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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54- Zhang JQ, Li XL, Peng Y, Guo X, Heng BC, Tong GQ. Reduction in exposure of human embryos outside the incubator enhances embryo quality and blastulation rate. Reprod Biomed Online. 2010;20(4):510-5.
55- Zaninovic N, Irani M, Meseguer M. Assessment of embryo morphology and developmental dynamics by time-lapse microscopy: is there a relation to implantation and ploidy? Fertil Steril. 2017;108(5):722-9.
56- Valbuena D, Valdes CT, Simon C. Introduction: Endometrial function: facts, urban legends, and an eye to the future. Fertil Steril. 2017;108(1):4-8.
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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3- Roque M, Lattes K, Serra S, et al. Fresh embryo transfers versus frozen embryo transfer in in vitro fertilization cycles: A systematic review and meta-analysis. Fertil Steril 2013;99:156-162.
4- Silber S. Foreword. In: Michael J. Tucker and Juergen Liebermann. Vitrification in Assisted Reproduction.Second ed. Boca Raton FL:CRC Press; 2016
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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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Authors: Javier del Río, Noemi Díaz and Belén Gómez
What is cryopreservation?
The first successful in vitro fertilization (IVF) treatment was in 1978. Since that, there have been a remarkable number of advances in assisted reproductive technologies (ART). Initially, all available embryos were transferred in IVF treatments owing to its low success rate. However, improvements on clinical and laboratory aspects led not only to increased pregnancy rates, but also to increased risk of multiple pregnancies. To prevent this, fewer embryos are transferred and leftover embryos are cryopreserved for potential future cycles use (1).
The first pregnancy resulting from transferring a thawed cryopreserved human embryo was reported in 1983 in Australia (2), and the first live birth following embryo cryopreservation was reported in 1984 in The Netherlands (3). Subsequently, the need for an effective cryopreservation program arose from rapid development and improvements of assisted reproductive technology protocols (1).
Cryopreservation is a method that requires cells and embryos to be exposed to non-physiological ultra-low temperatures (from -20°C to -196°C) (Fig.2). It aims to achieve “cryogenic suspension of life” through multiple steps, although this puts the elements at risk of damage or “cryoinjury” during temperature changes and phase transitions. These damages could be chilling injury or ice crystal formation, for instance, as a result of the water exchange between the intra- and extracellular compartments, consequence of dramatic changes in osmotic potential (osmotic shock). Therefore, vitrification requires the use of cryoprotectants to avoid the formation of ice crystals in the cells. Two types of cryoprotectants are necessary: permeating and non-permeating. Mixing both at different relative concentrations reduces intracellular ice formation by removing water from inside the cell. Additionally, it creates an osmotic gradient that helps restrict water movement across the cell membrane, thereby preventing osmotic shock (4).
There are two typical methods used for cryopreservation: slow freezing and rapid freezing to achieve vitrification.
Vitrification is a term used to describe the transformation of a solution into glass by a dramatic increase in viscosity. This method requires to minimize the time for the sample to be exposed to temperature ranges associated with chilling injury and ice crystal formation. As slow freezing, vitrification causes cell dehydration using cryoprotectants. However, unlike that, there is no attempt to maintain equilibrium on both sides of cell membrane (4).
The time frame required to reach ultralow temperatures by vitrification is very brief, almost instantaneous. But, the main concern is the need for using high concentrations of cryoprotectant solutions. These might lead to osmotic shock and it could be toxic to cells, affecting embryo survival. Nevertheless, it is possible to limit toxicity by mixing different cryoprotectants, thereby decreasing their relative concentration and the exposure time of embryos to the solution (5).
How efficient is the vitrification?
This technique seems to be more attractive than slow freezing because it does not require expensive equipment. It uses small amount of liquid nitrogen and it is a simpler technique to perform once the embryologist has gained enough experience in it (6).
A recent research performed by Viladimoiv et al. suggests advantages arising from the freezing and thawing process; the authors hypothesize a theory about “cryo-treatment of the embryo”. According to these authors, as a result of freezing or thawing of the embryos there is a decrease in reactive oxygen species levels, in the rate of mitochondrial DNA mutation and cells detoxification is carried out. Also, the authors describe another mechanism involved in restoring the mitochondrial activity (“jumping effect”) which is part of the physiological process of implantation. However, current available data cannot confirm the hypothesis yet (7).
Advantages and disadvantages of fresh and frozen cycles
Nowadays, fresh embryo transfers (ET) are the most common choice in IVF cycles (8). Nevertheless, in the last years, controlled ovarian stimulation has increased the uncertainty on the possible adverse effects of the ovarian hyperstimulation syndrome (OHSS), and also on possible deleterious effects on the endometrium and implications in obstetric and perinatal results (9).
In spite of this, recent developments in cryopreservation of oocytes and embryos have led to substantial improvement in IVF outcomes. This resulted in a significant increase in the number of cycles with frozen embryo transfer (FET), which subsequently led to the enhancement of live births rate (10).
What are the advantages of a frozen cycle?
Ovarian hyperstimulation syndrome
The first strong argument for FET strategy is the prevention of OHSS, that results from an increase in vascular permeability (11,8). OHSS is a medical condition affecting the ovaries of some women who take fertility medication to stimulate oocyte growth. OHSS arguably remains a major cause of morbidity in IVF treatment (10).
During a fresh cycle, a woman has to undergo hormonal treatment to regulate her menstrual period, to stimulate the development of multiple oocytes (superovulation), and to encourage their maturation (11, 12). However, in a frozen cycle (FC) the patient does not have to go through ovarian stimulation or egg retrieval depending on their circumstances (13). Many people find that FETs are less stressful than fresh cycles because they do not have to worry about oocytes production or whether there will be viable embryos, since those procedures have already been done (9).
Deleterious effects on the embryo
The optimization of vitrification protocols has reduced the deleterious effects that this process may produce in embryos. Also, it have been observed similar survival and embryo development in FCs compared to fresh cycles (10). Moreover, best quality embryos, morphologywise, can be stored and transferred in a future cycle in better conditions. These data have allowed for an increment of success rates and the confidence of sanitary personnel and patients over FCs (5).
The implantation process, one of the crucial steps in the success of ART, requires a reciprocal interaction between the embryo and the endometrium during a small period of time called window of implantation. This interaction involves the embryo, along with its inherent molecular program of cell growth and differentiation, as well as differentiation of endometrial cells into an adequate uterine receptivity (11). Some patients may find easier to turn to FCs, since dealing with the whole process of medication during a normal cycle for ovarian stimulation may result psychologically and emotionally overwhelming. In this regard, FC may also provide a better outcome (3).
