Embryo Transfer in In-Vitro Fertilization: Factors Affecting Successful Outcome

Abstract

Embryo transfer is the last component of series of events in the process of in vitro fertilization treatment. Events happening at the embryo transfer stage if not well managed could jeopardize the entire process with unpleasant consequences. It is considered to be a critical determinant of clinical outcome in the entire in vitro fertilization procedure. Consequently, factors affecting its efficiency are very vital in the determination of clinical pregnancy, implantation, ongoing pregnancy and live birth rates. Some factors have been clearly seen to be beneficial to the positive outcome of in vitro fertilization, while the benefit of the other factors is still very questionable or have been dismissed. The most important of all factors are ultrasound guided embryo transfer and the use of soft flexible catheters for embryo transfer. Others are removal of cervical mucus before the transfer process, accurate placement of the embryo within the endometrial cavity and immediate ambulation after embryo transfer.

1. Introduction

Among all the events involved in the process of In Vitro Fertilization (IVF) treatment, embryo transfer (ET) is considered one of the key steps. It is in deed the last procedure in the process of IVF. In vitro fertilization success rate is largely dependent on three main factors viz.: the embryo quality, the development and receptivity of the endometrium and the efficiency of the transfer process i.e. the embryos transfer technique [1]. It is estimated that 30% of in vitro fertilization failures, can be attributed to poor embryo transfer techniques [2].

Embryo transfer has evolved over the years both in timing and technique involved. At the inception of IVF treatment, embryo transfer was done on day 2, later day 3 embryo transfer gained prominence and now the blastocyst [day 5] transfer has dominated the IVF practice world over.

The original position for embryo transfer as described by Steptoe was in the knee-chest position [3]. However, the supine/dorsal lithotomy position is now preferred. Blind embryo transfer was the order of the day till ultrasound-guided transfer started gaining prominence. Patients were kept on bed rest for hours and days post ET however the usefulness of this practice is now highly questionable.

The procedural technique of embryo transfer has also seen some changes with associated practices which may or may not be beneficial. In recent times the embryo transfer technique varies. American Society for Reproductive Medicine [ASRM] practice committee in 2017 outlined four transfer modalities [4].

Direct transfer: The catheter is loaded with the embryo(s) and without any form of immediate preceding trial, the transfer is performed.

Trial followed by transfer: A trial or regular embryo transfer catheter (completely connected outer and inner sheath) is used immediately before the real transfer. At the immediate trial, the catheter is passed to and just through the internal os ensuring no resistance and withdrawn thereafter. The embryo transfer catheter is then loaded and the actual transfer is performed.

Afterload transfer: The outer sheath of the embryo transfer catheter is separated from the inner catheter. The inner catheter is pulled back to the extent that only about 1 cm of its tip is protruding through the outer sheath. The two (outer and inner) are held together while advancing through the cervical canal until the inner catheter passes through the internal os of the cervix. At this point, the outer sheath is positioned at the top of the cervical canal and stabilized there while the inner catheter is withdrawn. The embryo is loaded into the inner catheter which is threaded into the endometrial cavity through the outer sheath for the expulsion of the embryos.

Trial transfer converted into an after-load transfer: This is applicable when trial transfer is difficult. In a difficult trial transfer, once the inner catheter passed the internal os, the outer sheath is separated from the inner catheter and moved forward to the top of the cervical canal at the same time the inner catheter is gradually withdrawn. Just like the after-load transfer, the inner catheter is now loaded with the embryo(s) and threaded through the outer sheath for expulsion into the endometrial cavity. Embryo transfer is done usually without any form of analgesia or sedation and it’s done in an atraumatic manner. Difficulty at embryo transfer is to be avoided as much as possible. The proficiency of the personnel carrying out the embryo transfer is also important.

