatch repair, post replication repair and DNA double strand break repair (19). These DNA repair processes can take place during spermiogenesis (20), in the fertilized oocyte (21) or during early embryonic development (22). The capacity to repair DNA damage depends on the type of DNA damage present in the sperm; should it fail, the embryo may not develop or miscarriage may eventually occur (14).
DIFFERENT TECHNIQUES TO ASSES DNA DAMAGE
- Sperm Chromatin Structure Assay (SCSA):
This technique, first proposed in 1980 by Evenson and coauthors (23), uses flow cytometry to measure sperm DNA integrity. It tests the vulnerability of DNA to acid-induced denaturation by exposure to acridine orange media (AO) (24). AO binds to both double-stranded and single-stranded DNA, but fluoresces in different colour, being red or green, respectively. The DNA fragmentation index (DFI), which represents the percentage of spermatozoa with red fluorescence, is analyzed and represented on a histogram. Green fluorescence in turn shows the percentage of mature undamaged sperm (24).
- TUNEL test:
This test detects SSBs and DSBs by labelling 3’-free ends of DNA fragments (25) with fluorescent dUTP nucleotides in a reaction catalyzed by the terminal deoxynucleotidyl transferase. The evaluation process can be performed using light and fluorescence microscope or flow cytometry (26, 27).
- Single cell gel electrophoresis assay (COMET):
It enables the detection of both SSB and DSBs. It consists in the lysis of sperm cells with detergents and high salt-density components. After lysis, the sample is introduced into a gel and subjected to an electrophoretic field resulting in the migration of both DNA and proteins. DNA fragments move faster than intact DNA; as a consequence, fragmented DNA from sperm cells migrate through the gel leaving a comet-like trace, whereas DNA without breaks is seen as a dot. Samples can be analyzed by fluorescence microscopy. The ratio between comet-like trace and dots defines the degree of fragmentation of the sample.
- Sperm cromatin dispersion (SCD) test:
SCD test allows for the differentiation between sperm with fragmented DNA and intact DNA. This technique denatures DNA that contains breaks using an acid solution. Subsequently, spermatozoa are treated with a lysis buffer to remove membranes and proteins and, in so doing, a peripheral halo of DNA loops is formed around the sperm. The observation of a large halo means the sperm nucleus presents low levels of DNA fragmentation. On the contrary, a small or absent halo is indicative of highly fragmented DNA (Fig. 5). Results can be evaluated using either fluorescence or bright-field microscopy (29).
- γH2AX test:
Not only is γH2AX labelling assay used to assess DNA fragmentation in sperm cells, but it has been also used in order to evaluate DNA damage in tumour cells during radio- or chemotherapy, thus providing a potential prediction marker (30). Once DSBs occur, histone H2AX becomes phosphorylated at Ser139 (γH2AX), playing a role in signalling the break sites to be repaired (31). The presence of γH2AX in this context is commonly assessed by flow cytometry, being the percentage of γH2AX higher among infertile men.
LIFESTYLE AND POTENTIAL HARMFUL FACTORS FOR DNA
Among the six mechanisms above mentioned that can produce DNA damage, oxidative stress and ROS are the most common (17), which is mainly due to current lifestyle and habits.
The term ROS includes highly oxidative radicals like OH, non-radical species like O2- and H2O2, as well as reactive nitrogen species. Spermatozoa produce ROS themselves, since these are required for several functions such as sperm motility, capacitation, acrosomal reaction, zona pellucida binding (32) and chromatin compaction (17). Because ROS concentration can be harmful, it is important to guarantee the action of antioxidant mechanisms in order to maintain cell homeostasis (33). Therefore, decompensation in ROS final concentration, due to either excess production or to failure in antioxidant mechanisms, would eventually lead to oxidative stress and, as a consequence, to DNA damage (34-36).
Alcohol and smoking
Several studies have demonstrated both alcohol and tobacco to be responsible for the significant increase in ROS at sperm production (37,38). Although some studies have shown reduced efficiency of antioxidant mechanisms in smokers and people with a high intake of alcohol (39), contradictory results have been reported as well (40). These studies showed an unbalance in favour of ROS despite the increased antioxidant activity, suggesting the antioxidant mechanisms were somehow unable to protect from oxidative damage (40).
The various chemical products found in tobacco such as cadmium, lead (17) and nicotine, and alcohol itself most likely seem to result harmful for semen parameters, since they have been reported to cause DNA damage (40).
A recent study by Jin-Chun Lu and collaborators has shown data from a correlation between sperm DNA fragmentation index and obesity (41). Samples from 1010 subfertile men from 18 to 50 years old were analysed using SCSA. Although the authors found no correlation between obesity and sperm index fragmentation, they did find a positive correlation between sperm fragmentation and seminal plasma triglyceride and total cholesterol levels.A separate study on these matters concluded there is no correlation between body mass index and DNA integrity. The authors stated obesity might affect lipid metabolism and reproductive hormones, leading to a decreased sperm quality (42).
Varicocele is an enlargement of the veins located within the scrotum. It is postulated that varicocele causes a rise in temperature that leads to higher DNA fragmentation ratio and ROS production (43). About 15% of adult men suffer from this condition (44), but authors suggest that varicocelectomy can improve semen parameters after 3 months (8), which is the expected period for de novo sperm production in humans.
Exposure to environmental contaminants has been demonstrated to be the main cause for male infertility, even over genetic defects (45). These substances, known as endocrine disruptors (ED) (Table 1), can negatively affect the endocrine system by mimicking natural hormones in the organism like estrogens and androgens, and so they can block interactions between natural hormones and their receptors (46). Exposure of an individual to ED may cause important specific epigenetic modifications in sperm (that are eventually passed down to several generations), as well as severe fertility disorders (45).
Electromagnetic radiation: mobile phones radiation and Wi-Fi
It has been suggested that the electromagnetic radiation (EMR) emitted by some electrical devices such as mobile phones or Wi-Fi suppresses the natural antioxidant mechanisms and increase the oxidative stress, which consequently increases infertility (50). Although studies in both animals and humans have demonstrated the detrimental effect of EMR, there is still controversy. Some studies show that the prolonged use of mobile phones decreases sperm motility (51), concentration (52), viability and morphology (53), while others have found no effects on sperm concentration (reviewed in 54). Moreover, results have been reported on the increase in DNA fragmentation in human sperm associated to exposure to Wi-Fi networks (51).
It is important to maintain testicular temperature 2-4ºC lower than the body temperature for spermatogenesis to progress normally. This justifies the existence of several thermoregulatory mechanisms found in the organism, such as the characteristics of the scrotal sac, the musculature and the vasculature (18).
Fertility is directly affected by a rise in temperature due to the increased DNA fragmentation index (55) and sperm cell apoptosis (18). Several factors can cause hyperthermia, namely: the use of tight clothes, saunas, laptops and mobile devices closely located to the testes (18), or even an episode of high fever or varicocele (55). Moreover, the severity of damage varies with duration, frequency and intensity of heat (18).
Spermatozoa present low levels of antioxidants and DNA repair enzymes. Therefore, the presence of these factors in the seminal plasma is critical. As it has been demonstrated, not only subfertile men have been observed to present lower levels of antioxidants than fertile men (35, 56), but they have been also found to exhibit higher levels of ROS (36).
Antioxidants present in semen include vitamins C and E, zinc, selenium, carnitine and carotenoids (57). These components can be found in the food (table 2), meaning that controlling the dietary antioxidant intake, oxidative stress can be minimised or avoided and, as a consequence, DNA damage, too (17). Besides the above-mentioned antioxidants, polyunsaturated fatty acids are also an important source of antioxidants. They are classified into omega-3, omega-6 and omega-9 fatty acids and, with the exception of omega-9, they need to be incorporated into the diet through the intake of vegetable and fish oils.
The first step to check male infertility in a couple undergoing infertility issues is the completion of a conventional semen analysis established by the WHO. This kind of test evaluates several parameters such as motility, vitality, morphology, concentration and total volume of the ejaculate.
Because of the evidenced involvement of sperm DNA damage in infertility, its study has been gaining significance and, consequently, it is usually indicated in certain cases such as implantation failure or recurrent miscarriages. Techniques like SCSA, SCD, TUNEL, COMET and γH2AX test are normally used to analyze sperm DNA integrity. These approaches are helpful to discriminate between SSBs and DSBs or other types of DNA damage.
Due to the current lifestyle and habits, high production of ROS and oxidative stress are the most common mechanisms responsible for DNA damage. Among such habits and lifestyle it is worth mentioning alcohol and smoking, environmental contaminants, electromagnetic radiations and heat sources.
An efficient method to prevent DNA fragmentation is to resort to the intake of specific products as an antioxidant dietary therapy. Thus, a proper diet rich in such components may be able to reduce the incidence of sperm DNA fragmentation, and, consequently, to also improve male fertility.
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Authors: Iris Martínez Rodero and Raquel Pillado González
"Selection of the best spermatozoon for ICSI is critical and it is directly linked to fertilization rates, optimal embryo development and implantation rates"
It is estimated that approximately 10% of couples seek help to overcome infertility problems. Data from the American Society of Reproductive Medicine points to male factor as the reason behind one third of couples’ infertility problems (1). The remaining two thirds seem to be equally distributed between female-related problems and a mixture of unexplained causes and combinations of male and female factors, respectively (1).
As continuation of our previous post on sperm selection, we revisit the topic from the perspective of the numerous techniques currently available to be specifically used prior intracytoplasmic sperm injection (ICSI). This procedure is the advised treatment for most cases of male factor infertility; in fact, ICSI accounts for 70-80% of cycles performed globally (2). ICSI candidates often present low sperm count and/or increased probability of genetic abnormalities, compromised DNA and morphological defects of spermatozoa. Therefore, selection of the best spermatozoon for ICSI is critical and it is directly linked to fertilization rates, optimal embryo development and implantation rates (3). Traditional sperm selection procedures (swim-up and density gradient centrifugation (DGC)) are based just on motility rather than molecular features. Consequently, in order to choose the best spermatozoa, researchers have developed different non-invasive methods to process and select sperm for ICSI (Table 1) (2).
SPERM SELECTION TECHNIQUES
Sperm selection prior to fertilization is a crucial step for IVF success, especially in cases of low number of available oocytes to inject (4). Several techniques are currently utilised for this purpose, each of them relying on different features in order to select the optimal sperm.
BINDING ASSAY TECHNIQUES
Physiological intracytoplasmic injection (PICSI)
This technique is based on the fact that hyaluronic acid (HA) plays an important role in selecting functionally competent sperm during in vivo fertilization (5). Not only HA is the main component of the cumulus matrix that surrounds the human oocyte, but also a natural selector of developmentally mature sperm. Furthermore, experimental data have shown that it can be used with a similar effect in vitro. HA-binding spermatozoa in vitro exhibit complete plasma membrane remodelling, decreased levels of cytoplasmic inclusions and residual histones, nuclear maturation and reduced risk of chromosome imbalance (i.e. chromosomal aneuploidy) and chromatin diseases (4). Currently, there are two options available to perform this technique. First, plastic culture dishes with HA hydrogel microdots attached to the bottom. After using this method the desired spermatozoa remain bound to the microdots by the head, while flagella beat in place (4). Secondly, the use of an alternate HA-containing viscous medium, method known as “Sperm Slow” because the spermatozoa appear “slowed down” (4).
Since PICSI is considered to better assess the physiological potential in order to select sperm for ICSI, some authors claim it should replace the use of conventional ICSI and potentially toxic polyvinylpyrrolidone (PVP) (used to slow down sperm movement). Nevertheless, significant improvement of IVF outcomes using HA-based sperm selection is still to be confirmed in larger studies (6, 7), reason why the use of PICSI is currently limited to punctual cases in which significant improvement of clinical outcomes has been proved (8). Such cases include total fertilization failure by ICSI, high levels of DNA fragmentation, impaired embryo development, failure to implant in the uterus and recurrent miscarriages (9, 10).
Fertilization failure after ICSI can sometimes be explained by problems in sperm nucleus decondensation and chromatin abnormalities related to DNA fragmentation (11). Sperm with such DNA damage and/or chromosome unbalance are avoided by PICSI/HA binding negative selection (3).
Although it is a controversial issue, several authors have found a significant improvement of embryo quality and implantation ability when performing PICSI vs conventional ICSI, as well as a reduced number of miscarriages (3, 11, 12, 13, 14). The decreased incidence of miscarriages seems to be linked to the incapacity of sperm with genetic defects to form a normal pro-nucleus after conventional ICSI, thus generating aneuploid embryos with high levels of fragmentation during cleavage. Such aberrant embryos normally end up in pregnancy loss (14). Since PICSI allows for selection of sperm with properly compacted chromatin, embryos derived from these spermatozoa are less likely to present defects in quality (15). This translates into a higher implantation potential and a lower incidence of miscarriage, which eventually improves the reproductive outcome (12, 13).
Magnetic activation cell sorting (MACS)
In 2008 Said and colleagues proposed a protocol combining a new method, denominated magnetic activation cell sorting (MACS), and density gradient column (DGC) in order to obtain higher-quality sperm samples (2). MACS consists in mixing up the semen sample with annexine V (A5) (known to have high affinity with phosphatidylserine (PS)) coupled to magnetic microspheres. The sample is then exposed to a magnetic field in an affinity column (16). A5-bound sperm have been linked to PS externalisation, a well-known sign of apoptosis due to the loss of membrane integrity. MACS should be performed prior to DGC because PS externalisation naturally occurs during sperm capacitation by DGC, and so MACS may inadvertently discard good-quality sperm (17). During the last decade, several studies have corroborated the efficient use of this protocol in order to obtain sperm samples with a low DNA fragmentation index and higher fertilization potential (2, 16).
Because MACS reduces the percentage of apoptotic sperm selected from the sample, this technique is mostly indicated in cases of high rates of DNA damage or predisposition to suffer from it (18). Such cases include: patients with altered sperm parameters, cryopreserved spermatozoa from cancer patients (19), previous fertilization failure, recurrent implantation failure and recurrent miscarriage (20, 21).
Several clinics have implemented MACS in their daily routine, claiming selection of sperm through MACS to improve their ICSI outcome (22, 23, 24). However, such statement remains controversial, since other authors have reported no actual beneficial effect after using this technique (16, 25).
This method mimics natural sperm selection using the zona pellucida (ZP) from an immature sibling oocyte (26). The sperm sample is processed by DGC and then incubated along with the ZP for two hours. ZP-bound sperm are then eligible for ICSI. Although various studies have linked the ZP binding assay prior to ICSI to improved embryo quality and implantation rate, it is still not feasible to employ this method on a routine basis due to the extra amount of work and time required. Therefore, it is currently recommended to remain restricted to patients with poor outcomes in previous ICSI cycles, or to those who exhibit sperm with damaged DNA or abnormal morphologies (2).
MICROSCOPY BASED TECHNIQUES
Intracytoplasmic morphologically selected injection (IMSI)
Studies have shown that sperm classified as morphologically “normal” at conventional optical resolution and magnification (x200-x400) may carry ultrastructural defects. Many of these abnormalities have been linked to hidden chromosomal defects, high levels of DNA fragmentation, abnormal centriolar function, etc. (1, 2). These aberrations may impede fertilization, lead to post-fertilization arrest or even disrupt embryo development (2). IMSI is an approach developed from a method of sperm evaluation, first described in 2002 by Bartoov and colleagues and denominated “motile sperm organelle morphology examination” (MSOME) (27). In order to apply this evaluation technique, observations need to be performed at x6000-6600 magnification (1). Performance of sperm selection along with MSOME requires the use of specific equipment. This should be an inverted light microscope with high-power optics intended for differential interference contrast and enhanced by digital imaging, which allows the embryologist to assess sperm morphology in real time. Sperm selection is carried out in a dish different from the one used for ICSI; it is a glass-bottomed dish that allows for the best optimal quality. Semen samples for IMSI are subjected to discontinuous DGC beforehand, but the exact procedure may vary depending on the quality of the sample (2).
Sperm selection based on IMSI—MSOME findings helps to discard spermatozoa with mitochondrial dysfunction or DNA damage (4). In this regard, IMSI-MSOME has been proved useful for oligo-asthenoteratozoospermic patients (28) and for couples with recurrent implantation failures, reducing miscarriage rates by 50% (29). Nevertheless, simpler techniques are available for selecting sperm with low DNA fragmentation such as PICSI and MACS, so IMSI-MSOME is not yet extendedly used (4). In fact, this technique did not have the expected impact due to its downsides: high cost and long time for selecting sperm for ICSI. Depending on semen quality, selection may take around 60-120 minutes, which could negatively affect cells given that sperm nuclei may vacuolize after 2-hour exposure to warm media (30).
Polarized light microscope
The different anisotropic properties of spermatozoa through its protoplasmic texture provoke polarized light to be refracted at different speeds. These differences between refractions are known as retardance or birefringence (31). Viable human spermatozoa are naturally birefringent, while this characteristic is absent in pathological, dead or necrotic ones due to changes in the molecular structures of the cell and/or organelles (nucleoprotein filaments, axoneme, mitochondria, etc.) (32, 33). Examined under polarized microscope (PM), a normal spermatozoon will exhibit a non-luminous acrosome and luminous and normal-sized compact nucleus and flagellum (Giulia Collodel 2010). Multiple studies support the correlation between sperm head birefringence and DNA fragmentation, fertilization rates and higher embryo quality (2). The increase of a sperm head retardance is positively correlated with DNA damage, the optimal value for sperm head retardance oscillates between 0.56 nm and 0.91 nm. (32). Thanks to improvements in polarized light microscopy it is now possible to evaluate viability, motility, morphology and concentration of a sperm sample, all in a single step and with no need of exposure to potentially harmful dyes or environmental conditions (33).
MEMBRANE POTENTIAL BASED TECHNIQUES
Zeta potential method
Zeta potential refers to the negative electric potential observed between the outer medium and the sperm membrane surface. The sperm selection technique based on this potential was developed following the observation that sperm with damaged DNA exhibited a lower Zeta potential (34). This method consists in the use of positively charged test tubes to which morphologically normal spermatozoa containing intact DNA can bind (2). This promising, easy and time-efficient new method presents the major drawback of a low recovery rate (8.8%), which is especially troubling in cases of oligozoospermic patients, who represent a high percentage of ICSI candidates (34).