The importance of an adequate endometrial environment in ART is highlighted in those patients who resort to oocyte donation, where there must be a synchronization between donor and recipient in fresh cycles. Those cases that require an improvement in endometrial receptivity to stimulate implantation of these donor oocytes seem to obtain better results in frozen cycles or in the next fresh cycle (8).
Multiplet pregnancies are one of the major safety concerns of IVF due to the increased risk of neonatal and maternal complications. To achieve good results, to would be ideal to select the optimal single embryo to be transferred. Elective single embryo transfer (eSET) is the most effective way to reduce those risky pregnancies (14).
How can cryopreservation damage embryos?
Upon analyzing some ART studies and results, embryos are able to adapt and develop in a large range of culture media, showing different gene expression models in different environments. Cryopreservation causes stress in embryos and it is known as “hormesis”(5) (Fig.3).
However, if the conditions are too unfavorable or toxic, mitochondrial activity is suppressed below the threshold necessary for the development of the embryo, so that implantation in the endometrium will be affected (5).
Results of embryo transfer in fresh cycles vs. frozen cycles
The main current objective of IVF professionals is to improve pregnancy rates in both fresh and frozen-thawed cycles. It is clear that embryo and endometrial receptivity are important factors to promote pregnancy rate. Recently, many researches showed FET can enhance the embryo utilization rate and improve the success rate in contrast to other research lines (15).
In Roque et al. systematic meta‐analysis for 633 cycles in women aged 27-33 years old showed that FET resulted in a statistically significant increase in the ongoing pregnancy rate and clinical pregnancy compared with the fresh transfer group (8). Interestingly, the fresh group showed a higher miscarriage rate, but no statistical difference was found when compared with the frozen group. According to these data, it seems that the results of IVF-ICSI cycles can be improved by performing the FET especially in patients with normal or high follicular response. This advantage could be explained thanks to a more physiological preparation of endometrium. Several studies have also shown good results with cryopreservation of all embryos and subsequent FET in those patients most susceptible to develop OHSS (8, 16-19).
In contrast, Shavit et al. found lower rates of clinical pregnancy and live births in the vitrified-warmed blastocyst group. The difference in implantation and pregnancy rates could be attributed to a higher proportion of good-quality embryos in the fresh blastocysts transfer group. They suggest that in fresh cycles highest quality blastocyst is selected for transfer and the rest are usually vitrified. Thus, vitrified-warmed blastocysts may have slightly poorer grade after warming and prior to transfer (20).
In addition, it is necessary to take into account those cycles with frozen oocytes. Braga et al. found that warmed oocytes transferred in endometrial prepared cycles yield better clinical outcomes than fresh ETs. Indeed, they found that fertilization rate, embryo quality, and developmental competence was decreased in embryos derived from vitrified oocytes (12). Conversely, previous studies have suggested that the results of oocyte vitrification followed by ICSI are not inferior with regard to fertilization, embryo developmental competence, pregnancy rates, and live birth (21, 22, 23).
An interesting point found in Braga et al. research is that even with lower embryo developmental quality, warmed oocytes transferred in endometrial prepared cycles resulted in higher pregnancy and implantation rates compared with transfer in fresh cycles. This finding strongly suggests that controlled ovarian stimulation impacts endometrial receptivity, which may be a possible cause of implantation failure after ovarian stimulation (12). Indeed, some studies have suggested that pregnancy rate is inversely related to serum progesterone levels on the day of hCG administration (24-27). It has been demonstrated that elevated progesterone levels on hCG trigger day negatively influence the pregnancy, regardless of the oocyte quality. Raised concentrations of progesterone in the late follicular phase are likely to influence the secretory changes of the endometrium, leading to an asynchrony between embryo and endometrial dialogue, which may result in reduced implantation rate (12).
Another issue to consider is the obstetric and perinatal outcomes of frozen-thawed cycles. Maheshwari et al. quantified in a meta-analysis the obstetric and perinatal risks for singleton pregnancies after FET and compared it with those after fresh embryo transfer (28). They indicated better perinatal outcomes in singleton pregnancies after the transfer of frozen‐thawed embryos when compared to fresh IVF embryos. This could be explained by antepartum hemorrhage, very preterm birth (delivery at <32 weeks), preterm delivery (delivery at <37 weeks), small for gestational age, low birth weight (birth weight <2500 g), and perinatal mortality significantly lower in women who received frozen embryos than those transferred with fresh embryos (29, 28).
It is important to note that most studies comparing perinatal outcome of singleton births conceived after fresh and cryopreserved ETs included both single and multiple ETs. Therefore, part of the adverse perinatal outcome may be attributed to the vanishing twin phenomenon, which occurs in up to 10% of multiple ETs resulting in a singleton live birth (20).
What can we conclude?
Elective embryo cryopreservation followed by single FET has attracted increasing attention and has been regarded as a potential innovation of IVF treatment. Choosing the well-selected embryo could further increase the chance of live birth of a eSET, which is of high clinical significance. However, there are great gaps in the literature about the risk/benefit ratio of this strategy, which includes multiple steps of treatment (30).
The good outcomes in FC might be associated with having a well‐balanced embryo‐endometrium interaction in FC, and also with lacking controlled ovarian hyperstimulation, which may adversely affect endometrial receptivity during fresh IVF cycles. In addition, when hormone replacement cycles were applied in FETs, estrogen and progesterone were given in physiological doses to mimic natural cycles, while supraphysiological doses of gonadotropins were given in fresh cycles (31).
On the other hand, other authors find fresh cycles as the best choice, especially in patients who resort to oocyte donation. In fact, it seems that there is a higher proportion of good-quality embryos in fresh blastocysts compared to vitrified-warmed blastocysts, which may have slightly poorer grade after warming and prior to transfer. (8, 20).
In conclusion, each case must be individualized in relation to clinical characteristics of the patients and to oocyte, seminal and embryo quality. By doing so, results will be optimized in each cycle and the chances of achieving a live birth will be highly improved.
1. Wong KM, Mastenbroek S, Repping S. Cryopreservation of human embryos and its contribution to in vitro fertilization success rates. Fertil Steril. 2014;102(1):19-26.
2. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature. 1983;305(5936):707-9.
3. Zeilmaker GH, Alberda AT, van Gent I, Rijkmans CM, Drogendijk AC. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil Steril. 1984; 42(2):293-6.
4. Sparks AE. Human embryo cryopreservation-methods, timing, and other considerations for optimizing an embryo cryopreservation program. Semin Reprod Med. 2015;33(2):128-44.