Embryo transfer efficiency is a major rate-limiting step to IVF success [2]. Consequently, performing a smooth and technically sound embryo transfer is paramount. A host of factors have been associated with successful embryo transfer, some of them have been proven to be beneficial while others are of questionable importance. We shall take a look at some of those factors and review their importance in a successful embryo transfer process. Factors for consideration are; Ultrasound-guided embryo transfer, The type of transfer catheter used, proficiency of personnel carrying out the embryo transfer, Site of embryo placement within the endometrial cavity, loading discharge interval, Mock/trial embryo transfer, full bladder, flushing or removal of cervical mucus, bed rest, embryo glue and other factors.

2. Factors affecting successful outcome of embryo transfer

2.1 Ultrasound scan guided embryo transfer

Traditionally embryo transfer was done blindly placing the embryo at an imaginary depth within the endometrial cavity. It is thought that blind embryo transfer could have a limitation in that, there is a great variation in uterine size and depth between patients. Therefore, using a standard depth of 6 cm as is the case in most situations may not be very representative. The blind embryo transfer could also be fraught with the possibility of coil back of catheter tip resulting in depositing the embryo in the cervix.

Over the years, ultrasound scan was introduced to aid the process of embryo transfer. Initially, it was difficult to see the tip of the catheter during the transfer process. This led to the development of the echogenic tip catheter.

There is great debate on the usefulness of embryo transfer under ultrasound guidance. The majority of these studies looked at the trans-abdominal ultrasound scan [5, 6, 7, 8, 9]. Other ultrasound techniques used are the transvaginal ultrasound scan (TV—US) and more recently the three-dimensional ultrasound imaging (3D—US) and uterine length measurement before transfer (UL MbET). In UL MbET transvaginal ultrasound scan is used to measure the cervical length and the distance between the internal os and fundal endometrium, then using the clinical touch (CT) method the operator discharges the embryo about 1.5 cm short of the fundus.

The advantages of ultrasound-guided embryo transfer are related to the relatively low cost of the technology, the likelihood of accurate placing of the embryo in the desired location within the endometrial cavity and the opportunity for patients and operators to watch the entire process, possibly reducing anxiety levels [10, 11]. On the other hand, the disadvantage of using ultrasound guidance during embryo transfer include; The need for additional manpower, prolongation of embryo transfer duration and possible inconvenience to the patient of filling the bladder [8].

Some earlier studies showed that ultrasound-guided embryo transfer does not offer any advantage in the hands of experienced operators compared with blind transfer. That ultrasound-guided embryo transfer may be of probable benefit in the early training period [6, 7]. However, Sallam [12] accorded the Drakeley outcome to the fact that different catheters were used for the USS embryo transfer and clinical touch embryo transfer, thus the bias in outcomes.

Tang et al. [5] reported a significant improvement in implantation rate following ultrasound-guided embryo transfer, however, they noted that the extent of improvement in the pregnancy rate may depend on the specific techniques and methods of embryo transfer, used in individual centres. A meta-analysis of eight randomized trials [13] showed that ultrasound-guided embryo placement improves implantation and pregnancy rates.

Cozzolino et al. [14] did a systematic review and meta-analysis of 38 eligible studies on ultrasound-guided ET and came to the following conclusion;

Meta-analysis of RCTs comparing trans-abdominal ultrasound (TA—US) guided embryo transfer versus clinical touch: Analysis of five thousand, five hundred and three (5503) patients showed a significantly higher pregnancy rate in the trans-abdominal ultrasound embryo transfer group compared to the clinical touch group. The ongoing pregnancy and live birth rates were also significantly higher in the trans-abdominal ultrasound embryo transfer group. There was no significant difference between the two groups in ectopic pregnancy rate and miscarriage rates.

Meta-analysis of RCTs comparing Transvaginal ultrasound (TV-US) vs trans-abdominal ultrasound (TA-US) guided embryo transfer: Four studies were included in this analysis with a total of six hundred and thirty-five (635) patients. There was no significant difference between the two techniques in clinical pregnancy rate, ongoing pregnancy and live birth rate, ectopic pregnancy rate and miscarriage rate.

• Transvaginal ultrasound (TV- US) guided embryo transfer was compared with clinical touch embryo transfer in an observational retrospective study involving eight hundred and forty-six (846) patients [15]. This study found significantly higher implantation and pregnancy rates in the former group compared with the latter.