During sperm maturation in the epididymis, capacitation and acrosome reaction, the sperm membrane undergoes specific modifications (35, 36), among which the addition of sialic acid residues is noteworthy. High concentrations of this residue reflect normal spermatogenesis and sperm maturation (37), as well as higher negative charge compared to immature or abnormal sperm (38). Taking advantage of such trait, negatively-charged spermatozoa are selected as they migrate towards the anode during electrophoretic sperm separation (39). Similarly to the Zeta-potential method, micro-electrophoresis enables the isolation of sperm with no DNA damage, since they present higher negative net charge (40). Although micro-electrophoresis remains an experimental method, the percentage of negatively charged sperm is directly associated with fertilization rate and blastocyst development and inversely associated with embryo arrest (41).
ABSOLUTE IMMOTILE SPERM SELECTION TECHNIQUES:
There are some cases of severe male factor infertility, such as total necrozoospermia, in which absolute immotile sperm are prevalent. In such cases, testicular sperm extraction (TESE) seems to be the only actual solution; however, obtaining motile and/or viable sperm for ICSI may result an impossible goal (42). Difficulties to select viable sperm can also occur in cases of severe cryptozoospermia and asthenozoospermia. Therefore, several techniques have been developed in order to differentiate immotile and non-viable sperm (2, 43, 44, 45).
Hypo-osmotic swelling test (HOST)
Studies have shown that in cases of severe male infertility normal sperm morphology will still likely have a high DNA fragmentation index. HOST is a method to estimate chromatin integrity (2), based on the degree of swelling of the cytoplasm and curling of the tail in live sperm when exposed to hypo-osmotic conditions. Different swelling patterns correspond to different degrees of chromatin integrity; by identifying these patterns, live spermatozoa with normal membrane function and low DNA fragmentation can be selected (2, 43).
Laser assisted immotile sperm selection (LAISS)
LAISS helps to differentiate between viable and dead spermatozoa by evaluating curling of the flagellum, as detected in live sperm when hit by the laser. The use of LAISS has reported results comparable to HOST; however, the former is much quicker than the latter, and its effect can be immediately observed without the need of continued evaluation, contrary to what is required for HOST (43).
Mechanical touch technique or the sperm tail flexibility test (STFT)
Although HOST is the most used test to differentiate live immotile spermatozoa from dead ones, evidence suggests that it may not be totally reliable (46, 47). Furthermore, solutions used for hypo-osmotic shock can be harmful for spermatozoa. In 2003, Soares and coauthors published their results using a simple and low-cost technique for selecting viable immotile sperm. They observed acceptable fertilization rates after injecting spermatozoa with flexible tail from total immotile sperm samples. Based on these results, the authors proposed STFT as a means to discriminate live from dead sperm; STFT consists in touching the flagellum with the ICSI pipette to check flexibility. Spermatozoa with flexible (non-stiff) flagellum may be considered alive and therefore eligible for ICSI (44).
Methylxanthines: Pentoxifylline (PTX)
Use of Pentoxifylline (PTX) enhances sperm motility by inhibiting the breakdown of cAMP which is essential for sperm motility (48). PTX is a 3’5’-nucleotide phosphodiesterase inhibitor that might result highly toxic for the oocyte or the embryo (2). Nevertheless, due to the reported increased fertilisation rates for immotile sperm after exposure to PTX (48), this method is still recommended for thawed testicular sperm samples, though only in cases of 100% immotile sperm, and always for short periods of time (2, 48).
In 2014 Neri and colleagues proposed the use of ATP/MgSO4 solution for immotile spermatozoa that did not respond to motility enhancers (49). It is especially recommended for surgically retrieved or thawed sperm samples (49). The exposure to ATP/MgSO4 stimulate sperm kinetic machinery provoking viable, but immotile spermatozoa flagella to twitch (2). The same team reported that 64.6% of spermatozoa from an only immotile sperm sample exhibited flagellar movement after exposure to ATP/MgSO4 (49).
ICSI is a highly efficient technique, which reaches up to 85-90% fertilisation rates. Two main causes can explain fertilisation failure by this technique: the lack of appropriate sperm for injection or failed oocyte activation. Having covered the latter in a previous publication, the present post aims to review the current available techniques for the improved sperm selection for ICSI. Among these, IMSI, PICSI and MACS are widely studied, and their efficiency extensively accepted. Several novel approaches have been also developed. HOST, polarised microscopy and micro-electrophoresis are suggested to improve fertilisation rates, although multicenter randomised control trials will be needed before their general implementation to IVF routine. On the other hand, ZP binding assay and Zeta potential method are still experimental techniques that are yet to demonstrate their clinical advantages and feasibility. Finally, the best method for selecting live spermatozoa from totally immotile sperm samples remains a controversial issue. Whereas HOST seems to be the most extended one, simpler and easier techniques have appeared in the last decade that may also prove highly useful for an effective good-quality sperm selection.
Authors: Belén Gómez Giménez and Edel Rocher
"The assessment of embryo development in vitro suggests that collapsing of the blastocoel might be indeed related to a certain degree of success"
One of the main factors related to embryo selection for transfer is the extension of embryo culture up to blastocyst stage. This approach has been demonstrated to improve clinical outcomes after in vitro fertilization IVF (2). Consequently, in the last couple decades a specific blastocyst grading system has been applied to assess embryo morphology during blastocyst stage (appearance of the inner cell mass (ICM) and the trophectoderm (TE)) [read our post on blastocyst assessment]. But there are other aspects about blastocyst development to focus on, such as the study of collapse and re-expansion in vitro and how it could affect implantation and pregnancy rates.
BLASTOCYST COLLAPSE UNDER NORMAL CIRCUMSTANCES
The term "collapse" refers to the contractions observed in the blastocyst that respond to a series of physical phenomena related to its proper development. Once morula stage has been reached, the forming blastomeres begin to pump ions through the Na+/K+ machinery (3). This entails an implicit osmotic response that results in the accumulation of water in the cavity thus formed, the blastocoel. The progressive accumulation of water causes the blastocyst to grow in size from early stages, consequently increasing the hydrostatic pressure between the TE and the zona pellucida (ZP) until the blastocyst hatches (4, 5).
The efflux of the blastocoel fluid through loose cell bindings in the already formed TE causes the aforementioned contractions or "collapse". This phenomenon was originally called "blastocyst breathing", due to the sequence of collapse and re-expansion events observed in the rupture of the ZP during blastocyst hatching (6). However, the mechanisms of blastocoel collapse and TE recovery after the rupture in vitro still remain unclear (7).
INFLUENCE OF THE NATURAL COLLAPSE OF BLASTOCYSTS ON REPRODUCTIVE OUTCOMES
When considering the effect of blastocyst collapse on future outcomes, certain aspects must be taken into account:
1. Number of collapsing episodes
Nowadays, there are still few studies on the frequency of blastocyst collapse. Marcos and coauthors (2015) focused on this aspect for the first time and reported almost a 20% of single collapse episodes in a total of 715 blastocysts, whereas only about 1.5% exhibited multiple collapse events (8). Independent authors have further studied this topic; even though they all have shown significant variations in the percentages of both single and multiple collapse events during blastocyst stage (8, 9), results do suggest a correlation between the number of collapsing episodes and the future outcome for the blastocyst (7, 8, 9).
2. Effect of collapse on blastocyst hatching
Research on certain mammalian species had shown that blastocysts with smaller contractions (collapse/re-expansion events) were more likely to reach the hatching stage than those with large strong contractions (10, 11, 12). Results in patients showed that differences in hatching rates were not statistically significant between blastocysts with and without collapse (28.7% vs 31%, respectively) (7). However, hatched embryos with previous collapse episodes exhibited lower implantation rates than those with no collapse (35.1% vs 48.5%, respectively) (8). Taken together, these results indicate the process of collapse and re-expansion may not have a direct influence on blastocyst development up to the hatching stage, but they seem to negatively affect the success rate of such blastocysts after implantation in the uterus.
3. Relationship between standard morphological evaluations and blastocyst collapse
Morphological assessment of embryos has also been used in order to figure out the main cause for blastocyst collapse. This evaluation has been performed considering the number of blastomeres, level of fragmentation and quality in days 2, 3 and 5 (blastocyst stage). Nevertheless, the comparison between all these standard morphological features did not yield significant differences that could relate to the event of blastocyst collapse (6, 8). Whereas further evaluation is a usual practice for non-hatched blastocysts, these studies showed no results regarding day 6. Although comparisons including this factor may potentially reveal connections between collapsing of blastocysts and their early developmental history, no data are currently available on this issue.
4. Prediction value of morphokinetic variables
The evaluation of morphokinetic variables has proven to be useful in selecting embryos for transfer, given their potential to predict the successful development of the embryo. These variables have been associated with blastocyst formation, implantation potential and aneuploidy status (13, 14, 15), measured in different critical steps of embryo development (16).
In spite of the increasing amount of studies focused on the relation between morphokinetic values and embryo implantation rates, only a few studies can be found that link these variables with the occurrence of blastocyst collapse (7, 9). Marco et al (2015) showed a significantly slower development of those embryos that had not collapsed, compared to those that did, considering development times from 2-cell stage up to blastulation (t2 - tB, respectively) (7). These results are found difficult to combine with those by Bodri and colleagues (2016); although the authors did relate blastocyst collapse to a progressively decreased live birth rate, they were not able to state this feature as a valid predictor of embryo survival on its own (9).
As it has been previously discussed, blastocyst collapse is a natural phenomenon linked to normal blastocyst development. Even though previous data have shown no direct correlation between this feature in vivo and implantation rates, recent and current studies in vitro suggest that the occurrence of blastocyst collapse has indeed a significant effect on the clinical outcome of the embryo, as explained below.
Currently, vitrification is a widely spread practice in laboratories, aimed to preserve cells/tissues/organs at ultralow temperatures (-196°C). It is well known that the traditional slow freezing approach for cryopreservation frequently leads to ice crystal formation that may damage the cell/tissue, thus decreasing its quality. This associated issue has been avoided by the addition of high concentrations of cryoprotectants and a significant rise in the speed of temperature drop. Such a combination increases the viscosity of the solution and turns it into a glass-like structure (17). Supporting evidence of the success of this technique are numerous studies that demonstrate that the quality of vitrified embryos is comparable to the quality of fresh ones [find more about the success of frozen blastocysts on our previous post here].
A number of researchers have concluded that blastocoel size at the moment of vitrification has an effect on embryo survival and implantation rates. The authors postulated that a large fluid-filled cavity in expanded blastocysts may inhibit sufficient permeation of the cryoprotectant into the blastocoel, thus allowing ice formation and decreasing the chances of survival (18, 19).
There exist several ways to reduce the size of the blastocoel in expanded blastocysts (D5-D6) through artificial shrinkage (AS). Some of them are summarised below:
In 2002, Vanderzwalmen and collaborators achieved AS by introducing a needle into the blastocoel until blastocyst contraction was observed (18). However, and even though the authors reported an increase in survival and implantation rates compared to intact blastocysts, the increase in pregnancy rates was not statistically significant. Similar results were achieved by Son et al (2003), who induced AS prior vitrification, obtaining about 90% of blastocyst survival. Having implanted almost a third of the total, 48% of those turned into a clinical pregnancy (21).
However, these studies were regarded as invasive, due to the injury the method caused in the ZP and the TE. In 2004, Hiraoka et al reported AS of the blastocoel by mechanical pipetting using a fine hand-drawn glass pipette. Even though this method also harms the TE, consequences are less severe due to the needles used. These authors also observed increased survival and pregnancy rates after AS (22).
Figure 2. Artificial shrinkage (AS) of expanded blastocyst with the micro-needle: (a) holding the expanded blastocyst with the holding micropipette. (b) Insertion of the micro-needle inside the blastocoel at a point away from the ICM. (c) Puncture through the blastocoel and gradual removal of the micro-needle. (d) Beginning of shrinkage 10 s after puncture. (e) Partial shrinkage 30 s after puncture. (f) Complete shrinkage 1 min after puncture. Magnification is ×400 (23).
Laser pulse has been also previously used for AS. Application of this method has reported survival rates to reach 97% and pregnancy rates up to 60% (23). In a randomized study, Van Landuyt et al (2015) found no significant increase in implantation rates after applying the pulse prior vitrification, but they did report higher post-warming blastocyst survival rate and quality in collapsed blastocysts (19). After AS, blastocysts were more likely to reach the hatching stage than those not subjected to the procedure. Similar results were obtained by Darwish et al (2016), who observed improved blastocyst survival, clinical pregnancy and implantation rates after removal of the blastocoel fluid (24). In addition, artificially collapsed blastocysts before vitrification have been recently found to re-expand more rapidly after warming (25). However, differences in live birth rates were not significant and the sample size used in the study may have been too small, so these results should be taken carefully.
Other methods have been employed for AS, too. In fact, compared studies have been performed to assess differences on the protocols that may result in improved clinical outcomes. Laser pulses applied on cell-to-cell junctions on the TE and exposure of blastocysts to hyperosmotic sucrose solutions both provided evidence of increased probability of fast-developing embryos and higher implantation rates (1). Even though the authors found no significant differences between methods, the latter was suggested based on the cost/benefit ratio.
These and similar results suggest that AS of human expanded and hatching blastocysts is actually a useful approach to improve clinical outcomes, regardless of the methodology employed. However, and even though it seems that collapse of the blastocoel might be indeed somehow linked to higher implantation and/or pregnancy rates, conclusions should be still drawn carefully.
Blastocyst collapse has been found to be an inherent trait of embryo early development. Its occurrence seems to be related to the physical normal progression of the embryo, and depending on the normal microenvironmental conditions, starting at the zygote stage and expanding up to the last moments before hatching and implantation. Whereas there is no compelling evidence of a link between blastocyst collapse and implantation success in natural pregnancies, the assessment of embryo development in vitro suggests that collapsing of the blastocoel might be indeed related to a certain degree of success. Comparisons between different AS methods have revealed different behaviour of blastocysts after implantation, and even an increasing number of collapsing episodes may be related to a decreased implantation potential.
It is also worth to be noted the importance of vitrification in the routine practice in the laboratory; even though this process optimizes cryopreservation of cells and tissues, the integrity of the embryo may result compromised. Should this be true, blastocoel collapse may become more evident after warming of the embryos, or simply these blastocysts are more prone to collapsing, thus existing correlation rather than causation between both events.
Because blastocyst collapse has been payed attention to in laboratories for just a few years now, thorough and more insightful studies are required to clarify a real connection between this feature and the outcomes of clinical pregnancies.
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(8) Esbert M, Marconetto A, Soares SR, Quera M, Molina J, Florensa M, Ballesteros A, Meseguer M. Does the blastocyst collapse respond to a biological need? The analysis of 1,952 embryos by time-lapse can give an answer. Fertil Steril 2017; 108 (3, Supplement):e157.
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(17) Armitage WJ. Cryopreservation of animal cells. Symp Soc Exp Biol. 1987;41:379-93.
(18) Vanderzwalmen P, Bertin G, Debauche C, Standaert V, van Roosendaal E, Vandervorst M et al. Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod. 2002;17(3):744-51.
(19) Van Landuyt L, Polyzos NP, De Munck N, Blockeel C, Van de Velde H, Verheyen G. A prospective randomized controlled trial investigating the effect of artificial shrinkage (collapse) on the implantation potential of vitrified blastocysts. Hum Reprod. 2015;30(11):2509-18.
(20) Bedient CE, Khanna P, Desai N. Laser Pulse Application in IVF. In: InTech. Lasers - Applications in Science and Industry. Las Vegas. 2011. p. 204.
(21) Son WY, Yoon SH, Yoon HJ, Lee SM, Lim JH. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod. 2003;18(1):137-9.
(22) Hiraoka K, Kinutani M, Kinutani K. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Hum Reprod. 2004;19(12):2884-8.
(23) Mukaida T, Oka C, Goto T, Takahashi K. Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum Reprod. 2006;21(12):3246-52.
(24) Darwish E, Magdi Y. Artificial shrinkage of blastocoel using a laser pulse prior to vitrification improves clinical outcome. J Assist Reprod Genet. 2016;33(4):467-71.
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Authors: María Caballero & Lidón Carretero Vilarroig
"The main cause for unsuccessful ICSI seems to be failure in oocyte activation mechanisms"
Fig 1. Zona-free mouse oocytes prepared for IVF. Staining with Draq 5 for DNA (blue) and phalloidin-Alexa Fluor 488 for filamentous actin (green) has been performed. (A) An oocyte with two pronuclei (arrows) and a polar body (asterisk) is shown, meaning activation has already begun after fertilization. (B) A spermatozoon (arrow) is observed bound to the oocyte not having penetrated the cortical actin. The equatorial plate in the meiotic spindle (asterisk) suggests the oocyte remains arrested at MII (polar body has presumably been lost during oocyte denudation), which indicates that oocyte activation has not yet been produced, given fertilization has actually not been achieved (modified from ).
Fertilization involves the fusion of male and female gametes. However, for this to occur both cells must undergo certain morphological and physiological changes in order to be able to fuse. The sperm cell must undergo a process known as capacitation, which implies a series of changes regarding motility and plasma membrane composition (among others), essential to acquire the ability to fertilize. The oocyte in turn must not only complete several molecular changes involved in maturation, but also a combination of processes initiated by the sperm entry that culminate in the so-called oocyte activation (OA), which prepares the newly formed zygote for embryogenesis (2). In the context of in vitro fertilization (IVF) cycles, oocyte activation failure (Fig. 1) has been observed to occur in about 1-3% (3), resulting in visibly identifiable non-fertilized oocytes. In order to overcome this problem, assisted oocyte activation (AOA) has recently gained interest for those couples with a history of fertilization failure through intracytoplasmic sperm injection (ICSI).
To better understand oocyte activation failure and AOA, here we review gamete maturation, focusing on oocyte activation mechanisms and where/when main problems may arise that cause failure in this process.
GAMETE PREPARATION FOR FERTILIZATION
Fresh ejaculated sperm is still not capable to fertilize. In order to be able to interact with the oocyte, the spermatozoon needs a final maturation step known as capacitation. This process takes place into the female reproductive tract, culminating in the acrosome reaction (4). During capacitation, the sperm cell undergoes biochemical changes related to the acquisition of fertilization ability, mainly involving the molecular reorganization and hyperpolarization of the plasma membrane, as well as hyperactivation (5). Changes in membrane organization are necessary in order to increase its fluidity, which is achieved by removing cholesterol. The mechanisms related to the efflux of cholesterol are not well understood yet, but albumin and high-density lipoproteins located in the female genital tract have been recently pointed out as the main responsible actors for this process (6).