5. Konc J, Kanyó K, Kriston R, Somoskői B, Cseh S. Cryopreservation of embryos and oocytes in human assisted reproduction. Biomed Res Int. 2014;2014:307268.
6. Loutradi KE, Kolibianakis EM, Venetis CA, Papanikolaou EG, Pados G, Bontis I, et al. Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertil Steril. 2008;90(1):186-93.
7. Vladimirov IK, Tacheva D, Diez A. Theory about the Embryo Cryo-Treatment. Reprod Med Biol. 2017;16:118–125.
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9. Gurbuz AS, Gode F, Ozcimen N, Isik AZ.Gonadotrophin-releasing hormone agonist trigger and freeze-all strategy does not prevent severe ovarian hyperstimulation syndrome: a report of three cases. Reprod Biomed Online 2014;29:541-544.
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11. Lessey BA. Endometrial receptivity and the window of implantation. Baillieres Best Pract Res Clin Obstet Gynaecol. 2000;14(5):775-88.
12. Braga D, Setti A, Figueira R, Azevedo M, Iaconelli A, Lo Turco E et al. Freeze-all, oocyte vitrification, or fresh embryo transfer? Lessons from an egg-sharing donation program. Fertil Steril. 2016;106(3):615-622.
13. Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Hudson C. Clinical rationale for cryopreservation of entire embryo cohorts in lieu of fresh transfer. Fertil Steril. 2014;102:3-9.
14. Tobias T, Sharara FI, Franasiak JM, Heiser PW, Pinckney-Clark E. Promoting the use of elective single embryo transfer in clinical practice. Fertil Res Pract. 2016;2(1):1-9.
15. Shen C, Shu D, Zhao X, Gao Y. Comparison of clinical outcomes between fresh embryo transfers and frozen-thawed embryo transfers. Iran J Reprod Med. 2014. Jun;12(6):409–14.
16. Griesinger G, von Otte S, Schroer A, Ludwig AK, Diedrich K, Al-Hasani S, et al. Elective cryopreservation of all pronuclear oocytes after GnRH agonist triggering of final oocyte maturation in patients at risk of developing OHSS: a prospective, observational proof-of-concept study. Hum Reprod. 2007;22(5):1348-1352.
17. D'Angelo A. Ovarian hyperstimulation syndrome prevention strategies: cryopreservation of all embryos. Semin Reprod Med. 2010;28(6):513-518.
18. Griesinger G, Schultz L, Bauer T, Broessner A, Frambach T, Kissler S. Ovarian hyperstimulation síndrome prevention by gonadotropin-releasing hormone agonist triggering of final oocyte maturation in a gonadotropin-releasing hormone antagonist protocol in combination with ‘‘freeze-all’’ strategy: a prospective multicentric study. Fertil Steril. 2011;95(6):2029-2033.
19. Devroey P, Polyzos NP, Blockeel C. An OHSS-Free Clinic by segmentation of IVF treatment. Hum Reprod. 2011;26(10):2593-2597.
20. Shavit T, Oron G, Weon-Young S, Holzer H, Tulandi T. Vitrified-warmed single-embryo transfers may be associated with increased maternal complications compared with fresh single-embryo transfers. Reprod Biomed Online. 2017;35(1):94-102.
21. Trokoudes KM, Pavlides C, Zhang X. Comparison outcome of fresh and vitri- fied donor oocytes in an egg-sharing donation program. Fertil Steril. 2011; 95:1996-2000.
22. Herrero L, Pareja S, Aragones M, Cobo A, Bronet F, Garcia-Velasco JA. Oocyte versus embryo vitrification for delayed embryo transfer: an observational study. Reprod Biomed Online. 2014;29:567-72.
23. Rienzi L, Romano S, Albricci L, Maggiulli R, Capalbo A, Baroni E, et al. Embryo development of fresh ‘versus’ vitrified metaphase II oocytes after ICSI: a prospective randomized sibling-oocyte study. Hum Reprod. 2010;25:66-73.
24. Xu, B., Li, Z., Zhang, H., Jin, L., Li, Y., Ai, J. et al, Serum progesterone level effects on the outcome of in vitro fertilization in patients with different ovarian response: an analysis of more than 10,000 cycles. Fertil Steril. 2012;97 (1321-7.e1-4).
25. Wu, Z., Li, R., Ma, Y., Deng, B., Zhang, X., Meng, Y. et al, Effect of HCG-day serum progesterone and oestradiol concentrations on pregnancy outcomes in GnRH agonist cycles. Reprod Biomed Online. 2012;24:511–520.
26. Bosch, E., Labarta, E., Crespo, J., Simon, C., Remohi, J., Jenkins, J. et al, Circulating progesterone levels and ongoing pregnancy rates in controlled ovarian stimulation cycles for in vitro fertilization: analysis of over 4000 cycles. Hum Reprod. 2010;25:2092–2100.
27. Hamdine, O., Macklon, N.S., Eijkemans, M.J., Laven, J.S., Cohlen, B.J., Verhoeff, A. et al, Elevated early follicular progesterone levels and in vitro fertilization outcomes: a prospective intervention study and meta-analysis. Fertil Steril. 2014;102:448–454.e1.
28. Maheshwari A, Pandey S, Shetty A, Hamilton M, Bhattacharya S. Obstetric and perinatal outcomes in singleton pregnancies resulting from the transfer of frozen thawed versus fresh embryos generated through in vitro fertilization treatment: a systematic review and meta-analysis. Fertil Steril. 2012;98:368–77.e1.
29. Qiao J, Zhang L, Yan L, Zhi X, Yan J. Female Fertility: Is it Safe to "Freeze?". Chin Med J (Engl). 2015;128(3):390.
30. Wei D, Sun Y, Liu J, Liang X, Zhu Y et al. Live birth after fresh versus frozen single blastocyst transfer (Frefro-blastocyst): study protocol for a randomized controlled trial. Trials 2017; 18(253): 1-7.
31. Zhang L, Yan LY, Zhi X, Yan J, Qiao J. Female Fertility: Is it Safe to “Freeze?” Chin Med J. 2015;128 (3):390-7.
Authors: Shuyana Deba and Roberto de la Fuente
In assisted laboratories, semen samples are usually obtained after masturbation and the ejaculate is collected into a container. However, semen recovered from non-spermicidal condoms can be of higher quality because of the time producing the sample influences in the amount of seminal emission before ejaculation (2). Moreover, according to the World Health Organization criteria, laboratory manual for the examination and processing of human semen, the results of laboratory measurements of semen quality will depend on: completed collection of the sample, products of the accessory glands (that will dilute the concentration of epididymal spermatozoa), time past since last sexual activity, abstinence period and the size of the testis. All these factors, among others, will have an influence on the total sperm count per ejaculate (2).