• Two dimension was compared with three-dimension trans-abdominal ultrasound-guided embryo transfer by Saravelos et al. [16] in a randomized controlled trial and found no significant difference in ongoing pregnancy rates, implantation rates, clinical pregnancy rates, ectopic pregnancy rates and miscarriage rates. Similarly, Li et al. [17] did not find any difference in pregnancy rate using either of the techniques in a single-blind, single-centre RCT.

In a large randomized controlled trial involving one thousand six hundred and forty-eight (1648) patients, Revelli et al. [18] compared TV UL MbET with transvaginal ultrasound (TA—US) guided embryo transfer (ET) and found comparable implantation rate, clinical pregnancy rate, and ongoing pregnancy rates between the two groups. However, there was a significant reduction in discomfort intensity score and proportion of patients with moderate to severe discomfort at embryo transfer in the UL MbET group.

In the evaluation of patient discomfort; comparing transvaginal ultrasound with transabdominal ultrasound-guided embryo transfer, Bondri et al. [19] reported uterine cramping rate to be comparable in the two groups. This study also noted 41% of light, 16% of moderate and 6% of severe discomfort associated with bladder distension in the TA US group, but Porat et al. [20] reported no significant difference between the two groups with regards to the degree of uterine cramping and pain during ET.

In recent times Karavani et al. [11] noted that transvaginal ultrasound scan (TV US) use was significantly associated with better visualization of the endometrial cavity and location of embryo transfer. There was also a significant reduction in anxiety, discomfort and pain using the transvaginal ultrasound (TV US) method.

Hassan et al. [21] compared TV US versus TA US-guided ET in obese women with BMI ≥ 30 Kg per sqm in a randomized controlled trial involving 800 participants and found significantly higher clinical pregnancy rates in the transvaginal ultrasound group compared to the trans-abdominal ultrasound group (37.8% vs. 30.8% P = 0.044). However even though the live birth rate was higher in the transvaginal ultrasound group (50.2% vs. 44.8%), the difference was not statistically significant. Pain associated via the visual analogue scale (VAS) was significantly less for the transvaginal ultrasound group compared with the trans-abdominal ultrasound group. Likewise abdominal discomfort.

The use of transabdominal ultrasound scan to assess the endometrial cavity and other pelvic structures and for the guidance of the embryo transfer procedure has been recommended by the ASRM as there is good evidence based on several RCTs that it improved clinical pregnancy and live birth rates [4, 22].

2.2 Type of transfer catheter used

There are generally two categories of catheter used commonly for embryo transfer, the soft and the stiff (rigid) catheter. Theoretically, it is thought that the stiff catheter is more likely to cause trauma during embryo transfer, while the soft catheter can easily meander its way through the curve of the cervix into the curvature of the endometrial cavity in an atraumatic fashion. This translates to less risk of plugging the catheter tip with blood, mucus or endometrium. The drawback for use of soft catheters is that sometimes they are more difficult to insert and could require assistance with the stylet.

To improve the ease of the ultrasound-guided embryo transfer, the echogenic tip catheter was introduced in clinical practice. A comparison of the Wallace soft echogenic catheter (sureview catheter) and the conventional soft Wallace catheter revealed comparative implantation and clinical pregnancy rate. However, the sureview catheter simplified the ultrasound-guided transfer process [23, 24].

Studies have compared the soft and rigid catheters in embryo transfer. The superiority of the soft catheter over the rigid catheter in terms of success rates of in vitro fertilization was demonstrated [25]. Many studies have favored the use of soft catheters over firm catheters [26, 27, 28, 29]. The use of soft catheters has relegated firm catheter use. Consequently, firm or rigid catheters are not used any longer as the first choice in modern-day practice.

The ASRM practice committee [22] has concluded that there is good evidence to recommend the use of soft embryo transfer catheters to improve in vitro fertilization embryo transfer pregnancy rates. However, there are limited data on live-birth rates and specific types of soft catheters used (Figure 1).

Figure 1.

(a and b) soft tip catheter.