Sperm membrane reorganization leads to the exposure of some important channels and receptors, which increases the permeabilization of sperm to certain ions. Some of the most important channels are CaSper and NBC, Ca2+ and Na+/HCO3- channels, respectively. Calcium influx plays an important role in the process of capacitation, contributing to acrosomal reaction and aiming to the exocytosis of the acrosomal vesicle (8).
Another milestone of capacitation is hyperactivation, referred to as the acquisition of increased and differential sperm motility. This is activated by phosphorylation of protein kinase-A (PKA) in a (HCO3−)- and (Ca2+)-dependent manner, being these ions transported through the cell membrane via CaSper channel and NBC cotransporter, respectively (9). Additionally, tyrosine phosphorylation levels of a multitude of proteins rises significantly in the sperm while in the female tract. Such an increment causes an increase in the activity of the adenylate cyclase, which in turn causes intracellular cyclic adenosine monophosphate (cAMP) levels to rise, ultimately resulting in a differential oscillating movement of the flagellum (hyperactivation). This particular pattern of flagellar beating provides the sperm cell with the proper motility to move forward towards the ovum (Fig. 2).
Finally, when the sperm cell reaches the oocyte, acrosome reaction (AR) is triggered, upon interaction with the zona pellucida (ZP). AR refers to the regulated exocytosis of the acrosome content (including several crucial enzymes) and exposure of surface antigens necessary for oocyte-sperm recognition. The acrosome and plasma membranes fuse, tipping out the content and enabling lateral contact between the sperm head plasma membrane and the oolemma. This is followed by fusion of the plasma membranes of both gametes and delivery of the sperm nuclear material into the oocyte.
Oocytes begin meiosis during foetal development and arrest at a special diplotene stage of prophase I called dictyotene or dictyate, so women are born with a limited number of primary oocytes. There are two mechanisms used by the oocyte to achieve meiotic arrest. The first one can be explained due to the low activity of M-phase promoting factor (MPF), promoted by the oocyte itself via endogenous production of cAMP (10). The oolemma exposes G-protein receptor (GPR3) leading to G-protein-coupled GPR3 and GPR12 activation (11), which in turn stimulates cAMP, a cyclase responsible for AMP synthesis. The second mechanism uses the cumulus-oocyte complex (COC) as a barrier that prevents meiosis reactivation. COC cells produce guanosine 3′, 5′-cyclic monophosphate (cGMP), which enters into the oocyte through gap junctions. Once inside the oocyte, cGMP inhibits the phosphodiesterase responsible for cAMP hydrolysis (PDE3A or cGMP-inhibited phosphodiesterase), which increases cAMP activity, thus inhibiting MPF (10) (Fig. 3). Primary oocytes remain at meiotic arrest until puberty, when the pituitary gland releases luteinizing hormone (LH) during the menstrual cycle and before ovulation. LH acts as a molecular key and activates MPF. LH receptors are located on the surface of granulosa cells, so they transfer the meiotic arrest release signal into the oocyte. Nevertheless, after the resumption of first meiotic division, the maintained activity of MPF promotes the oocyte arrest at metaphase II (12,13).
As a result of the sperm penetration into the oocyte, the female gamete undergoes a process called oocyte activation (OA). This refers to the resumption of the transcriptional activity of the oocyte (inactive until fertilization), essential to allow the subsequent embryo development to progress (14). OA is characterized by the triggering of several events, such as the resumption of meiosis, pronuclei formation, second polar body extrusion, cortical granule exocytosis and genetic material replication, among others (15,16).
All these events are the result of an increase in intracellular calcium (Ca2+) concentration, which takes place upon gamete fusion. In mammals, this Ca2+ release is known as Ca2+ oscillations, due to the fact that this molecule is gradually released over time. Every species shows a specific pattern of frequency and duration for these oscillations (17).
The “sperm factor”
The idea of the sperm containing some kind of OA trigger has been on the table for decades. Evidences showing that sperm extracts are able to fertilize eggs from different species (even phyla) suggested the existence of a common factor that would act as a universal activator of the oocyte (18, 19). Independent observations from different models revealed that the so-called “sperm factor” could effectively be a soluble calcium releasing agent, role that has been attributed to several molecules to date, including phospholipases, second messengers and others. Even involvement of adenosin diphosphate ribose (ADPr) has been discussed in this context, since it has been shown to trigger calcium release through a nitric oxide-based pathway (20, 21). Other molecules such as citrate synthase (22) or post-acrosomal WWP-domain binding protein (PAWP) (23) have been considered potential candidates for years, after their involvement in triggering OA had been demonstrated. However, several pieces of evidence have shown that the key sperm factor responsible for intracellular Ca2+ release and the subsequent oocyte activation may be a testis-specific PLC isoform named PLCζ (24). Although PLCζ is mainly located in the spermatozoon head, it remains unclear if the triggering isoform localizes in the post-acrosomal, acrosomal or equatorial region (17). Once PLCζ is delivered into the ooplasm, it induces hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP2), a membrane-bound substrate located in an intracellular vesicular membrane. The hydrolysis generates two second messengers: inositol triphosphate (IP3) and diacylglycerol (DAG). Thereupon, release of Ca2+ is induced by binding of IP3 to its receptor (IP3R), localized in the endoplasmic reticulum (ER) membrane. This molecular activity causes the characteristic Ca2+ pattern responsible for triggering subsequent events explained below (Fig. 4) (18, 25). Although IP3 causes the first Ca2+ oscillations, DAG has been reported to interact with protein kinase C (PKC), thus increasing its activity. PKC also seems to be involved in events such as the production of Ca2+ oscillations, meiotic resumption or triggering of cortical granule exocytosis (26).
The role of oocyte in activation
Increased levels of intracellular Ca2+ have two consequences (Fig. 5). First, it enhances Ca2+ production by stimulating PLCζ and generating higher levels of IP3 (26). Secondly, it causes hyperpolarization of the plasma membrane, which results in the opening of specific channels present in the oolemma such as the store-operated Ca2+ entry (SOCE), the TRP family proteins and the plasma membrane (Ca2+)-ATPase (PMCA). Opening of these channels activates the influx of Ca2+, which is essential to keep Ca2+ oscillations and reload the intracellular stores (22, 28).
Fig. 5. Representation of the two consequences of Ca2+ increase in the fertilized oocyte. First, high Ca2+ concentration stimulates PLCζ activity and causes the hyperpolarization of the oolemma, which results in the opening of channels like SOCE, TRPM7, TRPV3 and PCAM. Secondly, whereas part of the released Ca2+ is retained by mitochondria, the rest enters into the ER through SERCA or leaves the oocyte through PCAM and SOCE channels. ER: endoplasmic reticulum. N: nucleus. M: mitochondria. CG: cortical granules. Ca2+: calcium. PB: polar body.
Regarding intracellular Ca2+ release, it is necessary to keep a balance to prevent prolonged exposures to the ion that may be detrimental to the egg. Although part of this Ca2+ will be pumped back to the ER by sarcoplasmic/endoplasmic reticulum calcium-ATPase (SERCA) or the protein SOCE system, mitochondria are the actual major regulators of intracellular Ca2+ homeostasis, this means mitochondria retain Ca2+ during fertilization, so that they will act as a passive buffer regulating Ca2+ release. Ca2+ in turn activates oxidative phosphorylation (ATP synthesis). Moreover, it has been demonstrated that ATP generation may regulate intracellular Ca2+ release by making the IP3 receptors more sensitive to (Ca2+)-mediated activation (29). The remaining Ca2+ would then be expelled from the cell through PMCA and the Na+/Ca2+ exchanger (Fig.5) (30).
"[...] the female gamete undergoes a process called oocyte activation (OA) [...] characterized by the triggering of several events, such as the resumption of meiosis, pronuclei formation [...] and genetic material replication, among others."
Egg activation events
As previously mentioned, the increase of intracellular Ca2+ concentration triggers certain key events that enable embryo development:
Cortical granule exocytosis and changes in the ZP
Once fertilization has occurred, the ZP needs to be modified in order to prevent binding and penetration of additional spermatozoa (“polyspermia”). This is achieved by releasing the content of the cortical granules (CGs), specialized secretory vesicles located in the periphery of the oocyte (31). First, CGs are translocated to the plasma membrane by two calmodulin-dependent proteins, CaMKII and MLCK. CaMKII is responsible for releasing the CGs attached to the cytoskeleton, whereas MLCK promotes CG translocation by phosphorylating the motor protein myosin II (14).
Upon translocation, CG fuse with the oolemma in a calcium-dependent manner and deliver their enzymatic content to the perivitelline space (PS). The so secreted glycosidases, proteases and cross-linking enzymes modify the structural network of the ZP ("zona hardening"), thus hindering the entry of multiple sperm (14).
Resumption of meiosis
As aforementioned, MPF is directly involved in maintaining the MII arrest of human mature oocytes (32). MPF is a heterodimer composed of cyclin B and Cdc2 kinase, a regulatory and a catalytic subunit, respectively (33). Following fertilization, Ca2+ oscillations induced by PLCζ activate a phosphorylation cascade that eventually results in the destruction of cyclin B, the concomitant MPF deactivation and the subsequent resumption and completion of meiosis (14).
Pronuclei formation and second polar body extrusion
With the completion of meiosis, the secondary oocyte produces the second polar body and reorganizes its own chromosome complement in the form of a visible and spherical pronucleus. Formation of the paternal pronucleus in turn is dependent on maternal proteins; since the sperm genetic material is highly condensed due to its association with protamins (14), such condensation needs to be loosened in order to make DNA replication and gene expression possible. Maternal histones then replace protamins and the paternal pronucleus becomes appropriately remodelled. Then, maternal and paternal pronuclei fuse together in a process known as singamy or karyogamy (34).
Maternal RNAs and zygote genome activation (ZGA)
Upon egg activation, embryo development commences. However, during early stages, the embryo genome remains transcriptionally inactive. Therefore, the maternal mRNAs and proteins present in the oocyte take charge of early development (14).
Maternal mRNAs are modified to regulate gene expression at the postranscriptional level, which includes edition, splicing, translation and degradation (35). Subsequently, during the so-called maternal-to-zygotic transition (MZT) nuclear genome becomes transcriptionally active. Two main molecular activities are involved in this period: (1) maternal clearance, which means the deletion of maternal mRNAs and proteins necessary for oocyte maturation and first stages of embryogenesis; and (2) gene expression with new instructions for embryo development. This process is activated by the maternal genome and it is known as zygotic genome activation (ZGA) (14). ZGA is not a sudden event, but it has been found to occur in a wave-like fashion. Similar studies in mice and humans have shown that ZGA commences as early as the 1-cell stage; concomitant with maternal mRNA degradation, a minor zygotic gene expression is observed, followed by the major ZGA and the mid-preimplantation gene activation (MGA) (compaction and cavitation processes are also accompanied by waves of gene expression activation) (36, 37).
ICSI FOLLOWING OOCYTE ACTIVATION FAILURE
Between 10-16% of couples worldwide are unable to have children without draw on assisted reproduction techniques (38). Both conventional IVF and ICSI have been proven to be highly successful in ART treatments worldwide, however, the use of ICSI has been reported to be of 66% in 61 countries between 2008 and 2010 and it continues increasing, even in cases with normal semen parameters (39). This success may be explained by to the fact that ICSI shows the highest success rates (around 97-99%) (38), and also because it represents a useful method to rescue fertility for men with suboptimal semen parameters, or even for couples whose fertilization rates after conventional IVF are close to zero (3).
Despite the low failure rates, ICSI cycles do sometimes fail. There exist several possible causes that may explain failure of ICSI cycles, such as technical factors, failed sperm head decondensation, oocyte spindle defects or poor sperm chromatin condensation (40). Nevertheless, the main cause for unsuccessful ICSI seems to be failure in oocyte activation mechanisms, due to either sperm or oocyte defects (17, 39) (Table 1).
Sperm factors associated with oocyte activation failure
Several independent studies have found deficient PLCζ levels when both morphologically normal and abnormal sperm (such as in cases of globozoospermia) from different patients were analysed using mouse oocytes (41, 42). Results have shown reduced or absent levels of PLCζ in the sperm head to be associated with two mutations found in the active domain-coding regions of the gene. Although a deficiency in PLCζ has been associated with most cases of men infertility, other molecules might also be involved in oocyte activation failure. Evidences exist that have linked PAWP, a sperm-specific protein located in the sperm perinuclear theca, to the process of OA. Previous studies have shown this protein to induce pronuclei formation and meiosis resumption when injected into swine mature oocytes. The authors suggested one of PAWP domains to interact with PLCγ, a PLC isoform present in the oolemma, thus contributing to the generation of Ca2+ oscillations (23). This suggests that defective levels of the protein or defective interactions with oocyte factors may eventually lead to oocyte activation failure (23).
Oocyte factors associated with oocyte activation failure
However, neither PLCζ nor any other sperm factor deficiency is always the main responsible for ICSI failure. This points to the existence of oocyte factors that contribute to such failure, as well. Unfortunately, evaluation of human oocyte-related problems is usually quite difficult due to ethical issues and restrictions. As a consequence, the number of available oocytes destined for research is actually low. Therefore, an in-depth study of these potential oocyte-related factors is still necessary nowadays (39).
Despite such problems, there are some actual hypotheses about oocyte-related factors that might be involved in activation failure. As it has been previously exposed, PIP2 plays an essential role, since it is the first element to interact with PLCζ. Some studies have suggested that either a small number of oocyte vesicles containing PIP2 or even a low amount of PIP2 in them could lead to oocyte activation failure (43). Once PIP2 is hydrolysed, IP3 binds to its receptor. Certain genetic mutations in IP3R may block this interaction, thus preventing Ca2+ oscillations to occur. Such mutations may also be responsible for abnormal protein folding or abnormal protein trafficking (39). Moreover, high levels of PLCζ are likely to lead to overproduction of DAG, which may contribute to oocyte activation failure by creating unusual patterns of Ca2+ oscillations (26).
Even when Ca2+ oscillations occur normally, it is important to keep a balance within and outside the oocyte; any mistake in homeostasis regulatory mechanisms may compromise OA (39). As the main regulators of homeostasis, mitochondria function needs to be guaranteed; defective mitochondrial activity due to a low number of these organelles may alter ATP production and generation of Ca2+ oscillations, leading to defects in OA (44). On the other hand, homeostasis is also regulated by the correct function of specific membrane channels. For instance, SOCE is responsible for the introduction of Ca2+ into cellular stores (45). Both components of this system, STIMI and ORAII, may also result mutated, in which case their compromised function would negatively affect the normal occurrence of Ca2+ oscillations (39).
There also exist other oocyte proteins that could lead to activation failure. For instance, the above-mentioned CaMKII is a calcium/calmodulin-dependent protein kinase II that induces cyclin B1 degradation, allowing resumption of meiosis (46). CaMKII is dependent on Ca2+ oscillations, so defects in calcium homeostasis might impair the proper function of the protein and, in consequence, the meiotic progression (39). Additionally, other PLC isoforms present in the oolemma and different from PLCζ like β, γ or δ also seem to have a role in Ca2+ oscillations; in particular isoform γ has been found to interact with PAWP (29, 47).
ASSISTED OOCYTE ACTIVATION
Being oocyte activation failure the main cause for fertilization failure in ICSI cycles, some private clinics perform a special technique to test whether sperm is responsible for failure in oocyte activation. This test is known as MOAT test (Mouse Oocyte Activation Test) (48), and it consists in performing ICSI using sperm sampled from the patient and mouse oocytes. Sperm samples with proven fertility are used as positive controls, while the negative control is represented by injection of culture medium. Based on the results, patients are classified into one of three groups depending on the oocyte activation rate: (i) 0-20% (non-activation group); (ii) 21-84% (intermediate activation group); and (iii) 84-100% (sperm-related infertility refused). In addition, a calcium pattern analysis is also performed. Calcium plays a very important role in all forms of cell signaling. As already explained, calcium contributes to acrosomal reaction and enables the spermatozoon to penetrate into the oocyte (49). When the spermatozoon fertilizes the ovum, it triggers the generation of calcium waves leading to OA and embryonic development (50). A recent article published by Ferrer-Buitrago et al discusses whether calcium analysis can predict the response to AOA in patients with a history of failed fertilization after ICSI. Even though the sample size was limited, the results shown suggest that calcium analysis may actually foretell the response of ICSI-AOA, only in patients with abnormal capacity to generate Ca2+ oscillations (51).
There have been several protocols used for AOA depending on the mechanisms used to trigger Ca2+ oscillations, which can be classified into electrical, mechanical or chemical (52). The first one actually involves electroporation of the oocyte, which generates micropores in the cell membrane allowing Ca2+ influx (53). The mechanical approach consists in oolemma breakage by vigorous aspiration of the ooplasm. The spermatozoon is then introduced into the oocyte, followed by energetic ooplasm aspiration and re-injection during ICSI. This way extracellular Ca2+ from the medium is introduced into the oocyte, resulting in its activation (54). Nevertheless, chemical-based protocols are the most extended techniques. Treatment with ionomycin, calcimycin, ethanol, strontium chloride (StCl2) or calcium ionophores can restore normal calcium oscillations (55). Among the later, the use of A23187 is extended about patients with globozoospermia, whose sperm lack the acrosome and may carry a mutant form of PLCζ. According to the protocol by Rybouchkin et al., oocytes are exposed to the ionophore in the IVF medium after ICSI, and cleavage stage can be assessed the next day (55, 56). Several cases of successful pregnancy have been reported following this ICSI-AOA approach. Few years ago, Hye Jin Yoon and coauthors published results from 185 ICSI AOA cycles using a calcium ionophore ending up in thirty-eight healthy babies with no congenital birth defects (57). The same year, Kim et al. (58) reported five successful pregnancies following ICSI-StCl2 treatment that resulted in the birth of eight healthy children. These results point to AOA as a new therapeutic tool for couples experiencing complete fertilization failure after ICSI.
Although ICSI cycles show a high percentage of success (97-99%), there is still certain probability of not achieving fertilization. Among the different causes responsible for this, the most common is the oocyte activation failure.
For fertilization to succeed, it is important that both female and male gametes undergo a series of changes. In the case of spermatozoa, they need to be capacitated before oocyte penetration. On the other hand, upon fertilization, the mature oocyte needs to be activated in order to start embryo development. This process, known as oocyte activation, is characterized by internal Ca2+ oscillations produced by sperm factors like PLCζ, which is liberated into the ooplasm after fertilization. Ca2+ oscillations trigger the following events: (1) resumption of meiosis, (2) pronuclei formation, (3) second polar body extrusion, (4) cortical granule exocytosis and (5) maternal mRNA replication. This complex process needs a control in the homeostasis that will be basically regulated by the mitochondria present in the oocyte and by some channels in the ER and the oolemma.