Contrary to what is found in natural fertilization after ejaculation, there are no actual barriers to enhance semen capacitation in vitro. As a consequence, in assisted reproduction laboratories sperm must be separated from the seminal plasma within one hour after ejaculation. The purpose of semen processing is to increase the concentration of high quality spermatozoa, and the method choice will depend on the nature of the sperm, that will in turn determine which reproductive procedure will be performed (2).
Reports over the last two decades have made emphasis on the need for the optimal method for sperm selection to compile as many as possible of the following conditions: non-toxicity, ease to perform, inexpensiveness, suitability for high-throughput sample processing, accuracy in selecting the best subpopulation of sperm and ability to discard other cell types and seminal plasma as well as other substances that may harm the sperm (cryoprotectants, bioactive complements, motility enhancers) (3). Unfortunately, no known method to date successfully meet all the above, and so the selection of the most appropriate method normally depends on the specific procedure to follow.
“Conventional” techniques like the nowadays common washing swim-up (WSU) and differential density gradient centrifugation (DDGC) procedures are normally used for a variety of assisted reproductive procedures, although it is clear that not all of them will have comparable efficiency in selecting high quality sperm. Some techniques are better indicated for certain conditions. For instance, high concentrations of highly motile sperm from normospermic men will show a good performance after WSU, and this will be suitable for regular IVF. On the contrary, severe oligospermic or asthenospermic individuals will require semen selection by using other techniques. The introduction of ICSI in the regular clinical practice enhanced the fertilization rates in the 90’s (4), but did not significantly increase live birth rates because of failures in early development, mostly due to the high incidence of structural chromosomal aberrations (5). This is a direct consequence of the inability of ICSI to specifically detect good quality spermatozoa.
In the following paragraphs we discuss the appropriateness of different techniques for distinct assisted reproduction treatments, namely: in vitro fertilization (IVF), intrauterine insemination (IUI) and intracytoplasmic sperm injection (ICSI).
Sperm preparation techniques for IVF
In the last few years several studies have discussed about the best technique for sperm capacitation in vitro. Recently, Volpes and collaborators compared the effects of four methods used for sperm preparation, namely, direct swim-up, pellet swim-up, density gradient and density gradient followed by swim-up (6). The authors evaluated levels of DNA fragmentation using the sperm chromatin dispersion test for samples meeting the following criteria: minimum volume of 2 mL, minimum sperm concentration of 10 million/mL and a minimum motility of 35%. The study showed lower DNA fragmentation after pellet swim-up and after density gradient followed by swim-up. However, the study highlighted the limitation that clinical outcomes from IVF/ICSI procedures were not correlated with DNA fragmentation in the sperm (6).
A different study compared sperm processing by WSU and DDGC in normospermic individuals, analyzing sperm motility, concentration, and morphology recovery rates (7). Conclusions were that, on the one hand, DGC was appropriate for males with low sperm concentration, since it yielded higher sperm concentration than the WSU technique. On the other hand, the latter facilitated a more efficient morphology-based sperm selection. Nevertheless, the main limitation of the study was the small number of patients and the fact that all of them were normospermic (7).
In 2016, Yamanaka and coauthors attempted to determine the efficiency of combining both DGC and WSU techniques in reducing the number of sperm with abnormal nuclear morphology under the microscope (8). Results showed that combination of the two approaches was better than one alone; both DNA fragmentation levels and sperm motility were improved compared to results after just DGC. Data also demonstrated that the combination of both techniques was efficient in enriching the sample with sperm with normal head and flagellum morphology (8).
Intrauterine Insemination (IUI)
A study by Karamahmutoglu on the most effective sperm preparation technique for IUI compared WSU vs. DGC (9). Even though data showed higher IUI success rates after having performed the DGC approach, no significant difference was found in the "mild male factor" subfertile group (with sperm count in the range of 5-15 million/mL). Moreover, other factors were affecting fecundity success rate, such as female age, number of cycles and type of infertility (9). These observations on the efficacy of DGC and WSU methods for IUI have recently been confirmed in a similar study by Butt and Chohan (10).
On the particularities of ICSI
Sperm selection for ICSI is commonly carried out by the embryologist’s own judgement based on morphological criteria. This results in inconsistent decision-making and often selection of poor quality sperm, since semen samples are considered morphologically normal with just 4% of normal-looking spermatozoa (2). Therefore functional, physiological and molecular traits of spermatozoa cannot be evaluated by ICSI, and so unnoticed DNA abnormalities (even specific causes of sterility) might be passed on to the offspring by the selected spermatozoon. Thus it is easy to understand why the embryology and reproduction community has been trying to develop new strategies to successfully select the best spermatozoa regarding phenotype, functional characteristics and genetic and molecular integrity.
Advanced methods for sperm preparation
There are currently three main groups of methods to facilitate selection of high quality sperm, based on morphology, electrical charge and sperm surface maturity and organization (11, 12):
A simpler and cheaper technique uses the electrokinetic potential of the sperm (19); the electric potential difference of the sperm membrane decreases with capacitation, which is used to pipette washed sperm into positively charged tubes, so that negatively charged (mature) sperm can be retrieved afterwards. However, the total sperm recovered by using this technique is low, which represents an important limitation (11).
PS externalization to the outer sperm membrane is a typical apoptotic feature, which allows the cell to bind to magnetic beads conjugated with Annexin-V. This made it possible to develop a magnetic-activated cell sorting system (MACS) (25); the sperm suspension is incubated with the microbeads so that those apoptotic spermatozoa will bind to the beads, which will be subsequently retained within the MACS column in a magnet. Non-apoptotic sperm will then flow freely to be collected. Even though this technique enriches the sample in healthy sperm, it does not discard leukocytes or germ cells, and thus it must be combined with DDGC (26). A variation of the approach known as Annexin V glass wool (annexin V-GW) eliminates potential side effects of free magnetic beads (27), but still needs to be combined with repeated DGC cycles, which is actually not appropriate for oligospermic individuals (11).
The approach followed for semen preparation and selection for every patient/couple needs to be chosen upon a series of factors that mainly refer to the cause of infertility. The wide range of situations found regarding this topic makes it necessary to adopt a specific strategy every time. This will ultimately define the treatment to be applied and the techniques to be carried out subsequently.
Recent advances have developed new and better ways to detect good quality spermatozoa, minimizing DNA fragmentation and optimizing the rates of good morphology or motility, for instance. However, it is important to remember that there exists no particular strategy that always relates to the optimal clinical outcome. On the contrary, each situation must be considered in the light of the patient’s needs and characteristics, and so the technique for sperm preparation must be chosen accordingly.