Oocyte activation failure is caused by the absence of Ca2+ oscillations or by alteration of their frequency. This failure can be related with both gametes. Regarding sperm, deficiencies in PLCζ have been reported to be directly associated with the absence of Ca2+ oscillations. As for oocytes, and even though studies are scarce due to ethical restrictions, certain hypotheses have proposed activation failure to be associated with deficiencies in mechanisms of homeostasis. This actually means failures in mitochondria and related channels, and also with some molecules like PIP2, DAG, PLC isoforms and IP3R.
In order to avoid oocyte activation failure, assisted reproduction clinics have developed certain protocols for assisted oocyte activation, being those with calcium ionophores the most widely extended in fertility centres.
Authors: Javier del Río, Belén Gómez-Giménez & Iris Martínez
"The relationship between blastocyst morphology and implantation has been investigated mainly according to expansion, trophectoderm and inner cell mass"
The main goal of in vitro fertilization (IVF) is the birth of a single healthy child. However, the consequences and the effects of assisted reproductive techniques on children’s short- and long-term health have always been a source of discussion. Although IVF techniques and protocols have dramatically improved, the overall success rates are still relatively low, and assisted reproduction units still face the challenge of improving pregnancy rates (1). For this purpose, transfer of a single human embryo at blastocyst stage is becoming more common in the practice of assisted reproduction (2). It allows a better synchronization between the endometrium and the embryo and the possible selection of embryos with a higher implantation potential (3).
Several morphology- or kinetics-based approaches have been described to select the best blastocyst in order to increase pregnancy rates. However, the yielded results are conflicting and the outcome is a matter of never-ending and controversial debates, specially regarding blastocyst stage (4, 5).
BLASTOCYST ASSESSMENT SYSTEMS
The relationship between blastocyst morphology and subsequent blastocyst implantation has been investigated according to various criteria. Traditionally, morphology has been evaluated after embryo compaction (6). The significance of examining the embryo after compaction is the ability to examine it after embryonic genome activation. Furthermore, the obvious benefit of looking at the blastocyst is the possibility to examine both cell types. The extent to which the trophectoderm (TE) develops will reflect the embryo’s ability to attach and implant in the endometrium, whereas development of the inner-cell mass (ICM) is obviously crucial for the progress of the foetus (7).
There have been described several assessment systems to predict the success of blastocyst implantation. However, Gardner's grading system seems to be a better predictor of pregnancy rates (8, 4, 7). Following this method, blastocysts are initially scored from 1 to 6 based on their degree of expansion and hatching status, and ICM and TE grading is then assessed from A to D depending on their morphology (9).
It was felt that expansion was important for cavity formation. This process requires both extensive energy utilization through the Na+/K+ ATPases on the basolateral membrane of the TE and formation of effective tight junctions between TE cells to form a barrier. Therefore, expansion seems to be a reflection of embryo competence (7).
Recently, Richardson et al. proposed a simplified blastocyst grading system. These authors demonstrated both its prognostic potential and the inter- and intra-observer variability. This grading scheme was able to effectively predict clinical outcomes in terms of implantation, clinical pregnancy and live birth. Slight variation existed both between and within embryologists grading the embryos but, overall, consistency in their analyses was similar to, if not better than, those associated with more complex grading systems (10).
However, most of the grading systems that are currently used for assessing viability of IVF embryos are subjective, relying on visual inspection of morphological characteristics of the embryos that are qualitatively evaluated. Grading based on qualitative criteria is imprecise, and it inevitably results in inter-observer variability and in intra-observer to some extent, as well (10).
MORPHOLOGICAL ASSESSMENT AND OUTCOME RESULTS
As it has been exposed, there is a need for increased knowledge about the relative impact of each morphology parameter at the blastocyst stage (and their potential correlation) on predicting the probability of successful implantation and pregnancy (1, 2, 11).
Shapiro et al. compared up to 25 parameters in order to develop predictive models of clinical pregnancy within a set of blastocyst transfer cycles (12). Among these variables, blastocyst diameter seemed to be the most significant predictor of clinical pregnancy in the multivariate models. The authors concluded that embryos developing into expanded blastocyst stage on day 5 were approximately twice as likely to implant, compared to those for which expansion was delayed until day 6 (13, 3). This is supported by Van den Abbeel and coauthors, who found that high scores of blastocyst expansion and hatching stage, ICM and TE grade were all significantly associated with increased pregnancy and live birth rates after fresh transfers (11). The finding that the expansion and hatching stage is the most important parameter when selecting a blastocyst for transfer (11) is in contrast with some retrospective cohort studies that suggest TE grading to have the strongest predictive power for treatment outcome in fresh transfers (14, 15).
On the contrary, Basak Balaban et al. exposed that quantitative measurement of blastocysts and ICM is not a practical way to assess blastocyst quality, arguing that two-dimensional measurements of three-dimensional global structures can be misleading. The reasoning is that the size of a blastocyst may vary depending on the time the blastocyst is assessed under the microscope, and this may easily confuse grading (9). For this purpose, Almagor et al. tried to provide an easily measurable assessment of the ICM and evaluate its correlation with pregnancy rates in a series of single blastocyst transfers. They found a high ICM/blastocyst ratio associated with significantly increased pregnancy rates. Thus, they proposed this measure to be used as an additional strongly predictive parameter of successful implantation (16). Recently, Bouillon et al. have confirmed that clinical pregnancy and live birth rates were significantly higher for blastocysts with good TE and ICM quality, and so it was concluded that both rates decreased with morphology (4). Even though some blastocysts with non-optimal morphology are able to implant, it has been suggested that when selection is made among suboptimal blastocysts, preference should be given to those with a normal ICM (6).
However, the current goal for researchers is to establish the optimal perinatal outcome of singletons according to blastocyst morphology. This has been recently analyzed by Bouillon et al., who found no increased rates of adverse obstetric and perinatal outcomes after transfer of blastocysts with poor morphological features (4).
Figure 2. Examples of blastocyst grading: (a) 3AA blastocyst; (b) 3AB blastocyst; (c) 3BA blastocyst; (d) 4AA blastocyst; (e) 4AB blastocyst; (f) 4BA blastocyst; (g) 4CC blastocyst; (h) 5AA blastocyst; (i) 5CA blastocyst. For details of the EH stages and ICM and TE grades, see Materials and methods from Van den Abbeel (11).
BLASTOCYST ASSESSMENT BY TIME-LAPSE TECHNOLOGY
As previously explained, the most accepted blastocyst grading system is Gardner’s (17), based on the degree of blastocyst expansion and the morphological appearance of both the ICM and TE. However, since embryo development is a dynamic process, conventional grading practices may not detect subtle differences in morphology, which changes significantly over a time span of only a few hours (18). In order to obtain a complete picture of morpho-kinetic events occurring during embryo development a time-lapse system is needed. This technology offers continuous monitoring of embryos rather than just a limited number of discrete observations annotated through conventional assessment. Besides, time-lapse allows embryos to be cultured uninterruptedly, thus getting rid of embryo trafficking from and into the incubator (19). Nevertheless, the actual new and unique contribution of morpho-kinetics is the ability to predict how likely is for a zygote to reach the blastocyst stage in vitro. Several algorithms based on parameters detected by time-lapse, such as early divisions of cleavage-stage embryo, have recently been developed in IVF laboratories to predict blastocyst formation (20). In addition, some authors have made an effort to take time-lapse usefulness further, for instance, to predict the ploidy status of pre-implantation embryos (21, 22).
Implantation potential of blastocysts can be evaluated by means of time-lapse during its development. In this regard, three main events are currently being investigated: duration of both compaction and blastulation plus number of blastocyst collapse events (19, 23, 24).
Duration of compaction
After several cell divisions during the initial stages of embryonic development, the intercellular boundaries become obscured in a process called compaction, which maximizes the intercellular contact and gives rise to the morula (25). Although the compaction of embryos has not received sufficient attention in the IVF field, some studies have focused on the relationships between compaction patterns and embryo developmental potential. Embryos that begin to compact before the eight-cell stage exhibit aberrant in vitro development. Conversely, embryos that complete compaction on day 5 have a lower ability to develop into high-quality blastocysts than those that compact on day 4 (26). These results suggest that the compaction patterns of embryos can facilitate the prediction of their ability to develop both in vitro and in vivo.
An interesting work on this issue has been recently published by Mizobe and collaborators (23). The study retrospectively examined the outcome of 299 embryos from 243 patients, which were transferred at blastocyst stage. The whole early development was analysed by comparing morpho-kinetic parameters between implanted and non-implanted embryos, and measuring the time length of specific events, particularly of embryo compaction. Compaction length was calculated by using values of beginning and end of compaction. Beginning of compaction was considered as the time point when the intercellular boundaries became diffuse somewhere in the embryo, while fully compaction was defined as the point when blastomeres were finally unified into one cluster. Compaction length was significantly shorter in blastocysts resulting in pregnancies compared to those that failed to do so. These results indicate a correlation between the length of compaction and implantation potential. This finding is in agreement with the results from previous studies, which observed that the compaction patterns of embryos affected the rates of good-quality blastocyst formation and implantation (26, 27, 28). By contrast, some studies have reported that compaction time of embryos does not affect clinical pregnancy rates (29, 30).
Duration of blastulation
Blastulation is the process through which a morula becomes a blastocyst. Two different structures will arise to form the blastocyst out of the compacted blastomeres of the morula. The first sign of blastulation is compaction and differentiation of the outer blastomeres, forming the TE. This compaction gives the structure a watertight condition, allowing the fluid later secreted to be contained (31). Then, a different group of blastomeres normally located at the centre of the morula start to get closely attached to each other by the formation of Gap junctions, thus facilitating cell communication. It is these cells that differentiate into the ICM (the future embryoblast) and acquire a polarized location at one edge of the embryo. Such polarization creates a cavity, the blastocoel, and gives rise to the structure termed blastocyst. The trophoblasts (TE cells), in turn, continuously pump fluid into the blastocoel, which results in an enhanced size of the blastocyst. This increased volume leads the embryo to hatch through the zona pellucida (32).
A recent study conducted by Mumusoglu analysed whether time-lapse morpho-kinetic variables differ among those euploid blastocysts that result in ongoing pregnancy after single embryo transfer (24). For that purpose, 129 patients who had been transferred a single embryo after an ICSI cycle with PGS were considered. Embryos were cultured in a time-lapse incubator up to the moment of TE biopsy, and 23 time-lapse morpho-kinetic parameters were annotated. After biopsy, blastocysts were vitrified and transferred within the next cycle. When comparing all time-lapse parameters, only blastulation time was statistically different: it had lasted shorter in successfully implanted blastocysts than in those that had not implanted. Blastulation time was calculated as the interval from initiation of blastulation up to full blastocyst formation (33, 34). Even though only a few studies have genetically tested euploid blastocysts (21, 22), all of them have pointed out that faster-developing euploid blastocysts might exhibit higher implantation potential. Even so, further large-scale studies are needed in order to confirm such an association (24).
Blastocyst collapse events
The phenomenon of blastocyst collapse is actually the shrinkage caused by the efflux of the blastocoel fluid due to the loss of cell bindings along the TE. When blastocysts expand, fluid gradually accumulates in the blastocoel -mediated by the sodium pump (Na+/K+-ATPase) (35), resulting in an increased pressure on both the TE and the zona. In parallel, TE cells produce lysins that are involved in the zona weakening and hatching. Formerly to implantation, the embryo needs to leave the zona behind, place adjacent to the endometrial epithelium and then make first contact with the uterus (36). Thus, embryo hatching from the zona is thought to be related to collapse-expansion cycles.
By using a time-lapse monitoring system, it has been observed that many of the human blastocysts that reach stage 5 of expansion experience one or more collapse events of the blastocoel cavity, producing a separation of part (if not all) of the TE cells from the zona (19). In a study conducted by IVI Valencia and IVI Murcia clinics (19), blastocyst collapse was analysed to determine its potential influence on reproductive outcomes and whether it may serve for prognostic purposes. 460 patients and data from over 500 blastocysts known to have implanted were included in the study. Blastocyst collapse was considered to have occurred if the separation between TE and the zona pellucida was higher than 50% of the volume. Blastocysts that had experienced just one collapse event were found to present a significantly reduced implantation potential when compared to those transferred after having experienced none. The authors proposed that the molecular mechanisms underlying this association could be related to the mechanical stress suffered from by the embryo, which could result in an excessive energy consumption that would adversely affect the consequent development (19).
Figure 4. Drawing tools used with Embryovieverw for blastocyst collapse evaluation. First, a line was drawn across the embryo diameter (A). Then, the two circumferences that define the contracted blastocyst and the inner surface of the zona pellucida were outlined (B) [for more details, go to Materials and methods from Marcos (19)].
In spite of the data discussed above, the negative association between blastocyst collapse and implantation potential is not yet clear. In a report by Bodri and colleagues (37), blastocysts were classified according to the number of collapses: embryos with no collapses represented 54% of the total, 22% of the embryos had experienced one single collapse, and multiple collapse events occurred in 24% of the blastocysts. Whereas the live birth rate was observed to decrease as the number of embryo collapse increased, multivariate analyses suggested blastocyst collapse not to be a significant predictor. Rather, it was found to be a confounding factor, along with other morpho-kinetic variables such as time up to two-cell division completion and female age. Therefore, it was concluded that blastocyst collapse patterns should not be evaluated alone without stronger predictors of reproductive outcomes being taken into account (37).
TIME-LAPSE AS A MEANS TO EVALUATE EMBRYO QUALITY
As previously exposed, the use of time-lapse technology is recently common in embryology laboratories because of its noticeable potential for enhancing embryo selection. Using these technologies, Desai et al. analysed possible kinetic differences between embryos with limited potential and those that accomplished in vitro blastocyst formation and/or implantation (38). Certain parameters such as time of pronuclear formation and cleavage stage were found to be different in embryos reaching blastocyst stage vs. poor-quality embryos. Moreover, a large number of embryos were found to present multinucleation and reverse cleavage, but they were able to form a blastocyst with optimal criteria for freezing (38), which resembles previous reports on the dynamic nuclear formation of blastocysts by Ergin and coauthors (39).
With respect to blastocyst formation, Motato et al. (2016) proposed two models to classify embryos based on their probability of reaching blastocyst stage and implantation (40). However, the study was limited by parameters such as subjective criteria from different clinics with different culture media (40). Consequently, it would be reasonable to keep on research on this subject in order to achieve a consensus regarding embryo classification and implantation potential (40).
CONSIDERING DAY-2 AND DAY-3 EMBRYO MORPHOLOGY BEFORE DAY-5 TRANSFER
Even though blastocyst stage is currently widely accepted as the optimal moment for embryo transfer, cleavage stage has been traditionally regarded as the right moment in global practice. In fact, it still continues to be so in some laboratories, and early transfer into the uterus has been proposed to be advantageous to the embryo due to the limited time exposed to the in vitro environment (41). However, there exist two main arguments supported by extensive scientific literature to explain why blastocyst transfer after extended culture has advantages over the traditional cleavage-stage transfer:
First of all, when the embryo arrives to the uterus in natural conditions it has already reached morula stage, which corresponds to, at least, day 4 of in vitro culture (42). This means blastocyst stage is the most physiologically compatible stage for transfer, since it allows a better synchronization between embryonic stage and endometrial receptivity (43) [you can read more about the optimal day for embryo transfer in our previous article here].
Secondly, several studies have reported higher implantation potential for blastocysts compared to cleavage-stage embryos (6, 41), the first transferred blastocyst being reported in 1995 (44). Furthermore, some authors have postulated that a large proportion of morphologically normal day-3 embryos are actually chromosomally abnormal or mosaic, which may contribute to the 80-90% rate of implantation failure observed after cleavage-stage embryo transfer (45).
EXTENDING EMBRYO CULTURE UP TO BLASTOCYST STAGE
Considering the need for further studies on the subject, and the fact that day-3 embryos can actually implant and develop successfully, does it really make sense to extend embryo culture up to blastocyst stage?
As above-stated, morphologically normal embryos may actually present chromosome abnormalities, which proves the insufficiency of morphological criteria to evaluate implantation rates (46). Because of embryo plasticity, the proportion of chromosomally abnormal cells varies within the culture; corrupted cells can be eliminated, thus resulting in a good-quality blastocyst developing from a poor-quality cleavage-stage embryo (47). Some studies have evaluated pregnancy rates derived from transfers of blastocysts with previous poor quality as cleavage-stage embryos, finding an approximate success rate of 45% after culture and freezing of embryos at an early stage for another cycle. The conclusion of this being a valid practice to avoid the repetition of IVF-ICSI treatments (48) agrees with recent findings showing that low-scoring day-2 or 3 embryos, which are not considered transferable, can still result in successful blastulation and end up in a live birth (49, 50).
All this said, the right question now would be: should day-2 and day-3 embryo morphology be considered before transfer at day 5 when blastocysts reach a similar good quality?
A recent retrospective study by Herbemont has suggested that only the quality of the transferred blastocyst may be predictive of the subsequent clinical outcome, whereas morphological aspects at day 2 or day 3 have limited interest (51). These same results had previously been observed by Guerif; even though early morphological parameters were relatively helpful to predict blastocyst development, their value to predict blastocyst morphology was limited, and so they provided no significant additional information that could prognosticate blastocyst implantation and live birth rates (6). A few years earlier and with the same goal in mind, Zech and coauthors carried out a prospective randomized study in which they compared ongoing pregnancy rates per single embryo transfer between day 3 and day 5. When good-quality embryos were available, pregnancy rates were found to be higher after blastocyst transfer. Therefore, the authors concluded that morphological criteria-based seleccion at day 3 may not be a suitable procedure when just one embryo is to be transferred out of a cohort of all morphologically good ones. Thus, and as stated by the authors, extending embryo culture up to day 5 may result in a better strategy in order to correctly identify and select those embryos with higher implantation potential, provided there is a sufficient number of top-quality eight-cell embryos available (52).
On the contrary, a study performed by Silber (2014) found that blastocysts arising from poor-quality embryos displayed lower implantation and pregnancy rates compared to good-quality embryos. These discrepancies could be due to different criteria used to score embryo quality (53). So, in order to minimise discrepancies between studies, the use of time-lapse is currently established as a common approach to evaluate embryo morpho-kinetics. In fact, reduction in the time of embryo exposure to the environment outside the incubator has been demonstrated to enhance both embryo quality and blastulation rates (54).