Authors: Inés Abad, Roberto de la Fuente and Sara Sanz
Fertility treatments are more and more common in our days, reason why it is important to perform these procedures accurately resembling in vivo conditions. Additionally, male factor may oftentimes be underrated, and yet it is 50% of the treatment. The following text aims to establish an updated comparison between in vivo and in vitro semen preparation methods. In the first part a general description of the processes of maturation and capacitation of sperm are presented.
Where does sperm maturation take place?
Once spermatogenesis is completed in the seminiferous epithelium, immature spermatozoa migrate towards the epididymis, the organ in which sperm maturation and storage take place. The epididymis is usually divided into three different parts: caput (head), corpus (body) and cauda (tail) (2).
Typical changes in sperm during maturation
1. Acquisition of progressive motility. Even though immature sperm have functional movement machinery, motility of these cells begins in the caput segment. Whereas beating intensity is similar throughout the whole epididymis, flagellar amplitude is modified within this path. This is due to changes on the sperm surface, such as acquisition of new proteins and molecular changes involving inactivation of Ser/Thr phosphatases (3, 4).
2. Migration of the cytoplasmic droplet (CD). This droplet migrates from the neck towards the annulus of the mammalian spermatozoa (in humans, the CD is more proximal, located at the neck as opposed to the distal position of the annulus). The role of this droplet is to regulate ion homeostasis. It contains K+, Cl- and water channels, which have been suggested to work in regulation of sperm volume during the different regions of the epididymis. It also accumulates Ca2+, which has a biphasic role controlling phosphorylation pathways in sperm cells. In immature spermatozoa, it has been hypothesized that high Ca2+ levels found in the CD maintain low levels of tyrosine phosphorylation (5).
3. Changes in sperm protein and lipid profile.
- Protein and lipid content
Even though changes in these profiles are not well understood, there are three complementary mechanisms that participate in completing maturation:
Perhaps the most important among these changes is the significant reduction in cholesterol content of the sperm membrane. This reduction involves a decrease in the cholesterol/phospholipid ratio that facilitates protein trafficking from and onto the membrane and enhances its fluidity, which will eventually play a role in triggering capacitation and fertilisation (8).
- Post-translational protein changes
Additionally, certain post-translational modifications of proteins have also been hypothesised to occur during sperm maturation. This is the case for oxidation of thiol groups, which promotes the formation of disulphide bonds (S-S) and stabilises components of both the head and flagellum (9).
Fig.2. Schematic representation of the main items during sperm maturation. (A) Principal functional and morphological aspects in immature caput spermatozoa. (B) Molecular characteristics of immature spermatozoa. (C) Main morphofunctional traits of cauda mature spermatozoa and (D) their molecular features (4) (*) Ability to fertilize the egg will ultimately depend on completing capacitation.
What is the composition of the ejaculate?
In mammals, semen is composed of two different phases:
Following ejaculation, semen is deposited to the anterior wall of the vagina, adjacent to the ectocervical tissues. From here on, for sperm to progress towards the egg through the oviduct or Fallopian tubes, semen must undergo liquefaction. This process usually takes about 20-30 minutes (11). Semen goes through the cervix and reaches the distal portion of the Fallopian tube, where sperm is stored and maintained by interacting with the endosalpingeal epithelium (12, 13).
In 1951, Austin and Chang individually observed that a certain period of time in the female tract was required for sperm before fertilization could take place (14, 15). Later, observations in multiple mammalian species confirmed these first notions, and certain studies showed a delay of at least 2 hours before sperm entry into the egg. This supports the hypothesis of sperm maturation before becoming fertile after ejaculation (16). Nowadays, such process is known as capacitation, as opposed to maturation in the male tract explained above.
Capacitation and fertility
Some of the factors involved in sperm capacitation are steroid hormones such as oestrogens and progesterone, both produced by the follicle. These steroids play different roles: they act as chemoattractants, facilitate triggering of hyperactivation, regulate trafficking of cGMP or modulate the potential for completing acrosome reaction (17-19).
Semen liquefaction following ejaculation is mainly modulated by prostate derived peptidase KLK3. In females, KLKs 5–8, 10–11, and 13–15 are expressed at very high levels in the cervix and vagina compared to other adult tissues (20, 21). Moreover, KLK1 and KLK3 transcripts are expressed at the highest level in human endometrium when circulating estradiol (E2) is elevated. These findings suggest that KLKs are expressed in the human reproductive tracts and that some of the KLKs in the uteri are regulated by E2. Abnormal E2 signalling in the female reproductive tract leads to semen liquefaction defects, associated with defective SEMG cleavage and sperm transport, which may result in some cases of infertility.
It is known that mice lacking ESR1 (one of the oestrogen receptors) in the epithelial cells are infertile (22), partly due to a reduction in the number of sperm able to reach the oviduct (23). However, the effect of ESR1 loss in the epithelial cells on sperm transport in the uterus has not yet been investigated. Similarly, other potential research lines could investigate liquefaction defects caused by diminished KLK activity in females or regulation of KLKs by molecular signalling in the female tract.
Once semen trespasses the cervix, sperm are known to achieve capacitation in an asynchronous fashion during the interaction with the epithelium, which results in a continuous flow of fertile spermatozoa towards the Graafian follicle (24). The ability to bind to the epithelium, in turn, may be indirectly related to the sperm DNA integrity, and so DNA fragmentation levels would be indicative of the fertility potential of the sperm (25).
Elements involved in sperm capacitation
Even though capacitation had traditionally been regarded as a two-step process, through which changes in the cell membrane would lead to the acrosome reaction (AR) (26), capacitation is currently considered as a continuous process that culminates in the AR. It would be difficult to describe all capacitation-related events separately because all of them are connected to each other in time. However, the most important changes in the sperm during the process are (27-37):
Sperm capacitation is a complex process with multitude of interconnected and highly regulated molecular pathways. One of the first events is the alteration of the permeabilization of the sperm plasma, so that the influx and intracellular concentration of certain ions are increased. The main molecules involved are probably Ca2+ and HCO3-; the net intake by the sperm cell triggers alkalinisation of the pH and the concomitant activation of the soluble adenylyl cyclase (sAC) (38, 39). As an immediate consequence, cAMP levels increase followed by activation of the protein kinase A (PKA) (40, 41).
The rise of cAMP causes redistribution of certain phospholipids and proteins of the membrane, and so exposing cholesterol, which accumulates in lipid rafts (42). The organization of these rafts promotes the removal of cholesterol and its translocation to extracellular acceptors like albumin (43). Also, increased cAMP activates PKA, which in turn activates SRC kinase (44).