Nevertheless, to answer the question previously postulated, a prospective randomized study would be needed that compare at least two similar good-quality blastocysts, one arising from a good-quality day-2/3 embryo, and to the other from a poor-quality one (51).
It is important to take into account that the main population features of different patients, such as paternal age, maternal BMI, parental smoking or cause of infertility may influence clinical outcomes. Moreover, certain methodological aspects also need to be considered, like blastocyst evaluation by the same personal (in order to minimize variation) or the consistent use of the same type of culture media (to avoid potential effects on birth-weight and other traits), just as previously suggested (4).
A universal embryo grading system needs to be validated, before widespread implementation in IVF laboratories. Also, it has not yet been clearly established which morphological feature of blastocysts (expansion, TE or ICM) is the most reliable as a predictive factor for post-transfer implantation success. Consequently, there is still a debate between authors about the true outcomes of single transfer of low-quality blastocysts (4).
Morpho-kinetics assessment, along with chromosomal screening, may ultimately help identify euploid embryos with the highest developmental potential (55). Since these features are susceptible of being affected by in vitro culture conditions, each embryology laboratory should define their own cut-off points in order to standardise time-lapse variables (24).
Finally, it should be taken into account the fact that embryo quality is not the only parameter with influence on implantation rates; endometrial receptivity is also involved, and it may be greatly determined by a variety of factors (56) [to learn more about endometrium status and receptivity, read our previous post here].
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Authors: Iñaki Arroyos, María Caballero & Raquel Pillado
"Up to date, clinical practice has proven that the use of in vitro culture for human embryos does not imply any major adverse consequences on the offspring"
- Why is it important to talk about IVF culture media?
Within the last 40 years, the improvement of assisted reproduction technologies (ART) has experienced rapid and great advances (2). This success is largely due to the important enhancement of the culture conditions, specially IVF media (2,3), since this is where the early embryo develops up to blastocyst stage (Figure 1) before being transferred into the womb.
However, it is currently known that in spite of this huge improvement regarding culture media, there still exist certain important associated-risks. These include foetal growth restriction, premature birth, low birth weight, congenital anomalies, perinatal complications and even epigenetic alterations (2,3,4).
Therefore, there is still a need to improve our current knowledge on IVF media components, concentrations and related factors, in order to dodge those potential negative effects.
- How did culture media evolve through history?
The development of culture media for human embryos has been possible thanks to many years of animal research (Figure 2). The first mouse embryo culture took place in plasma (5) and blood clot (6). Later on, development of synthetic media and simple culture media began, with improvements such as salt solutions supplemented with glucose, egg white and yolk (7) or Krebs-Ringer-bicarbonate (KRM) solution with glucose, bovine serum albumin (BSA) and antibiotics (8). However, embryos used at that time were collected from the Fallopian tubes at the 8-cell stage, thus being impossible to address development from the very 1-cell stage (2).
Elements such as the appropriate pH level and osmotic pressure, the effect of energy substrates, BSA and amino acids in the embryo culture were widely studied during the 60’s. Glucose was replaced by lactate and pyruvate upon Brinster's demonstration that the 2-cell-stage embryo did not use glucose as energy source, and that its presence at certain concentrations hampered the 2-cell embryo development to blastocyst (10).
The first success in human IVF was achieved by Edwards and Steptoe in 1969, who used a medium based on a modification of Tyrode’s solution (10). Two years later, using a more complex medium called Ham’s F-10 (made of inorganic salts, glucose, pyruvate, amino acids, vitamins and serum, among others), Steptoe and collaborators succeeded in culturing human embryos from the zygote to blastocyst (11). In 1978 and using this medium, Edwards and Steptoe got the first embryo transfer that resulted in the birth of the first IVF baby, Louise Brown (12). However, over the years, some elements from Ham's F-10 medium were shown to have adverse effects on embryo development, and so its usage was stopped (13).
Years passed and different modifications took place: the removal of proteins from the media, the use of amniotic fluid and, finally, the search of the “optimal” medium. For this approach, around 1970 the development of the following media was based on the “back to nature” vision (2). New media were developed according to the composition of the reproductive (tubal and uterine) fluids (as an example, the HTF medium was made only of inorganic salts, glucose, pyruvate, lactate, human serum albumin and antibiotics). Nevertheless, glucose and phosphate were reported to be toxic to cleavage-stage embryos (as mentioned above), glutamine replaced the glucose and ethylenediaminetetraacetic acid (EDTA) was added instead of phosphate. Blastocyst formation rates were not entirely satisfactory; subsequent studies found that amino acids and vitamins improved these rates because the toxic effect of glucose was avoided (reviewed in 13).
At the same time, sophisticated software was used to get a simplex optimization medium by getting the concentration of each medium component in vitro. Using mouse embryo assays, the potassium simplex optimized medium (KSMO) (14) and the KSOMAA medium (KSMO supplemented with amino acids (15)) were developed. These media are effective for the human embryo culture and, in fact, KSOMAA is widely used in human ART under the brand of Global (13).
WHICH MEDIA DO WE USE NOW?
Nowadays, the market of IVF culture media is widely expanded worldwide. Cook Medical, Cooper Surgical (producer of both SAGE and ORIGIO media), FertiPro, Gynotec, Genea Biomedx, InVitroCare and Kitazato are some of the companies that produce culture media for human IVF (2). Even though all commercial media present the same components (see Table 1) (2), all of them well known by every company, the concentrations and the most basic parameters for embryo culture such as the use of amino acids and glucose, temperature or gas composition, vary between brands (16).
- Different embryo needs (time/development stage)
It is important to bear in mind the nutritional requirements of the embryo during development. Several authors have found that the required energy substrates, amino acids and proteins concentrations differed according to the embryo stage. Based on these results, it was established that between days 1 and 3 (cleavage stage), glucose should be reduced or removed if there are no amino acids or EDTA in the medium, in order to avoid the adverse effects of glucose. Lactate, pyruvate, taurine and non-essential amino acids should be included as well, but essential amino acids should be omitted at this point. A protein source such as pure (or almost pure) albumin should also be added (16). From day 3 to day 5 (blastocyst stage), the glucose concentration is increased, as well as the concentration of 20 amino acids. Pyruvate concentration in particular is lower, and protein requirements maintained (16). Change of media at day 3 is appropriate, since the embryo undergoes a series of changes at this time, such as compaction and activation of the embryonic genome (17).
As a consequence, sequential media were developed. By contrast, the alternative use of single-step media is based on letting the developing embryos choose the nutrients they need. This way it is possible to minimise the stress caused by different culture environments (13,18). Both types of media are currently used by IVF clinics, even though sequential media seems to be the preferred option (18).
DIFFERENCES BETWEEN CURRENT MEDIA AND TRENDS
-Differences between sequential and single-step media
Previously, two different kinds of media have been introduced: sequential (or two-step) and single-step (or one-step) media. In order to use these media to culture embryos from zygote to blastocyst stage, there are three different protocols available (19):
 Sequential media protocol. Two media of different compositions are used sequentially. Medium is changed on day 3 of embryo culture.
 Single-step protocol. Uninterrupted culture using one medium throughout the 5 days.
 Single-step protocol with renewal. Interrupted culture using the same type of medium throughout the 5 days, including renewal on day 3.
Originally, all human embryos were cultured just until day 2-3 before transfer. However, extended culture from zygote to blastocyst has attracted more attention since 1997 (18). The studies by Gardner and Lane (reviewed in 19) or Pool (16) have been the main supporters of the use of sequential media protocols. There are four main arguments in favour of the use of two-step media culture (19):
 - The energy source required by the preimplantation embryo changes throughout development, from pyruvate and lactate to glucose, but glucose has inhibitory effects on early cleavage stages.
 - Ethylenediaminetetraacetic acid (EDTA) helps to bypass the two-cell block. Later on, it has an inhibitory effect on blastocyst development and on the inner cell mass (ICM).
 - Although evidence supports embryo development up to blastocyst stage with no amino acids (AA) present in the media, non-essential AAs (NEAAs), and specially glutamine (Gln) favour embryo growth during cleavage stage. Later on, during blastocyst stage, all 20 AAs stimulate the development of the inner cell mass (ICM). Furthermore, NEAAs also have an stimulation effect on the trophectoderm and the hatching process of the zona pellucida. However, an inhibitory effect on blastocyst development and viability has been observed in the presence of essential AAs (EAAs) during cleavage stage (20).
 - The chemical breakdown of L-glutamine (Gln) in aqueous solution is the main contributor to the accumulation of ammonia during culture, which can compromise embryo development. Nonetheless, Gln is also beneficial to overcome the two-cell block.
The four points mentioned above are easily addressed by using two-step media (21). However, supporters of the one-step medium protocol, in turn, argue that these problems can also be addressed through the use of one single type of medium.
First, the inhibitory effects of glucose on early cleavage stages is not absolute. Furthermore, glucose is present in the natural environment of the Fallopian tubes, and there is evidence of culture media with glucose that do not inhibit embryo development (2). The reason behind this is that the inhibitory effect of glucose is determined by the interaction with other substances present in the environment. These interactions can be determined experimentally and adjustments to the concentration of medium components can be made accordingly (19).
Furthermore, studies supporting the negative effects of EDTA during late developmental stages of the embryo refer to concentrations of 0.1 mmol/L; the optimal concentration needed to bypass the two-cell block is only between 0.005-0.01 mmol/L, which is insufficient to have any deleterious effect (20).
Some authors like Lane and Gardner have suggested leaving out EAAs from the media during early developmental phases (19). By contrast, and after studying the AAs net depletion during human preimplantation embryo development, others such as Leese argue that the most prudent choice would be the use of a mixture with all 20 AAs. This way, the embryo would be able to choose which ones to use by itself (20).
The problem of accumulated ammonium from decomposing Gln is solved by using more stable dipeptides of Gln, such as glycol-L-glutamine (GlyGln) or L-alanyl-L-glutamine (AlaGln) (19).
Despite the explanation for both types of media addressing the embryo needs throughout early development, there is still a remaining question: which culture medium is more efficient? Even though in recent years multiple studies have dealt with this question, the answer is less than clear.
In order to compare one-step protocols with sequential protocols, some studies compare ongoing pregnancy rates, clinical pregnancies or miscarriage rates (22). Other studies also include blastocyst formation per randomized oocyte/zygote (23), or the number and size of blastomeres in certain days, along with the final quality of the embryos (21). But the results of all of them agree in that there is no clear evidence to support either culture medium being better than the other. No significant difference was found between sequential and single medium for ongoing pregnancy rates, clinical pregnancies or miscarriage rates. Regarding embryo development, embryos cultured in one-step medium were found to present significantly more blastomeres (although unequally sized) and lower fragmentation rate in day 2 than those cultured in sequential media. However, no significant difference was observed in the percentage of good quality blastocysts between both groups (21).
-Trends in the use of culture media
Ever since the beginning, sequential media have been the most popular option. However, the popularity of single-step media has slowly risen within the last decade. This fact is evidenced by the increased offer of commercial media; whereas in 2008 there was only one commercial single-step medium and six sequential media (19), a few years later the number of single-step media available in the market had increased up to three times (20).
Not only does the non-renewal single-step media require a reduced level of embryo handling, but it also reduces the chances of damaging or stressing the embryo. This is particularly evident in time-lapse systems, whose medium-related costs are lower. On the other hand, both the one-step medium with renewal approach and sequential protocols avoid excessive accumulation of potentially harmful waste products in the medium. Without reliable evidence supporting one type of medium over the other, it is up to the clinics to decide which one is more suitable. Therefore, further research with larger samples will be needed on comparing the efficiency of embryo culture media.
CULTURE MEDIA AND PERINATAL OUTCOMES
-Perinatal outcomes and assisted human reproduction
Extensive animal research have provide evidence of the link between early embryo development environment and adult diseases. In the same way, in vitro culture may induce epigenetic changes in the embryo with long-term consequences, even if they are not obvious at birth or during early childhood (2). Consistently with these studies, it is known that newborns from assisted reproductive technologies (ART) often have poorer perinatal outcomes (see Table 2) compared to naturally conceived newborns. Usually, ART outcomes have been related to parental underlying medical conditions, preimplantation genetics diagnosis (if performed), cryopreservation and thawing processes, differences in hormonal treatments, laboratory conditions during embryo culture, culture media or the combination of all of them (24).
Up to this day, clinical practice has proven that the use of in vitro culture for human embryos does not imply any major adverse consequences on the offspring. Nevertheless, the population born through ART is still relatively young, being Louise Brown, the first IVF child, only 49 years old. Therefore, the possible adverse repercussions on late childhood or adulthood are still a subject of study (2).
-Perinatal outcomes and culture media
Within last decade, multiple articles have been published that reveal actual associations between culture media and perinatal outcomes (25,26). Nevertheless, there is certain controversy when choosing between the type (single-step or sequential media) or the brand of the compared media, as well as with sample sizes.
Several studies have shown no significant differences between children born after IVF and children conceived spontaneously or by intrauterine insemination (IUI) (24,25). However, there are available data that do show significant disparities in preterm birth rates between groups cultured with different media (25,26,28). Additionally, VitroLife medium seems to present a trend towards newborns large for gestational age (LGA) (25). This medium has been related to significantly higher pregnancy, clinical pregnancy and implantation rates. Simultaneously, Cook medium has been associated with lower birthweight means, higher LBW incidence and higher proportion of single embryo transfers (26). In other cases, when testing VitroLife against other media (SAGE), results indicated no significant differences for any rate; size for gestational age, LBW or even birthweight means between fresh and thawed embryos of both groups showed similar values (29). Lastly, when single-step media SSM and Global were compared, the former exhibited poorer performance than the latter, resulting in lower pregnancy and implantation rates (28).
It is clear from previous studies that some culture media underperform in comparison to other media of the same type. As above-mentioned, such lower performance affects important factors like birthweight means. LGA for instance, may cause problems during labour, and LBW has been associated with higher probability of abnormal growth, neurodevelopmental problems and increased incidence of diseases such as obesity or type-2 diabetes (30). Preterm birth, in turn, may lead to the need of incubators and may cause learning disabilities or visual/hearing problems. The most likely reason behind the differences between media efficacy is the disparity in their chemical composition. For instance, whereas VitroLife uses a stable dipeptide of Gln, Cook contains L-Gln, which may be the cause for the higher percentages of LBW observed (31).
APPLICATIONS AND NEW APPROACHES IN EMBRYO CULTURE
Approximately, two out of three IVF cycles fail to result in pregnancy, causing significant physical, emotional and financial distress for women undergoing infertility treatments (32). In order to prevent failures, centres have chosen to perform transfer of multiple embryos, which implies a high risk of multiple pregnancy. This strategy increases the chance of developing maternal and infant morbidity; infants from multiple pregnancies are more likely to present low birthweight, and the probability of mothers suffering from miscarriage is also higher. (33). In this regard, the most common maternal complications associated with multiple pregnancies include high blood pressure, preeclampsia, increased likelihood of caesarean section, venous thromboembolism, postpartum hemorrhage and gestational diabetes (34).
Considering these data, one major objective in reproductive medicine is trying to find easy, useful and clinically applicable methods to identify embryos with higher probability of implantation. Classic embryo morphology assessment is still the most established method to select embryos for transfer. However, this approach is subjected to the embryologist´s own criteria, and even though there are several scoring systems, it is not a method capable of giving reliable results (35). The recent time-lapse technology provides kinetic information of the embryos and allows the embryologist to have additional criteria for selection. But even with this improvement, more information is needed in order to find the embryo with the highest implantation potential.
Nowadays, the OMICS field tries to identify biomarkers for therapeutic and diagnostic development (36). Approaches in embryo assessment based on the analysis of culture media may consider proteomics and metabolomics. Proteomics involves the study of the proteoma, including proteins secreted by embryos in the media. By studying the protein profiles, researchers are able to find altered expression of specific proteins related to important embryo events, like blastocyst development or implantation (37). The use of metabolomics, in turn, allows for the measurement of changes in the level of metabolites present in culture media that are associated with carbohydrate metabolism and amino acid turnover. The concentration of these metabolites can be used as main indicators for embryo potential. Metabolites such as glucose or lactate can effectively be associated with embryo development.
Because metabolism is the final product of gene expression, the study of metabolism may be useful compared to the other OMICS, and cell function may be more accurately reflected (37). Additionally, not only metabolic flow is regulated by genetic expression, environmental stress and metabolites can be measured more precisely (39).
Future directions of OMIC research should not only be focused on trying to elucidate which embryo presents the most suitable profile for implantation, but also on trying to reduce the cost of required technology and turn it affordable for daily clinical practice.
- New approaches for embryo culture
Culture media composition has been modified in the past recent years in order to achieve better physiological conditions. By contrast, research on embryo biophysical requirements has not developed equally fast (40). When developed in vivo, not only embryos are exposed to changing fluid chemical composition, but to mechanical stimulation (41,42,43). In addition, changes in the type of culture platform may lead to modifications in the environment immediately surrounding the embryo through regulation of chemical gradients. Thus, research on different new technologies may assist in improving embryo development (44,45).
Types of culture systems
Microdrop systems have the benefit of the growth factors, with a typical drop setting of 10-50 μL. Ultramicrodrop is a variant that works with volumes of 1.5-2.0 μL, but it still presents severals typical issues related to working with small volumes (evaporation, osmolality, potential toxicity, embryo loss...) (55).
A different model is conformed by the use of volumes of sub-μL (nL) of media and a vertical channel-containing polydimethylsiloxane (PDMS) culture chip (56). This allows the embryo to benefit from both reduced media volume and space. On the other hand, these same features makes the embryo difficult to recover.
The Microwells system creates a microenvironment that offers a potentially increasing surface area and simultaneously a reduced space between embryos. The most popular microwell, called “well of the well” (WOW), was developed by Vajta (2000), and it has since been validated by several studies in several species (57). Initially, microwells needed to be made manually, but there currently exist available WOW systems made of polystyrene.
Microchannels are related to the idea of increasing the surface area adjacent to the embryo, rather than just a single point of contact within a Petri dish. A different approach of microchannels is using glass capillary tubes filled with media (58).