Eventually, SRC kinase activity triggers tyrosine phosphorylation, which results and a wide range of proteins been modified and relocated in capacitating sperm. This has been described in several species, including humans (45). The end result of capacitation is the acrosome reaction (AR), the process by which the content of the acrosome is released to the extracellular environment. In natural conditions, this environment is actually the cummulus cells, whose connections will be broken by the chemical reactions of the acrosomal content, mainly proteases like acrosin and hyaluronidase, also exposed to the right membrane domains during lipid redistribution (46). It is not surprising that mutations affecting any of these processes will result in multiple causes for infertility (47).
Findings like the one regarding post-ejaculated liquefaction, mutations on acrosome protease-encoding genes or other molecular mechanisms of sperm capacitation are crucial to progress in the field of reproductive medicine, and can lead to: (i) potential diagnostic tools for unexplained infertility cases, (ii) the development of a novel contraception technology to entrap sperm (48), (iii) or even revolutionary new methods for human sperm capacitation in the laboratory (49), which could significantly improve live birth rates for fertility treatments.
In the following post... different methods for sperm selection in the laboratory will be explained, paying attention to advantages and disadvantages under different circumstances. The importance of different sperm features like DNA fragmentation or morphology will be discussed in relation to the best sperm selection method to achieve optimal clinical outcomes.
Authors: Paula Brígido, Roberto de la Fuente and Javier Del Río
Assisted reproduction technology can help fertile couples to achieve successful pregnancies. Sometimes, reproductive desires of these couples are affected by the presence of a genetic disease in either partner. In such cases, couples are at a reproductive risk and find themselves in the need of assistance that only ART can provide.
Preimplantation genetic diagnosis (PGD) provides an alternative to prenatal diagnosis to detect the specific genetic condition or disease they suffer from, and allows them to avoid passing it on their offspring (2). It requires the analyses of the embryos generated by ART in the IVF laboratory, by means of accurate and sensitive methodologies such as embryo biopsy, genetics, single cell genomics and, of course, background on prenatal diagnosis and counselling from experts.
Clinical application of PGD dates back to the late 60’s, when blastocysts of research animals could be sexed (3) (note that this was already possible ten years before Louis Brown, the first IVF baby, was born in the UK in 1978). At the beginning of the 90’s, early human embryos were sexed before implantation and the first genetic analyses were performed to avoid children inheriting Mendelian diseases. By the end of the century, other nowadays considered basic genetic methodologies were routinely used for preimplantation diagnosis and PGD was applied as a normal procedure to guarantee healthy babies (4).
In the present post we aim to give an account of the importance of PGD and the current view of the main clinical approaches for its application.
WHEN IS PGD INDICATED?
Indications for PGD are multiple and emerge from different motivations. Firstly, the couple may have suffered from a number of terminations due to the embryo having inherited the genetic condition. It could also be motivated by the parents already having a child with a severe genetic disease. In this case they might be willing to avoid passing it on the next one or even looking for a suitable treatment, if possible. However, one of the parents (or both) may be worried about their family history, being aware of the presence of a specific genetic condition, regardless of the type of inheritance.
If the parents are carriers of any genetic disease, either an autosomal-dominant disorder like Huntington disease or an autosomal-recessive one like cystic fibrosis, they are at reproductive risk because the resulting embryo may be affected (the probability depending on the specific disorder itself and the way it is inherited) (see  for details on inherited conditions). But there are even cases in which motivation is not based on biological but in ethical or religious reasons. Certain families might have serious concerns about going on for abortion of an affected embryo. In such cases, application of PGD may circumvent this kind of ethical conflicts.
Broadly speaking, steps for PGD are as follows (2):
PGD vs. PGS
Preimplantation genetic screening (PGS) is the general term for a compound of approaches that aim to evaluate the genetic content of the cell, in contrast to genetic tests whose goals are to determine whether an embryo is affected by a specific genetic condition (PGD). Originally termed PGD-AS (preimplantation genetic diagnosis for aneuploidy screening), PGS was developed to confirm the ploidy status of the embryo, searching for possible aneuploidies. Available data suggest that most of miscarriages occurred during the first trimester are a consequence of some sort of aneuploidies (5), and that mainly selected chromosomes were involved in these structural abnormalities (6). Thus, the main approach developed for PGS was the fluorescence in situ hybridization (FISH) for such chromosomes.
Types of approaches for PGD in the laboratory
Current technical methodologies for preimplantational genetic analyses mainly lie in one of the following:
WHEN TO PERFORM BIOPSY
Typical biopsies for PGD (and PGS) are as described as follows:
DAY 3. CLEAVAGE STAGE BIOPSY
There is a controversy regarding utility of this type of biopsy. In the cleavage stage biopsy, embryos are biopsied at day 3 when individual cells can be differentiated. This technique entails aspiration of one to two blastomeres to obtain the embryonic genetic material for PGD analysis (13). Following genetic diagnosis, embryo transfer may be performed on blastocyst stage. Embryos are usually selected for biopsy based on morphological criteria. Unfortunately, these do not predict the development potential of the embryo, and so it could fail to progress until blastocyst stage. This would compromise the advantages of using the day-3 approach (14). On the other hand, performing biopsy on the cleavage stage allows embryos to be cultured in vitro until they reach the blastocyst. This means they can be fresh transferred (15), whereas embryos biopsied on day 5 must be vitrified and transferred in a subsequent cycle.
How many cells should be removed?
The number of cells to be removed in the biopsy is still a controversial issue. Aspirating one cell reduces the cellular mass extracted but it can imply the presence of mosaicism. Conversely, aspirating two cells can reduce the risk of mosaicism, but removing such cellular mass could have consequences on the implantation rate (14).
Reported data have shown a dramatic reduction of 39% in the implantation rate in cleavage stage biopsy (16). The authors related it with proportion of the embryo total cellular removed. Whereas around five cells pulled out of the embryo in the trophectoderm biopsy represent 2-3% of the total cell content (expanded blastocyst has 200-220 cells approximately), extraction of a single cell from an eight cell embryo supposes 13% of the total content (16).
What do experts say?