Vibration systems have been developed due to the estimation that, in vivo, embryos are exposed to vibrations of 5-20 Hz in response to the ciliated epithelium of the oviduct (62). Data from different studies have shown that even short periods of gentle vibrations during oocyte maturation or early fertilization events may be of benefit to embryo quality (63,64). Finally, controlled fluid systems have been developed based on the premise that embryos, just like other cell types, can detect the shear stress (an endothelial concept) of the fluid flow (65). Excessive stress could damage the blastomeres, affect signaling pathways and cause embryo degeneration (66).
Culture media have experienced a significant development in ART, from primordial cultures like simple salt solutions supplemented with glucose and egg yolk, including changes in embryo energy sources, until more complex solutions closer to in vivo reproductive fluids. This evolution of culture media has been possible thanks to research on embryo metabolism. This allowed to find out the nutritional requirements of the embryo for every stage, thereby making it possible to develop sequential media. The development of the alternate philosophy (“let the embryo choose”) has in turn offered other advantages, such as the reduction of stress caused by different microenvironments.
Both approaches have defenders and detractors, all of them with arguments to support one over the other. However, it is not yet clear which media offers the best “in vitro environment” for the embryos. Single media have gained supporters partly due to their advantages regarding reduced manipulation. This is related to time-lapse technology, even though the classical sequential media or the “renew single media” option are widely employed, too. The usage of these media is justified by the potentially deleterious effects of the accumulation of harmful waste substances.
On the other hand, different ART-related procedures, including embryo culture, seem to also affect the offspring, showing poorer perinatal outcomes in comparison to naturally conceived newborns. It remains unclear whether this is derived from the culture media used or from combinations of other parameters. However, current data indicate that media do not seem to entail any major adverse consequences on the offspring. Even so, caution should be exercised. Different chemical proportions and the quality of molecular composition could explain the differences observed in some parameters like LBW between distinct commercial brands. It is important to have more information about the role of culture media in adverse perinatal outcomes. For this purpose, not just the chemical composition but also the concentrations of commercialised media should be disclosed to facilitate research on this subject.
It seems obvious that modifications in the platforms used for culture media may influence embryo development. Dynamic systems are a new field of research, which may potentially help understand the physiological requirements of the embryo. However, it might result difficult to eventually improve these strategies due to extra expenses. In order to achieve so, such new devices need to count on the companies´ trust, since they are the actual responsibles for making it possible to spread technology throughout the clinical community.
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"The underlying cause for endometriosis is likely to present a multifactorial origin"
Authors: Javier del Río, Noemi Díaz & Edel Rocher
Endometriosis is defined as the presence of endometrial tissue outside the uterus, which induces a chronic inflammatory reaction. It is generally assumed that lesions arise through retrograde endometrial tissue loss during menstruation, coelomic metaplasia and lymphatic spread in immunologically and genetically susceptible individuals. Associated symptoms usually have an impact on the general physical, mental and social well-being (2,3,4).
This condition is found in women from all ethnic and social groups, predominantly in those of reproductive age, which may represent an estimation of up to 176 million women all over the world. The prevalence of endometriotic disease seems to be around 5%, normally reaching a peak somewhere between ages 25 and 35. Among women aged 15-49, an annual incidence of endometriosis of 0.1% has been reported (2,3,4).
Which symptoms may be indicative of the disease?
Women who suffer from endometriosis typically experiment abdominal pain and related symptoms such as dysmenorrhoea, dyspareunia, heavy menstrual bleeding, non-menstrual pelvic pain, painful ovulation, dyschezia and dysuria, as well as chronic fatigue. Endometriotic lesions are followed by denervation and reinnervation, which represent accompanying changes in the central nervous system (central sensitization), thus generating a chronic pain syndrome. The extent of the disease varies from a few, small lesions on otherwise normal pelvic organs to large, ovarian endometriotic cysts (endometriomas) and/or extensive fibrosis and adhesion formation, causing marked distortion of the pelvic anatomy (2,3).
As a consequence, establishing the diagnosis of endometriosis on the basis of symptoms alone may result difficult, not only due to their high degree of variation, but also because there is so much overlapping with other conditions such as irritable bowel syndrome or pelvic inflammatory disease. Therefore, it is common to come across a delay of several years between the onset of symptoms and a definitive diagnosis. In this respect, the revised scoring system of the American Society for Reproductive Medicine is normally employed to determine the disease stage (ranging from I, indicating minimal disease, to IV, indicating severe disease). This system is based on the type, location and appearance of the lesions, as well as the depth of the invasion caused and the extent of disease and adhesions (2,5,6).
However, endometriosis symptoms do not always correspond to observations from laparoscopic exploration. The severity of these symptoms increase with age, along with the probability of a proper diagnosis. Moreover, the incidence of endometriosis peaks in women in their 40s (7).
Which are the possible causes of the disease?
Even though the underlying cause for endometriosis remains uncertain, it is likely to present a multifactorial origin. This includes inflammation enhanced by genetic causes, which may be epigenetically regulated, as well as triggered by exposure to environmental factors (3). So, it seems evident why pathophysiology of endometriosis keeps being a subject of debate. It remains unclear whether endometrial and endometriotic cells are genetically different, or whether such differences are the consequence of distinct environments or due to preexisting immunological defects (8). However, several hypotheses have been proposed to explain the origin of the disease:
This was the first hypothesis proposed, put forth as the main cause for the pathogenesis of endometriosis. Retrograde menstruation appears when viable endometrial fragments are driven through the Fallopian tubes, possibly by a pressure gradient originating from dys-synergic uterine contractions. Once these fragments reach the peritoneal cavity they can implant, grow and invade pelvic structures. The likelihood of this event is influenced epidemiologically by any menstrual, reproductive or personal factor that may augment pelvic contamination by regurgitated endometrium (Figure 1). Further support for this aetiology is derived from studies of obstructed or compromised outflow tracts (9,10,11).
However, this event occurs in 76–90% of women with patent Fallopian tubes, and not all of them suffer from endometriosis (11).
The monthly regeneration of the endometrium after menstrual shedding and re-ephitelialization after parturition or surgical curettage supports the existence of a stem cell pool. The involvement of these cells in the formation of endometriotic deposits could be the result of abnormal translocation of the normal stratum basalis of the endometrium via retrograde menstruation. Some group of cells may deposit and survive in the peritoneal cavity after retrograde flow, and may reactivate during adolescence in response to ovarian hormones. However, there are currently no data on the amount of endometrial stem/progenitor cells in neonatal period compared to the adult endometrium (11).
This theory postulates that endometriosis originates from the metaplasia of specialised cells present in the mesothelial lining of the visceral and abdominal peritoneum. Hormonal or immunological factors are thought to stimulate the transformation of normal peritoneal tissue/cells into endometrium-like tissue. The theory may explain the occurrence of endometriosis in pre-pubertal girls. However, the usual driving force for endometrial growth, oestrogen, is not present in pre-pubertal girls, and therefore this condition may be different from the endometriosis found in women of reproductive age. Nevertheless, this theory is imperfect due to endometriotic lesions being found in areas outside the course of the Müllerian duct (4,11).
Genetic factors represent about half of the variation in endometriosis risk, with an estimate of heritability of 51%. Meta-analyses of the few genome-wide studies performed in the past few years have provided evidence of a robust association of endometriosis with seven risk loci. Among them, WNT4, CDKN2B-AS1 and GREB1 are of particular interest due to their gene-based ranking, known pathophysiology and proximity to SNPs of genome-wide significance. Therefore, these genes represent strong candidates for further studies on endometriosis (4,12,13).
Inflammation is an additional typical feature of endometriosis, since the presence of ectopic tissue in the peritoneal cavity is associated with overproduction of prostaglandins, cytokines and chemokines. Macrophages infiltrating the ectopic lesions express typical markers of alternative activation, favouring the growth of the lesions and promoting associated angiogenesis. Overproduction of reactive oxygen species (ROS) is also accompanied by a decreased level of antioxidants that usually eliminates these molecules. Thus, the resulting accumulation of ROS may contribute to the propagation and maintenance of endometriosis and associated symptoms (4,10,11).
Oestrogens fuel ectopic endometrium growth, and alterations in oestrogen signalling have been associated with the disease. In fact, it is known that oestradiol produced both in the steroidogenic organs and locally in the endometriotic implants through aromatase expression promote the growth of endometrial tissue ectopically (4,11). This ectopic tissue has been consistently shown to express different levels of oestrogen receptors (ER) α and β than eutopic tissue, being ERβ highly present in ectopic tissue (4). Deficient methylation of the promoter of the ERβ-encoding gene has been suggested to result in pathological overexpression of ERβ in endometriosis, which in turn suppresses ERα expression and diminishes oestradiol-mediated induction of the progesterone receptor in endometriotic cells. This mechanism is thought to contribute to resistance to selective actions of progesterone in these cells, which is manifested by perturbations in a number of downstream progesterone target genes (4,11).
Progesterone normally triggers a uterine endometrial response characterized by inhibition of oestrogen-dependent proliferation of epithelial cells, secretory maturation of the glands, and transformation of stromal cells into specialized decidual cells. Moreover, progesterone transiently induces a receptive phenotype in endometrial epithelial cells essential for embryo implantation [you can read more about the role of these hormones in the endometrium in our previous publication here].
As a consequence of progesterone resistance, genes critical to these events, such as prolactin for decidual response or glycodelin for embryo implantation, are dysregulated in the endometrium of affected women. Endometriosis-derived inflammation, in turn, could induce progesterone resistance by altering the signalling pathway of this hormone through mechanisms of competition or interference with proinflammatory transcriptional factors (4,11,14,15,16).
The observation that autoimmune diseases tend to be more common in women with endometriosis supports the hypothesis that pathogenesis of this condition may involve a defective immune response in these patients. Women suffering from endometriosis present higher concentration of activated macrophages, decreased cellular immunity and repressed NK cell function (11).
Endometriosis and infertility
The relationship between endometriosis and infertility has been debated for many years. Infertile women are 6-8 times more likely to suffer from endometriosis than fertile women (7). Despite extensive research no agreement has been reached, and several mechanisms have been proposed to explain the association between endometriosis and infertility. These mechanisms include distorted pelvic anatomy, endocrine and ovulatory abnormalities, altered peritoneal function and altered hormonal and cell-mediated functions in the endometrium. Major pelvic adhesions or peritubal adhesions disturbing the tube-ovarian liaison and tube patency can impair the release of oocytes from the ovary, inhibit oocyte pickup, or impede the transport of the ovum itself (7).
Women with endometriosis may exhibit endocrine and ovulatory disorders, including luteinized unruptured follicle (LUF) syndrome, impaired folliculogenesis, luteal phase defect, and premature or multiple LH surges. Moreover, a complex network of humoral and cellular immunity factors modulates the growth and inflammatory behaviour of ectopic endometrial implants, and so it may have adverse effects on the function of the oocyte, sperm, embryo, or Fallopian tubes (7,16).
Management of endometriosis by assisted reproductive technologies
There is currently no clear answer to the question of whether endometriosis has a negative impact on IVF outcomes. Several studies have previously suggested poorer outcomes in comparison to control cases, whereas other have shown no significant differences (17).
Clinical outcomes for different stages of endometriosis
Patients with endometriosis can present different levels of severity, which have a strong influence in the outcomes. Thus, it would be common to find a case of disease at a more severe phase with worse results than a different one presenting less extensive endometriosis (18,19).
In stage III/IV endometriosis the prognosis for IVF/ICSI treatments is lower compared to milder stages I/II. (19). In their retrospective study, Opøien et al. highlighted the fact that patients with a more severe form of the disease had fewer oocytes retrieved, despite the higher gonadotropin doses these women had been administered (20). This may be due to endometriosis affecting both oestrogen and progesterone mRNA receptors in granulosa cells (21). Likewise, patients suffering from stage III/IV endometriosis have been reported to show a decrease in implantation and clinical pregnancy rates (22).
Endocrinology of endometriosis
Measurements of circulating hormone concentrations have shown statistically higher levels of hMG/FSH in endometriosis patients than in control patients. However, both high-quality embryo and clinical pregnancy rates were found to be lower in such patients (23). Similarly, the high expression of PR-A and ER-α in patients with endometriosis might be a leading cause of ovarian dysfunction due to this condition. This means that stages III-IV patients required higher gonadotropin stimulation doses to prevent ovarian dysfunction (24).
Endometriomas represent an additional variable that may also affect ART outcomes. They have been reported to affect 17-44% of patients with endometriosis, who exhibit reduced ovarian response due to the low response to gonadotropin stimulation (25).
Such response has been widely discussed by different authors. Conclusions from these reports suggest a reduced number of retrieved oocytes in women with bilateral endometriomas, as well as the rates of top-quality embryos, implantation, clinical pregnancy and live birth, when compared to control groups (26,27,28).
Gonadotropin stimulation for IVF in the endometriosis context
Patients with stage III/IV endometriosis normally undergo stimulation by higher doses of gonadotropin, which consequently results in higher circulating oestradiol levels (24). D'Hooghe and coworkers performed an analysis of this kind of patients and demonstrated that the cumulative endometriosis recurrence rate (CERR) was lower after ovarian hyperstimulation for IVF than after lower-dose ovarian stimulation for IUI. This suggests that temporary exposure to high oestradiol levels during ovarian hyperstimulation for IVF is not a risk factor for endometriosis recurrence in women subjected to ART (29).
Previous reports from Benaglia et al. have shown that after 3-6 months of IVF cycles 77% of patients experimented an improvement of the condition, whereas about 11% reported worsening. Also, it should be noted that endometrioma size also remained stable after IVF cycles, which suggests they are not affected by gonadotropin stimulation (30).
IVF outcomes after medical therapy for endometriosis
Medical interventions have been demonstrated to have benefit in alleviating, if not eliminating, symptoms associated with endometriosis (31). The first thing to control is the prolonged use of GnRHa prior to initiation of gonadotropin stimulation for ART. The extension of GnRHa use up to about 3 months has been reported to exhibit higher implantation rates and significantly higher clinical pregnancy rates (32). This is due to the fact that endometriosis patients were more likely to present aberrant endometrial expression of β3 integrin, and that a 3-month course of GnRHa allowed to rescue up to 64% of the expression (33). Another approach employed to increase integrin expression is the of danazol as a post-treatment after the ovulatory cycle. This has been reported to result in both similar clinical pregnancy and live birth rates to those from patients with normal integrin expression (34).
A different medical therapy is the administration of oral contraceptives in women with endometriosis before the initiation of the actual ART treatment. This approach seems to improve clinical outcomes, keeping them comparable to women of similar age without endometriosis. In contrast, ART outcomes are markedly compromised in endometriosis patients who are not pretreated with this method. It is worth to mention that ovarian responsiveness to stimulation was not altered by 6-8 weeks use of oral contraceptives prior to initiation of ART treatment, including poor responders with endometriomas (35).
So far, it has not been possible to establish the optimal duration of medical therapy for endometriosis by means of comparative trials. As a consequence, ideal patients who could benefit from medical intervention have not yet been depicted. In spite of this, it would be reasonable to think that those patients suffering from more severe stages of the disease and/or with prior implantation failure might be the best candidates (36).
Infertility in women affected by endometriosis may be related to alterations in follicles, poor oocyte quality (...), or even decreased endometrial receptivity.
Impact of surgical management of endometriosis on IVF outcomes
Previous reports have shown that pre-cycle surgical intervention may result beneficial for the final outcome. Opøien et al. demonstrated that patients with stage I/II endometriosis treated with surgical resection presented higher clinical pregnancy rates than those who had only been subjected to diagnostic laparoscopy before IVF/ICSI (37). Similar data were obtained when evaluating results from a group of 825 endometriosis patients; those treated with surgical resection presented overall significantly higher pregnancy and IVF rates when compared to those who had been subjected to IVF alone, and also to those with no treatment whatsoever (38).
Implantation and pregnancy rates have also been reported to increase after resection in patients with deeply invasive endometriosis, although this group needed higher gonadotropin doses for stimulation, and yet fewer oocytes were retrieved (39). However, it is important to keep in mind that research is limited by a variety of factors, such as variations in surgical techniques (i.e., ablation vs. resection), degree of removal of the endometriotic tissue or differences in IVF laboratories (40). Interestingly, previous reports indicate that IVF implantation rates are not affected by the time intervals between surgical interventions for resection of endometriosis in the absence of endometriomas (41).
On the other hand, certain studies have reported the negative impact of surgical management of endometriomas on IVF treatments, like the inability to access follicles at oocyte retrieval following precycle resection of endometriomas, or the harmful effect on oocytes after exposure to endometrioma fluid. Nevertheless, most authors refuse these arguments (42), and few studies assert the benefits of surgery in endometriomas larger than 3 cm in order to treat painful symptoms or to facilitate access to the ovary (43).
Current data from clinical outcomes suggest that, under controlled circumstances, IVF cycles are not compromised by the presence of endometriosis. The exception to this finding is the fact that patients with ovarian endometriomas showed lower response to gonadotropin stimulation (17). Thorough meta-analyses through early studies revealed lower number of oocytes obtained after egg collection, as well as decreasing fertilization, implantation and pregnancy rates in patients affected by endometriosis after ART treatments (18). However, it is important to note that pregnancy rates were extremely low in those years.
Effects of endometriosis on ART outcomes
Since endometriosis patients have sometimes been studied simultaneously to other groups of patients such as women suffering from Fallopian tube-related infertility, it is not clear whether endometriosis actually affects implantation rates. Barcelo and coauthors’ explanation is the similar percentage of meiotic abnormalities in in vitro matured oocytes between endometriosis patients and the control group after ovarian stimulation (44). An additional factor that might contribute to confusion is the presence of adenomyosis, which is frequently found in endometriosis patients and could have a deleterious impact on the implantation process (45).
Altered ovulation and oocyte production, as well as increased inflammatory cells in the peritoneal fluid of endometriomas have been observed in endometriosis patients (46). In fact, infertility in women affected by endometriosis may be related to alterations on follicles, poor oocyte quality and related failures during subsequent embryogenesis, or even decreased endometrial receptivity. Such hypothesis is supported by the altered progesterone and cytokine concentrations found in the follicular fluid from these patients (44).
Embryos derived from affected women are likely to develop more slowly, compared to embryos derived from women suffering from tubal disease (45). It is worth to mention that women with moderate to severe endometriosis who receive oocytes from healthy donors seem to present normal endometrial receptivity and pregnancy rates. Conversely, when donor oocytes from endometriosis-affected women are transferred into healthy women, implantation rates are lower and embryo quality is reduced (46). Further studies are needed, though, in order to determine pregnancy rates from donors in different conditions and disease stages (16).
Delayed histological maturation or biochemical disturbances may lead to endometrial dysfunction, too. This is concluded from reports that show uterine implantation being affected by changes in receptivity on an endometriosis background (17). Endometrial expression of the adhesion molecule αvβ integrin has been observed to be reduced during the time of implantation in some endometriosis-affected women. Additionally, the synthesis of the endometrial ligand for L-selectin has also been observed to be affected in some patients (16). L-selectin is a trophoblast surface-coating protein, which may explain the altered implantation rates observed in such cases (47).
The observation of luteal phase disruption associated to endometriosis may be due to dysregulation of the progesterone receptor, as well as an effect on progesterone target genes that consequently results in a decrease in endometrial receptivity (17,47). Sperm quality and/or function is also reduced, which has been proposed to be due to inflammatory/toxic effects of the peritoneal fluid, along with a higher amount of activated macrophages. Not only are these effects harmful to oocytes and sperm, but toxic to the embryo (17).
Treatment of endometriosis-associated infertility
IVF is currently the most effective treatment for endometriosis-associated infertility (17). However, comparison of data on the effectiveness of IVF for endometriosis patients vs. patients suffering from other causes of infertility is not without controversy. Reports from the Society of Assisted Reproductive Technology (SART) have shown that the average delivery rate per retrieval for patients undergoing IVF-ET is 39.1% for endometriosis-affected women, as opposed to 33.2% for women with other causes of infertility (48).
Pre-treatment ovulation suppression is a possibility to be considered in order to enhance suppression of inflammatory cytokines, as well as to reduce the presence of disease signs prior to any form of ART. Nevertheless, further research will be required on patients with endometriomas to assess their effect on IVF/ICSI, and to elucidate whether pre-ART surgical intervention may increase success rates (48).
Potential treatments in the future
There is currently no consensus on whether performing surgery prior to undergoing ART is fundamental for achieving pregnancy. Either way, there is a priority in terms of age in endometriosis cases (17). There are, however, some novel medical therapies, such as immunoconjugate (ICON) and aromatase inhibitors. ICON targets aberrantly expressed tissue factor on endometriotic endothelium, causing regression of the established disease (most likely by devascularization), which seems to improve fertility rates (17,49). Aromatase inhibitors, absent under normal circumstances, are found to be present in ectopic endometrial tissue; this may have a direct impact on oestradiol levels and implantation rates in endometriosis patients (50,51).
Current treatment of endometriosis-associated infertility focuses on improving fecundity by removing or reducing ectopic endometrial implants, thus restoring normal pelvic anatomy (47). There are several possibilities that practitioners can adopt when facing this kind of events, depending on the particular case and the patient: expectant management, medical treatment and/or surgical treatment (48,52). The reality is that the optimal method of choice to treat endometriosis-associated infertility is an individualized decision that should be made on the basis of the specificity of the patient (18,50).
Despite lower ovarian response, reduced embryo quality and impaired implantation in moderate/severe cases, endometriosis patients have been able to obtain IVF/ICSI success rates similar to those with tubal factor-related infertility. As it has been widely explained, the combination of aggressive but controlled ovarian hyperstimulation, appropriate hypophysis suppression and efficient surgery before initiating cycles seemed to be crucial and significantly efficient for IVF/ICSI success on patients suffering from endometriosis (50,52). Within the last few years, scientific knowledge have made it possible to develop certain clinical improvements that have surely opened new possibilities for endometriosis patients.
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Authors: Iñaki Arroyos, Lidón Carretero Vilarroig & Belén Gómez Giménez
Cryopreservation is a basic procedure in the daily work with assisted reproduction techniques. It is routinely and successfully applied to sperm samples, spare embryos from in vitro fertilization (IVF) cycles and oocytes.
In the past, slow freezing procedures were carried out with no reliable clinical outcomes, especially in egg freezing programs. After the recent introduction of vitrification it is now possible to cryopreserve embryos and oocytes, and to assure patients that this process will not decrease the quality and the biological potential of the cells.
At present, there is a growing trend towards a “vitrify-all” strategy after IVF with a single embryo transfer of a warmed embryo in a subsequent cycle (1, 2). There have been reports of improved implantation and pregnancy rates with frozen embryo transfers as compared to fresh autologous embryo transfer, suggesting superior endometrial receptivity in the absence of ovarian stimulation (2, 3).
The mature oocyte is the biggest cell in the body and contains a high proportion of water. The amount of water compromises the viability of the ovocyte during the slow freezing because ice crystals can be formed. The spindle of the mature oocyte is very sensitive to damage from ice crystal formation, and so it may easily become disrupted. Also, the oocyte is extremely vulnerable to mild chilling, which may severely compromise its integrity, and even cause complete degeneration.
Innovation and improvement of vitrification was not easy in the beginning. It took a lot of research and experimentation on different protocols for the developers, as well as long time to set the right process. The procedure itself is complex, and it involves numerous critical steps for successful performance (4).
Egg freezing is an increasing phenomenon because of its many advantages. It may provide an easier solution in cases of legal and/or ethical issues between the parents, such as divorce or decease. Freezing eggs rather than embryos can also avoid dealing with the complexities of having an increasing number of frozen embryos in nitrogen tanks unclaimed by the progenitors. Moreover, certain medical treatments to battle cancer can jeopardize the ovarian function, and so an egg freezing program seems suitable for such patients.
Regarding social aspects, egg freezing allows women to postpone their motherhood. In addition, egg freezing also makes synchronization between donors and recipients much easier. Finally, this technique is suitable for patients who have low ovarian reserve and need PGD treatments: it allows patients to accumulate oocytes from several cycles, which increase the success of the treatments (5).
This technique offers the possibility of freezing sperm to preserve fertility in those men subjected to treatments such as chemotherapy or radiotherapy, which may compromise gametogenesis. Additionally, sperm cryopreservation makes it possible to create sperm banks, which offer a valuable alternative to azoospermic patients willing to father children.
Embryo freezing represents an essential method for any assisted reproduction centre, since it allows to save spare embryos from an IVF cycle and preserve them in liquid nitrogen for future uses.
Ovarian Tissue Cryopreservation
Ovarian tissue cryopreservation (OTC) represents another technique to preserve fertility in women. It has been carried out for more than 18 years now (6, 7) and it represents the main option available for women who require cancer treatment but cannot delay chemotherapy. Moreover, it is the only possibility for pre-pubertal patients (8).
Currently, embryo and oocyte cryopreservation are the only fertility preservation techniques that are considered to be non-experimental by the American Society for Reproductive Medicine (9). Unfortunately, both approaches require previous preparation and stimulation with gonadotropins for oocyte retrieval, which usually requires 2-4 weeks or longer (10). Furthermore, oocyte harvest typically involves the use of transvaginal ultrasound and needle oocyte retrieval techniques, which could require general anaesthesia. This delay is often not possible or appropriate for females requiring urgent therapy or in breast cancer patients, because high estrogen levels might have detrimental effects on the primary tumor. Additionally, not all patients have partners with whom to create embryos for cryopreservation (10). Most clearly, these techniques are not indicated for young and pre-pubertal female patients due to the inability of their immature hypothalamic-pituitary axis to produce mature eggs (11)
Under these circumstances, the possibility of cryopreservation of ovarian tissue (cortex) has become an urgent and highly-demanded technology for two types of young female cancer patients. First, those who must undergo advanced chemotherapy and/or radiotherapy. Second, those with non-oncological systemic diseases such as autoimmune or haematological conditions, that sometimes require chemotherapy, radiotherapy, or bone marrow transplantation (12).
OTC does not require ovarian stimulation, and it allows to preserve gonadal function in pre-pubertal and adult patients. This procedure offers a promising option for women at high risk of premature ovarian failure and sterility (12). Furthermore, transplanting ovarian tissue not only restores fertility but also restores endocrine function (13).
Ovarian cortical tissue contains the primordial follicles, which are located in the ovarian cortex, near the surface epithelium. These are the smallest female fertility unit, including 90% of the ovarian follicular reservoir. Although there are other types of follicles present in ovaries removed from the patient, primordial follicles are the ones that are considered for ovarian cryopreservation. Among other reasons, these are the most resistant follicles to cryoinjury due to of the small size of the oocytes, the reduced cytoplasmic content and the absence of meiotic spindle that could potentially be damaged (14).
OVARIAN CORTEX CRYOPRESERVATION TECHNIQUE
The most common steps to be followed for this approach are summarized below:
1. Biopsy. The removal of ovarian tissue is a relatively simple procedure. In order to ensure optimal penetration of cryoprotectants, approximately twelve small biopsies of ovarian cortical tissue (3x3x1 mm) are collected (15). This procedure can be performed by using minimally invasive techniques like laparoscopy, or even in conjunction with other open procedures such as primary tumor resection. Regardless of the technique, any area away from the hilum and lacking predominant follicles and luteal tissue is preferred for biopsy. The goal is to maximize the number of primordial follicles per specimen without compromising vascular supply to the remaining ovarian tissue. In order to optimise results, it is best to remove the ovarian cortex from the medulla, which helps extreme penetration of cryoprotectants into the cortical tissue (14). Normally, extraction of a single block of cortical tissue through partial excision technique can be performed in either one or both ovaries (11).
2. Histological examination. Ovarian fragments are immediately observed by a pathologist to simultaneously confirm the presence of follicles and the absence of a massive cancer infiltration (16).
3. Cryopreservation. Ovarian tissue can be cryopreserved independently of the menstrual phase, which represents no delay for the oncological therapy. Centres offering OTC allow for the procedure to be performed even the first day after the patient's first visit (17), and it can be carried out using one of two established techniques: slow freezing or rapid freezing (vitrification) (Fig. 3). The slow freezing method has already resulted in dozens of live births worldwide, whereas vitrified tissue has only led to a few reported live births to date (18, 19).
The well-established standard method for human OTC is slow equilibrium freezing. It begins with exposing cells to low concentrations of one of the cryoprotective agents (CPAs) (propanediol, dimethyl sulfoxide or ethylene glycol) in combination with human serum albumin, and temperature must be slowly decreased. In contrast, vitrification requires higher concentrations of CPAs, which reduces the risk of ice nucleation, and it is significantly faster. When the solution is rapidly cooled, the tissue turns into a glassy, vitrified state, avoiding extracellular and intracellular ice crystallization (19). You can read our previous article to know more about these techniques here. The current main problem of ovarian banking is to restore ovarian function and fertility using frozen/thawed tissue with both high efficiency and minimal risks (16).
4. Biopsies are stored in the vapour phase of liquid nitrogen at -176 °C, thus allowing for long-term storage of potentially large numbers of primordial follicles (15).
5. Re-implantation. The stored ovarian tissue can be re-implanted orthotopically (into the remaining ovary, ovarian fossa, or broad ligament), heterotopically (into the subcutaneous space of the forearm, subcutaneous tissue of the abdomen, anterior wall of the abdomen, just beneath the peritoneum, or in the rectus muscle), or as recent studies have reported, xenografted or matured entirely in vitro (15, 19).
Orthotopic transplantation may provide the possibility to achieve natural pregnancy; however, it requires abdominal surgery with general anaesthesia. In contrast, heterotopic is advantageous in cases of severe pelvic adhesions, distorted pelvic anatomy, and poor pelvic vasculature due to previous irradiation. Furthermore, it implies the possibility of creating long-term ovarian endocrine function with a less invasive surgical approach for transplantation, which does not require general anaesthesia, and thereby creating a more cost-effective option. However, this option may produce oocytes (and therefore, embryos) with reduced quality, as compared to orthotopic transplantation sites. This outcome is likely related to the suboptimal environment of heterotopic sites in regard to local factors. These include: external pressure, temperature, vascularization (decreased blood supply), oxygen tension and reduced paracrine factors, which could affect the possibility of sustaining the growth and development of normal follicles and oocytes (16). Thawed ovarian fragments do not need vascular anastomosis when transplanted into a well-vascularized tissue. Genes for angiogenesis factors seem to be upregulated in the ovary, compared to other tissues (16). However, there is a significant risk of the tissue suffering from hypoxia until neovascularization starts, normally following the first 48 hours after the procedure. Survival of primordial follicles (around 90% after thawing) ranges between 5 and 50% after grafting, the most crucial factor being the degree of ischaemic injury after transplantation (16). Although restoration of ovarian function has been reported for both approaches, live births following bilateral oophorectomy have only been documented from orthotopic transplantations (19).
Successful studies in experimental animals with live births following transplantation of cryostored ovarian tissue have been previously reported in rodents and sheep (20, 21, 22, 23). Ovarian tissue cryopreservation was first described by Hovatta et al in 1996 (6). The first human ovarian transplantation with cryopreserved ovarian tissue was later performed by Oktay in 2000 (24). Donnez and colleagues reported the first human live birth from orthotropic transplantation of frozen human ovarian tissue in 2004 (25), with another successful live birth achieved by Meirow in 2005 (26). Live births following cryopreservation of pre-pubertal ovarian tissue had not been described until the 2015 case report by Demeestere et al (27).
During all these years, authors have been discussing about whether vitrification is better than slow freezing. Some of them found apoptotic cells in vitrified ovarian tissue, meanwhile others showed higher primordial follicle density using the slow freezing method. Sanfilippo et al found no significant differences between both techniques (28).
The American Society of Clinical Oncology advises that OTC for fertility preservation is still considered an experimental technique (9). Consequently, it should only be performed in centres with the appropriate expertise. This means performance under the Institutional Review Board (IRB) approved protocols, which include follow-up for recurrent cancer (29). However, in some clinical situations it remains the only available option. Here we describe some reported cases in different scenarios: fertility wishes and puberty induction, autotransplantation and allotransplantation.
OTC is the only available option for fertility preservation before menarche. Demesteree et al (2015) reported a spontaneous pregnancy case of a woman who had undergone ovarian tissue transplantation shortly before turning 14 (27). She had been diagnosed with sickle-cell anaemia at the age of 5 and treated with hematopoietic stem cell transplantation at 11. Her parents and herself were offered OTC in order to preserve fertility. The patient started puberty at the age of 10 (breast development) and the procedure was performed when she was 13 years and 11 months. At the age of 25 she interrupted hormonal supplementation and underwent ovarian tissue transplantation. Two years later she got pregnant and gave birth to a healthy boy in November 2014.
Andersen 2012 et al described a case of ovarian tissue allotransplantation between identical twins at the age of 38 (30). One of them had undergone menopause at the age of 22, whereas the other had already had 2 children. The woman who was transferred the ovarian tissue had three natural pregnancies: eight months after transplantation, at age 42 and at 45. She gave birth to a healthy girl and two healthy boys.
A.K. Jensen et al recommend OTC to all young girls who present a high risk of developing ovarian insufficiency and/or infertility following high-dose chemotherapy and/or irradiation (31). The ovary serves a double function: folliculogenesis and production of sex hormones, which play a main role in the woman’s body such as the development of secondary sexual characteristics. Ernst et al (2013) described a case of puberty induction in a girl treated with chemo- and radiotherapy at the age of 9 (32). Due to the treatment, her ovary had been damaged, leading to a pre-pubertal stage with postmenopausal levels of FSH. Four and a half years after OTC, tissue was grafted and she recovered normal levels of FSH and oestradiol, which let the ovaries reach the pubertal stage.
Clinical effectiveness and outcomes
Since the lifespan of a piece of transferred ovary varies from a few to several years (33), hormone levels can turn to those corresponding to a pre-pubertal stage some months after transplantation. The effectiveness of this technique also depends on the woman’s own ovarian reserve, which is linked to the age at which her ovary tissue was cryopreserved.
In all cases reported in which pregnancy has been achieved, patients had only cryopreserved tissue of one of their ovaries, whereas the other one had been kept during the chemotherapy treatment. Thus, it has not been possible to determine whether pregnancy was a product of the cryopreserved and transferred tissue or to the remaining intact ovary. On the other hand, cases have been described in which infertility due to hormone failure could be restored by ovarian tissue allotransplantation, thus leading to healthy pregnancies (34).
Even though numerous successful births have been reported after OTC, re-implantation of malignant cells (potentially causing metastatic processes), along with grafted ovarian tissue in a subject previously treated for the same cancer, still remains a serious concern.
- It is well known that treatments that expose patients to gonadotoxic therapies such as alkylating agents, pelvic irradiation, and/or stem cell transplant could increase the risk for post-therapy infertility, because the ovaries are very sensitive to these cytotoxic drugs.
- Although currently OTC still remains an experimental technique, there has been an increasing amount of successfully case reports since 1996.
- OTC remains an alternative and the only suitable option to preserve fertility in some special cases such as young female cancer patients.
- It is necessary to make efforts in order to develop the best protocol to preserve ovarian tissue, as well as to study some unclear aspects such as the possibility of reintroducing the original disease (no cases reported, through), or to try to improve the survival of primordial follicles after grafting.
- Finally, it would be recommended to follow up individuals from live births after OTC in order to analyze any abnormal findings.
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The endometrium during the window of implantation: endocrine, immunological and morphological changes
Decidualization is the combination of changes on endometrium structure and hormone profiles, along with gene expression, cell differentiation and tissue modification, that allow for implantation of the embryo.
Authors: Inés Abad, Roberto de la Fuente and Sara Sanz
The menstrual cycle in humans lasts 28 days in regular terms, being the first day of menses considered the first day of the cycle. But in actuality, cycle refers to a series of intermingled events mainly related to three aspects: the ovary, the uterus (or specifically the endometrium) and the hormones participating in the process. As a consequence, one may refer to either the follicular or the luteal phase regarding to the ovary, the proliferative or the secretory phase of the endometrium or even to the hormone with highest blood levels. Any of them refers to a specific moment of the cycle (Fig. 2).
The window of implantation (WOI) is the term used for that phase in which the uterus is ready to receive the conceptus, around day 9 after ovulation (depending on when fertilization actually occurs). In an ideal 28-day cycle, the window of implantation is considered to start at day 20 and to last for about 5 days (3). During this interval, there is a series of physiological changes in the woman (see below) aimed to facilitate the implantation of the embryo and its survival and development in the endometrial microenvironment. Because many cases of infertility have been associated with a reduced receptivity of the endometrium, a proper WOI is currently considered a fertility-determining factor (4, 5).
Hormone fluctuation within the cycle
The entire menstrual cycle is controlled by levels of different hormones, some of the most important being the gonadotropins. These hormones are glycoproteins secreted by the anterior pituitary through GnRH (gonadotropin-releasing hormone) stimulation, which is strictly controlled by the hypothalamus (reviewed in (6)). The main gonadotropins involved in regulation of the menstrual cycle are FSH (follicle-stimulating hormone) and LH (luteinizing hormone).