Cleavage stage biopsy produces different opinions among embryologists because of the presence of mosaicism and the possibility of self-correction of aneuploidies from cleavage to blastocyst stage (17). On the contrary, studies using array-comparative genomic hybridization (array-CGH) technology to analyse genetic abnormalities in day-3 blastomeres and confirming it in trophectoderm biopsy showed concordance between day 3 diagnosis and day 5 reanalysis; Treff and coauthors showed more reliable results for SNP-microarray (96 versus 83 %) and also a lower mosaicism degree (31 %) for SNP-microarray samples in a study comparing array technology versus FISH technique (18). These data would support the suggestion of some authors, who proposed that the incidence of mosaicism may have been overestimated in previous studies due to technical inconsistency of the FISH technique (17, 18, 19). At present, this matter remains controversial.
Regarding pregnancy rates, in both types of biopsies higher pregnancy rates are obtained comparing with the control group, in which no biopsy was performed (14, 19).
To sum up:
DAY 5. TROPHECTODERM BIOPSY
The blastocyst stage is currently supposed to be an optimal time to perform biopsies for PGD/PGS. The combination of improved blastocyst culture, trophectoderm (TE) biopsy, refined cryopreservation techniques, and molecular assays, such as array comparative genomic hybridization that allows for 24-chromosome screening, have led to a renaissance of PGS. TE biopsy will not detect every circumstance in which the embryo is at risk of aneuploidy, but it will detect mosaicism more reliably than cleavage-stage biopsy (which cannot be relied on at all for this purpose) (20, 21).
Moreover, when diagnosing monogenic disorders in single blastomere cells using PCR-based protocols, there is a high risk of PCR failure due to either no amplification (allele dropout) or preferential amplification of one of the alleles, potentially resulting in a reduced number of unaffected embryos available for transfer. Increasing the amount of starting DNA template should in principle increase the sensitivity and reliability of genetic diagnosis. Therefore, the biopsy of multiple trophectoderm cells from the blastocyst rather than a single cell from cleavage stage embryos should potentially lead to improved PGD outcome for patients (14).
How many cells should be removed?
Research to determine the appropriate number of biopsied TE cells in blastocyst biopsies are limited. The exact number of biopsied TE cells is hard to count visually because cells are small and usually remain as a clump. In most studies using comparative genome hybridization or single-nucleotide polymorphism array technology for genetic testing, biopsied TE cells were used for genome amplification and their number was impossible to know. Moreover, some studies showed that removing four to five cells leads to better results. Therefore, the biopsied cell number should be higher in the blastocysts with better TE quality than those with worse characteristics (22, 23).
Can biopsies affect blastocyst development and its implantation?
Whereas it remains possible that biopsy of cleavage-stage embryos can critically arrest further development through reduction of cell mass, the low miscarriage rates and high term birth rates in the present series, as well as data presently under analysis, suggest that this is not the case for TE biopsy. It can be speculated that the damage to blastocyst development potential caused by TE biopsy would be less for blastocysts with a greater number of TE cells (21, 22).
Some experts assured that TE biopsy at the blastocyst stage had no meaningful impact on the developmental competence of the embryo as measured by implantation and delivery rates. This contrasts with the information above-mentioned on the significant reduction in the probability for an embryo to implant and progress up to delivery (16). When combined with TE biopsy and blastocyst vitrification, SNP microarray has resulted in high implantation and low miscarriage rates for some IVF patients (15, 16, 24).
Are there any limitations?
Owing to the limitations of genetic analysis, most of the biopsied blastocysts need to be cryopreserved by vitrification, and blastocysts with normal results would be transferred in the next frozen cycle. In addition, biopsy of numerous cells from blastocysts with grade B or C may cause damage to the embryo, leading to either its arrest or implantation failure. However, 1-5 cells may be the appropriate biopsied TE cell number to maintain the implantation potential (15, 22).
Also, the personnel experience of different embryologists is an influencing factor in this technique. The number of biopsied cells in the blastocyst biopsy is hard to quantify and largely dependent on the experience of embryologist (22).
To sum up:
WHAT CAN WE CONCLUDE?
The availability of new embryology and molecular techniques allow preimplantation genetic diagnosis laboratories to offer patients at genetic risk the transfer of developmentally competent embryos, unaffected by genetic disease. Cleavage stage biopsy allows for fresh embryo transfer after genetic diagnosis. However, there are reports of high levels of mosaicism when the biopsy is performed on day 3. Trophectoderm biopsy, in turn, provides sufficient material for an effective and more reliable diagnosis in embryos compared to those on cleavage stage. Moreover, it seems that it does not compromise embryo implantation and pregnancy rates in PGD cycles. The drawback for this option is the usual need for cryopreservation and transfer in a different cycle.
The offer of PGD in fertility centres has increased over the last decade, primarily due to the progress on the application of diagnostic methods. The choice for either development stage relates to successful outcomes in the clinic, which mainly depend on technical challenges and timing of the developing embryo. For the embryologists, both day-3 and day-5 approaches are supported by evidence, but it will be essential to consider every single aspect of them to evaluate the best option for the laboratory.
If you want to know more about this topic, visit our ‘VIDEOS’ section; we have added two videos about PGD and day 3 and 5 biopsy and a sequence of an accurate TE biopsy.
Authors: Shuyana Deba, Isabel Sánchez and Sara Sanz
Every procedure carried out in an IVF laboratory, from ovarian puncture and semen capacitation to the embryo transfer, must be performed under specific safety conditions. These standards must be followed to avoid a decrease in gamete/embryo viability (2).
From the in vitro culture, gametes and embryos are exposed to diverse artificial situations that do not take place in nature. In vivo, both fertilization and embryo development in the female reproductive tract occur in the complete absence of light. In this environment, other conditions include oxygen (O2) concentration of 2-8%, pH 7.2-7.4, 37⁰C and gradients of diverse nutrients to which the embryo is exposed (3). Changes in temperature, CO2 and O2 pressure, light exposure or volatile organic compounds may adversely affect embryo quality.
Also, manipulation of embryos by embryologists is as important as air quality and culture conditions. Each human being is covered by about 1012 bacteria (4), which could contaminate embryo cultures if embryologists do not use the necessary clothing, such as laboratory cap, footwear and mask.
Therefore, a daily quality control at different levels should be carried out to obtain good results in IVF cycles. We are going to analyze the effect of some of the elements that can affect germ cells and embryos.
In vivo, mammalian germ cells and embryos are not exposed to light, which might explain why they do not exhibit any protection mechanism against this factor (5,6). In addition, sperm do not have the capacity to repair DNA, unlike oocytes and embryos, which do present some mechanisms for DNA repair (7).
Light variables to be considered are intensity, duration and wavelength. It seems clear that photooxidation increases along with light intensity and duration. However, what type of wavelength would be the most harmful for embryos and germ cells? Energy increases when wavelength is shorter (8). Accordingly, artificial cool white fluorescent light has been demonstrated as the most stressful in mouse embryos. Incandescent light, in turn, seems to be less harmful, and the best outcomes are achieved when warm white fluorescent light is used (7).