The beginning of the cycle is regarded as the beginning of the follicular phase of the ovary and the proliferative phase of the endometrium. During this phase, cohorts of follicles at different stages progress and develop due to an increase in FSH levels. The largest follicles, called Graafian follicles (~18mm), present granulosa cells that are able to produce not only oestrogen, like early smaller follicles, but also progesterone (7). The granulosa cells are the cells that conform the follicle structure, nourishing and providing support to the oocyte. Whereas progesterone levels are relatively low and constant during the first half of the cycle, there is an increment in the blood levels of oestrogen (mainly oestradiol 17β) due to its production from the follicles. However, during the last days before ovulation, largest follicles also acquire the ability to produce progesterone, and so its levels increase significantly at that time. Oestradiol secretion creates a positive feedback that stimulates LH production; but increased levels of LH are also the result of progesterone-mediated regulation (8).
Follicle growth and development means that granulosa cells not only respond to FSH-stimulation, but also to LH, as well. As a consequence, levels of oestradiol that had been increasing from ~day 5 become so high that at the preovulatory phase they trigger a rapid and highly significant release of LH from the pituitary (LH surge) (9). The LH surge causes a negative feedback on the steroidogenic pathway from the ovary, and so there is a switch from the production of oestrogen to the production of progesterone (10) (Fig. 3).
The LH surge coincides with ovulation (~day 14) and promotes the final stages of egg maturation to occur. At this moment, the huge amount of cells left at the follicle after ovulation form the so-called corpus luteum (CL), highly active in the production of progesterone, mainly from the granulosa cells. Oestrogen production, in turn, is back to low levels although maintained relatively constant.
The rapid increase in progesterone produced by the CL has another consequence: a negative feedback that causes release of LH to cease due to inhibition of translation of the mRNA into protein, and also suppression of the growth of antral follicles due to the depression of FSH production. By the end of this phase, in the absence of conception, steroid levels are reduced at luteolysis. This results in the relaxation of the negative feedback originated by progesterone, and the restoration of positive feedback that leads to the production of oestrogens, and to FSH and LH levels reaching a plateau. As a consequence, preantral follicle development is resumed along with the next cycle (7).
Fig 3. Profile of the main hormones involved in the menstrual cycle, with details on the different phases. Pulsatile stimulation of GnRH is seen, triggering release of LH and FSH (green profile lines). The overall levels of steroids, E2 and P, are depicted as purple and yellow lines, respectively. This image shows the clear predominance of E2 (oestrogen) in the proliferative/follicular phase vs. the main action of P (progesterone) in the secretory/luteal phase. Modified from (7).
The endocrine basis of implantation
It has been mentioned earlier that oestrogen (E2) is a key player during the proliferative phase of the endometrium. Besides its role in promoting stromal cell proliferation, oestrogen also induces the expression of the progesterone receptor (PR) (11), which will be of utmost importance during the secretory phase.
The invasion of the endometrium by the blastocyst entails a rupture of the luminal epithelium in order to achieve implantation. For this to occur, it is necessary a series of changes in the uterine structure, as well as in the profile of several transcription factors and hormones. This way the mother adapts to the progression of the conceptus and, at the same time, controls its invasion. The development of all these changes is a process known as decidualization, which in humans starts by the mid-luteal phase, ahead of potential implantation (12).
The significantly increased production of progesterone by the CL makes it the main character of the second half of the menstrual cycle. The action of progesterone has different simultaneous effects: it reduces proliferation of epithelial cells (regulated by oestrogen) while inducing their differentiation (13), and it promotes stromal cells proliferation, stimulating the glandular secretory activity. There is also a potential increase in the action of specific immune cell types (like uNKs, see below) to respond to local production of chemokines (14). Additionally, it is known that progesterone regulates the contractile activity of the junctional zone (JZ), so that it allows the conceptus to develop in a safe and controlled fashion underneath the myometrium (15).
Progesterone acts partially by binding to its type A receptor (PR A) (16, 17), which in turn downregulates type B receptor (PR B), both being isoforms of the same transcriptional factor (18). The combined action of the two receptors in both stromal and glandular epithelial cells results in a modulated decidualization-like response of the endometrium in preparation for implantation. However, progesterone is not the only inducer of decidualization; in vitro progesterone only-induced differentiation of stromal cells would take up to 8-10 days under normal circumstances (19, 20). Rather, it is actually the cAMP pathway activation the main process that initiates decidualization (21, 22). In fact, many of the factors produced and secreted after ovulation like prostaglandins or gonadotropins, among others, stimulate cAMP in the stromal cells of the endometrium (22). Interestingly, cAMP levels detected from biopsies during the secretory phase are higher than during the proliferative phase (23, 24). After stimulation of cAMP, during the first stages of pregnancy, progesterone stimulates the expression of a group of interrelated genes in the endometrium, preparing the uterus for the implantation (25).
Hormone profile at the beginning of gestation
It has been exposed that during the luteal phase, the key acting hormone is progesterone, produced by the CL, the remnants of the Graafian follicle after ovulation. So, by the time the oocyte reaches the uterus, the endometrium is already prepared for implantation. However, if fertilization has not occurred, decidualization results useless and the physiological and endocrine profiles must be reversed.
Thus, the transition from luteal to follicular phase starts around 2-3 days before the end of the menstrual cycle, with the so-called intercycle rise of FSH. This characteristic increase in FSH or FSH window is responsible for the development and eventual selection of one of the follicles in the first half of the cycle. However, after the rise of FSH, oestradiol (and inhibin B) concentration increases again, inhibiting FSH secretion, and so FSH levels are maintained (26).
In humans, fertilization usually takes place in the distal third of the Fallopian tube. From there, the fertilized oocyte (now zygote) travels along the oviduct while undergoing a series of rapid divisions (cleavage), until it reaches the blastocyst stage. The blastocyst must now hatch from the hardened zona pellucida to be able to implant in the endometrium, a process that normally takes place between day 8 and 10 after ovulation (27).
Not only maternal hormones regulate the process of decidualization, but the embryo itself also contributes to preparing the right microenvironment for implantation. As early as the 8-cell stage, the embryo produces hCG (human chorionic gonadotropin) (28, 29), its first active regulator of implantation. From here on, hCG secretion by the syncytiotrophoblast increases progressively after implantation, reaching detectable levels in maternal blood by day 10 of gestation (which can be normally used for pregnancy tests) and peaking around the 10th week (30).
hCG has been known for decades and its current use is quite extended in reproductive medicine. In fact, it is employed to stimulate final follicle maturation before oocyte collection for IVF cycles (31), which along the external administration of progesterone for luteal phase support, helps to avoid potential problems of ovarian hyperstimulation syndrome (OHSS) derived from the use of LH. After fertilization, embryonic/placental production of hCG seems to be related with maintaining the role of the CL in secreting progesterone to sustain pregnancy (32). The need to rescue the CL from luteolysis is related to the luteoplacental shift, which occurs around the 6th week of gestation (33). This transition coincides with the decline in both progesterone and hCG serum levels detected following the 4th week and recovery thereafter (34). A crucial balance of hCG and progesterone must be reached between the CL and the syncytiotrophoblast to avoid luteal insufficiency, one of the main reasons for implantation failure during the first trimester (35).
IMMUNOLOGICAL CHANGES DURING THE WOI
The proliferative phase of the human endometrial cycle is characterized by the active growth of different cell types, including stromal, epithelial and vascular cells (36). On the other hand, the secretory phase supposes an increase of the glandular secretory activity and a significant increase in the number of immune cells. The major cell types in this phase are T cells, uterine NK (uNK) cells and macrophages (37).
In the secretory phase, the endometrium is prepared for nidation of a conceptus, so it undergoes a series of changes during which the immune system plays an important role. Mainly, the aim of this role is to create an environment of maternal tolerance towards the conceptus, and at the same time protection against infections within the uterus (37).
Immunological changes from proliferative to secretory phase
T cells, essential components of the immune system and originally formed from stem cells in the bone marrow, are known to help protect the body from infections. They comprise around 45% of leukocytes in the proliferative endometrium and their number stays constant throughout the secretory phase (38). However, their relative number decreases because of a large increase in uNK cells during this phase and early pregnancy (37).
During implantation, uNK cells comprise 70-80% of the total leukocyte population (39). The NK cells are a type of leukocyte or white blood cell that acts in the immune system as a first line of defense against foreign invaders. They are cytotoxic cells (40), whose function in the endometrium is to act as a source of important cytokines, including leukemia inhibitory factor (LIF), tumor necrosis factor (TNF)-α, interferon (IFN)-γ, granulocyte macrophage-colony stimulating factor (GM-CSF), and IL-10 (41). Cytokines are proteins that regulate the function of the cells that originate them on other cell types. They are primarily produced by activated lymphocytes and macrophages, and they are the agents responsible for intercellular communication, inducing the activation of specific membrane receptors, cell proliferation and differentiation, chemotaxis, growth and modulation of immunoglobulin secretion (40).
Gene expression in the WOI
Several genes related to the immune response are specifically regulated during the WOI. Worth to mention, among others: decay accelerating factor, indoleamine 2, 3 dioxygenase (IDO), interleukin (IL) 15, IL-15Rα, interferon regulatory factor (IRF)-1, lymphotactin, natural killer-associated transcript-2 (NKAT2) and granulysin NKG5. Some of these genes' functions are: to promote uNK cell proliferation, chemotaxis, to inhibit NK cell cytolytic activity, to inhibit cell growth and to inhibit the classical complement pathway. The functional diversity these genes represent likely serves the goal of preparing the endometrium for implantation, thereby adapting the maternal system to accommodate the immunologically ‘foreign’ fetus (42).
Lobo et al. demonstrated by semi-quantitative RT-PCR (Fig. 4) and Northern analysis the upregulation of these genes in secretory versus proliferative phase (37). The mRNA of all these genes, while expressed in proliferative endometrium, demonstrated increased expression in the secretory phase. However, and taking into account the fact that uNK cells increase in number during the secretory phase, it is likely that the apparent upregulation of the above-mentioned genes is actually reflective of an increase in cell number rather than a true increase in gene expression (37).
Fig.4. Graph bar showing semi-quantitative analysis of the expression of several genes, comparing proliferative (light bars) and secretory (dark bars) human endometrium. Gene expression analyzed by RT-PCR is normalized to GAPDH. There is a significant upregulation of all genes shown in the secretory phase (*P < 0.05, **P < 0.01). Modified from Lobo et al. (37).
Genes that regulate an increase in uNK cell number
IL-15 is a cytokine localized in the endometrium during the secretory phase of the human cycle. Its main function is to induce proliferation of uNK cells (43, 46). Some studies have demonstrated that IL-15 expression is upregulated in secretory versus proliferative phase (37, 44, 45).
Additionally, Lobo et al. demonstrated the upregulation of IL-15Rα subunit from proliferative to secretory endometrium. This subunit belongs to the receptor of IL-15, and it confers high-affinity binding of cytokine to its receptor (37).
IRF-1 is a transcription factor that regulates expression of IL-15 (45). IRF-1 expression has also been shown to increase during the secretory phase of the endometrium (37, 47).
Genes that inhibit NK cell cytolytic activity
Natural killer cells are classically considered to be cytolytic to non-self cells. Therefore, one would expect a decrease in their numbers from implantation through pregnancy to allow the fetus to be safely carried to term. Instead, there is a dramatic increase in NK cells during implantation. The answer to this paradox lies in the fact that uNK cells have effects that extend beyond their killer function, and that cytokines secreted by uNK cells are essential for implantation. In fact, NK cell-deﬁcient mice exhibit decidual cell degeneration and endothelial cell distortion or displacement from supporting cells (48).
It is believed that, at the maternal-fetal interface, human leukocyte antigen (HLA) class I molecules expressed on trophoblast cells interact with inhibitory receptors on uNK cells to protect the trophoblast from being cytolysed by uNK cells. An example of one of these inhibitory receptors is NKAT2, which shows an upregulated expression in the secretory phase (37).
Genes that inhibit cell growth
The uterine epithelium provides a physical barrier to infection. One mechanism for inhibiting such infections is through the secretion of endometrial peptides with bactericidal properties, which becomes especially evident at the beginning of implantation (49).
NKG5 is an isoform of granulysin that exhibits antimicrobial activity against a huge spectrum of pathogens (50). The expression of this gene is especially high in NK cells surrounding the glandular epithelium in secretory endometrium (37).
IDO is another example of genes that inhibit cell growth in the endometrium. It is an enzyme from the tryptophan catabolic pathway that depletes tryptophan in local tissue environments, thus suppressing proliferation of cells in the vicinity. IDO may not only inhibit T-cell proliferation, but it could also be bactericidal by this mechanism (51). An upregulation of this gene in secretory versus proliferative endometrium has been demonstrated (38).
MORPHOLOGICAL CHANGES DURING IMPLANTATION WINDOW
In the evaluation of women fertility, morphological studies are one of the most common procedures. These studies began with Noyes’ work, which describes the particular features of the endometrial histology, correlating it with the specific days of the menstrual cycle (52). In fact, these studies are still important and also improved and complemented thanks to the recent advances in technology.
Endometrial thickness and pattern
At present, there exists a data conflict between the various studies focused on endometrial thickness measurements by ultrasonography. Several papers agree in that, during an IVF cycle, the endometrium should be ≥ 7mm on the day of human chorionic gonadotropin (hCG) administration and ≥ 8mm thick on the embryo transfer day. Likewise, thickness < 6 mm has been reported to adversely affect implantation rate (53). Nevertheless, controversy arose when some studies documented no association between implantation rates and endometrial thickness. These articles claim that the important factor is the endometrial pattern, which changes throughout the entire menstrual cycle (54).
Regarding endometrial pattern, the structure of a triple line is correlated with a receptive endometrium. Ultrasonography as a tool to study the endometrial pattern has a high sensitivity (79-100%), but also an elevated percentage of false positives (57-91%) (53). However, it is possible to achieve pregnancy with a “non triple-line” pattern, although at a low frequency (55).
In the receptive endometrium, a complex secretory environment is established in order to active the blastocyst and to create both proper uterine receptivity and stromal decidualization. The structures in charge of creating this environment are the uterine glands.
Once ovulation takes place, P4 levels increase and the endometrium switches into a secretory phase. The endometrium is divided into two layers, the upper one formed by loose stromal and glandular tissue in its majority, with dynamic structure and function during the cycle. On the contrary, the lower layer is structurally stable and it is formed by spiral glands and a dense stroma. During the time in which the endometrium is receptive, glycogen vesicles are disposed below the nucleus of the glandular epithelial cells, and their content is transported by microfilaments to the apical region, where glycogen is actively secreted (56).
Uterine blood flow
During endometrial receptivity the arterioles acquire spiral form, and there is also a similar growth of the subepithelial capillary plexus, both at basal and functional endometrium. Regarding spiral arteries, it has been reported that when glomerular capillary length reaches a certain limit, a new capillary loop is generated in order not to increase the resistance to blood flow. Additionally, during the early and mid secretory phase the vessel length, which has achieved its maximum density during the proliferative phase, experiments an increase in the number of vessel junctions (57).
Recent studies have found that the existence of correct blood flow in the border area between the endometrium and myometrium plays an important role in endometrial receptivity. Uterine artery blood flow can be studied through the impedance, a term that includes the pulsatility index (PI) and the resistance index (RI) (58). Several research lines suggest that high blood flow resistance is associated with pregnant failures, whereas lower PI is a good indicator for pregnancy (Fig. 5).
Applebaum et al (59). define the vascularity zones as follows:
It has been shown that pregnancy rates increase if the vessels reach the zone of the endometrium and the subendometrial halo. This fact demonstrates the relation between endometrial perfusion and endometrial growth, since the endometrium becomes thicker because of the presence of vessels. On the contrary, the absence of blood in the endometrial and subendometrial areas correlates with low probability of pregnancy, in which case uterine resistance is higher (60).
Ultrasonography is also used in the study of uterine blood flow due to its non-invasiveness, real-time monitoring and repeatability. hCG administration day is normally the day for ultrasonography to be performed, because it is at this moment when highest sensitivity and specificity are achieved, although it can also be performed the day of embryo transfer (60).
Uterine junctional zone
The JZ is not only a distinct structure from the myometrium and endometrium, but it also presents functional differences (Fig. 6). There is supporting evidence for an important role of the JZ in the implantation process. On one hand, the movements of this layer allow the sperm to move rapidly to the dominant ovarian follicle. On the other hand, these contractions must decrease once fertilization has occurred. Otherwise, implantation might result impaired, reason why it is better to perform the embryo transfer during another cycle or, at least, at day 5-6 (58,61).
Human endometrial transcriptomics
Although morphological evaluation is very useful, more advantages are needed in order to study the window of implantation. Actually, transcriptomics can be used to analyze the expression of certain genes involved in the implantation process (62). Nowadays, two existing products that can be found in the market for studying receptivity using transcriptomics are “ER Map” and “Test ERA”. These products can be used in those cases in which the uterus is apparently normal, the endometrium presents the proper thickness and yet recurrent implantation failure occurs (3 or more in case of young women and 2 or more in elderly women), especially if the embryos transferred were of good quality (63,64).
The high versatility of the endometrial structure responds to the function of housing the conceptus for implantation at the right time of the cycle. It is essential that all the endometrial features adapt to the needs of both the embryo and the mother. In order to cover its nutritional requirements and, at the same time, to regulate this extraordinary changes of the endometrium, blood supply must be increased, and so there is a significant process of angiogenesis that involves restructure of the endometrium itself.
The combination of all changes needed to support early embryo implantation and further development is known as decidualization. This is triggered and controlled mainly by the action of a series of hormones, mainly progesterone from the mother and hCG from the conceptus. Production and dynamics of both hormones are in turn controlled and regulated by a network of molecular interactions, involving hormones, enzymes and other factors acting in delicate balance. This allows for adaptation to the actual and potential situations, with the goal of being always prepared during the WOI, should conception happen.
The menstrual cycle exhibits extreme complexity, given the high amount of factors involved in its regulation. Even though the aspects described in the present text (hormone profiles, gene expression and physiological transformation) have been studied for decades, further research is needed to fully understand the roles of other molecular factors. Unravelling complete interaction networks of these players may uncover potential roles in implantation, thus helping find new solutions to diverse causes for infertility.
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