How can light affect the quality of these cells?
Indirect effect: Culture and oil photooxidation can affect embryo development (8). In this case, modified components will damage the lipid membranes. Also, if HEPES- or riboflavin-containing media is exposed to light, it results in the formation of hydrogen peroxide, a highly cytotoxic substance (9). Additionally, light can heat up both the plasticware and the oil, resulting in more toxic and damaging components (8,9).
Direct effect: Light can potentially compromise the quality of gametes and embryos, by activating stress-related genes or by ionisation, which may also damage the DNA. This phenomenon would cause DNA fragmentation and mutation, as well as an increase in the apoptotic index and change in the number of mitochondria levels (10).
How can we avoid this effect? (7)
1) Reducing the exposure time.
2) Using warm white fluorescent light in the lab and green filters on microscopes.
3) Adding antioxidants in the media in order to mitigate damages from ROS.
4) Avoiding riboflavin, which is responsible of the phototoxicity in the media.
VOLATILE PARTICLES EFFECT
Since the 1990s, IVF laboratory indoor air quality has taken a high relevance. Thence, focused on creating an optimal environment, laboratories have become clean rooms where filtration of particles is performed by using high-efficiency particle arresting (HEPA) filters, and successful chemical air filtration is achieved by removing volatile organic compounds (VOCs) with solid-phase filtration (e.g., potassium permanganate-impregnated, carbon filters) (11).
Focusing on VOCs (hydrocarbon-based compounds that are emitted by industries, cleaning products, computers, and microscopes among others), several studies have demonstrated their harmful effect on embryos, initially reported by Boone laboratory on mouse embryo development (11). Moreover, VOCs have been shown to increase DNA fragmentation in human sperm, and they can also have detrimental effects on pregnancy rates (12).
Recently, a retrospective study by Munch et al. concluded that, without solid carbon filtration, fertilization, cleavage, and blastocyst conversion rates declined in fresh IVF cycles. Even more, results were found to be even worse in ICSI cycles, probably due to the lack of protective barrier provided by the cumulus cells (13). However, the authors did not observe the same results when embryos had been cryopreserved in an environment with carbon solid filtration but thawed in a laboratory deprived of such systems. The absence of significant changes in cleavage and blastocyst conversion rates, as well as in the proportion of good quality blastocyst developed after thawing suggests that embryos are affected in the peri-fertilization period (13).
Also, products like cosmetics emit VOCs, especially perfumes, colognes, and deodorants. They are highly toxic to embryo development in vitro, primarily due to evaporation of their solvent bases (14,15). After analyzing the results of studies determining the toxicity of VOCs, ideal levels should be below 0.2 ppm but preferably zero (12). Personnel must understand the principles of air quality control, including the function of airflows and airlocks, hygiene, dress code and the use of cleaning agents (16).
pH level depends on bicarbonate concentration of culture media and the CO2 concentration of the incubator. However, other factors like altitude and composition of culture media could affect the pH level, too (17,18). Embryos are able to develop over a range of media pH, considering that they possess an intracellular mechanism to regulate its internal level (17,18). However, it is important to control pH variations because they affect development (17). To control pH level outside the incubator some culture media contain buffers like HEPES or MOPS, but long exposure of embryos to these buffers is not recommended (17). Thawed denuded oocytes and embryos are specially sensitive to pH variations because they do not have an inner system to regulate pH (17). So, an increase in the pH of the medium can affect the physiology and development of oocytes and embryos. Thus, acidification of the medium can even affect the fetal weight and size (18).
As previously mentioned, CO2 is necessary to control pH level of culture media (17,18). The importance of CO2 was demonstrated in 1985, in a study carried out on hamsters (19). The authors cultured hamster embryos in different CO2 concentrations (5% and 10%). They found a higher rate of blastocysts in those cultured at 10% compared to 5%, which demonstrated differences in embryonic development. This results showed that CO2 level is an important factor for embryo culture (19). The capacity of CO2 to get through cell membranes allows for regulation of the inner pH levels in blastomeres. In other studies, it has been shown that the required CO2 concentration to achieve the optimal pH varies in different species. For instance, the required CO2 level in rats is 7.5%, whereas for humans it is 6.5% (19).
Some studies have compared different values of O2 concentration in the incubator and they show that a low level (5-6%) improves results when compared to an ambient level (21%). It has been shown that low O2 levels increase implantation, pregnancy and live birth rates (17,20). It seems that a low O2 level reduces ROS in the culture and the presence of volatile particles in the air, although the exact mechanism of action is still unknown (17).
Standard temperature generally used in IVF laboratories is 37⁰C (17,18). However, optimal temperature is unknown because in the female reproductive tract it could be slightly lower, about 36⁰C. On average, temperature of the Fallopian tube is about 1.5⁰C less, whereas the follicular liquid temperature can reach 2-3⁰C lower than core body temperature (17). It is important to control and prevent temperature variations because it can affect meiotic spindle stability and alter embryonic metabolism. It has been shown that an increase of 2⁰C during 20 minutes potentially alters the integrity of the meiotic spindle, which cannot be completely repaired when temperature is set back to 37⁰C. As a consequence of this increase in temperature, embryos express some stress-response genes that compromise development (18). Interestingly, a small decrease in temperature does not have any effect on oocytes, whereas a large difference can be severely harmful for the meiotic spindle (18).
CULTURE MEDIA EFFECT
Nowadays, there exist two kinds of culture media: one-step media and sequential media (with different compositions for days 0-3 and 3-6) (17,21). All culture media are similar in composition; they contain energy substrates like glucose, pyruvate or lactate, and both organic and inorganic salts, which must be balanced accordingly. Culture media also contain amino acids in different proportions. The exact composition of amino acids in culture media is unknown. One of the most important problems related to the presence of amino acids is the ammonium generated as a product of metabolism. Ammonium has negative effects on embryo and fetal development. To avoid this problem, some culture media contain glutamine, which reduces ammonium production (17,21,22). Also, culture media can be supplemented with macromolecules and other components like HSA, α and β globulins, growth factors, vitamins, lipids, nucleotides, cytokines and hormones (17,22).
What can we conclude?
There are many parameters that should be kept in mind in order to maintain the optimal conditions for both gamete and embryo development in an IVF laboratory. In vitro, cells and embryos are exposed to different stress situations that must be minimized. Therefore, a routine control at different levels needs to be performed, so that the environment in the laboratory is adapted to resemble the reproductive tract and the intrauterine conditions.
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