2.3 Proficiency of personnel carrying out the embryo transfer

The proficiency and clinical experience of the operator carrying out the embryo transfer may be of great value in the success of embryo transfer.

Angelini et al. [30] reported a significant variation of 36.1% versus 20.6% [P ≤ 0.01] clinical pregnancy rate between two operators all other conditions remaining the same. It is a common place in medical practice that perfection comes with clinical experience consequently the outcome of embryo transfer is expected to be better with experienced clinician than trainees.

2.4 Site of embryo placement within the endometrial cavity

The actual portion of embryo deposit within the endometrial cavity has been a subject of scientific debate. Even though there tends to be a gradual resolution based on scientific evidence, continued research on the subject matter is still very valid. Embryo transfer site has been imperative in the determination of pregnancy rate in In Vitro Fertilization. The catheter must not touch to fundal endometrium as this could irritate the endometrium and cause contraction.

The position of the embryo deposited during ET has been measured largely in two categories viz.: Absolute position measured the distance from fundal endometrium and relative position according to endometrial cavity length. Controversies exist regarding which measurement to adapt universally for the absolute position. Many studies including a meta-analysis have shown that embryo placement at 2 cm distance from the fundal endometrium was associated with higher pregnancy rate, ongoing pregnancy rates and live birth rate [31, 32, 33]. The relative position of the catheter tip that is favorable for a higher success rate of embryo transfer was discovered to be close to the mid-point as reported in a randomized controlled [34]. Comparing the site of embryo deposit using the absolute or relative distance measurement Kwon et al. in a randomized controlled trial found no difference in pregnancy and implantation rate when the site of the embryo transfer was fixed at 2 cm to fundal endometrium or transfer at mid-point of the endometrial cavity length (Figure 2) [35].

Figure 2.

Catheter tip placement.

2.5 Embryo loading and discharge interval

The time between embryo loading into the catheter and discharge of the embryo could be a factor in outcome parameters. Anything that creates difficulty at the time of embryo transfer could elongate the embryo transfer time. Lee et al. in a retrospective cohort study found that longer transfer time does not negatively influence clinical pregnancy rate, implantation rate or live-birth rate [36]. This study however noted that difficulty in transfer negatively affects the clinical pregnancy rate. Some other studies however reported a significantly lower pregnancy rate when there is a longer loading discharge interval [over 60 seconds in some cases] [37, 38].

Ease of the transfer procedure which culminates in less procedure time has been associated with higher pregnancy rates compared with difficult transfers [39, 40].

2.6 The time interval between the discharge of the embryo and withdrawal of the catheter

Variability in the practice of time interval between embryo expulsion and catheter removal exists. While some practitioners remove the catheter immediately, others traditionally wait for 30–60 seconds before the withdrawal of the catheter. There are few documented studies on this subject. Two studies [a randomized controlled study and a follow-up cohort study] have compared immediate withdrawal versus delayed withdrawal [up to 60 seconds] and found that delayed catheter withdrawal was of no benefit in increasing pregnancy and live birth rate [41, 42]. ASRM practice committee [22] concluded that there is fair evidence to recommend the withdrawal of the embryo transfer catheter immediately after embryo expulsion.

2.7 Mock/trial/dummy embryo transfer

Some practitioners practise mock/dummy embryo transfer in the cycle preceding the in vitro fertilization cycle or sometimes at the time of oocyte pick up. Mock transfer aims to estimate cavity depth, determine the direction of the cervix and uterus, and establish if there is any cervical stenosis. It is used to determine the anticipated difficulty in embryo transfer and correct the obstacles before the actual transfer and in the situation of extreme cervical stenosis plan alternative routes of embryo transfer such as trans-myometrial embryo transfer.

Very few studies have been conducted on trial/mock embryo transfer. Two notable studies showed clear benefits of mock embryo transfer [43, 44]. One study expressed concern that the position of the uterus could change between the time of mock and real embryo transfer, they observed some retroverted uterus at the time of mock transfer changing to anteverted at the time of real transfer [45]. They, however, advise that patients with retroverted uterus at mock embryo transfer should still present with a full bladder for real embryo transfer since a significant proportion will be converted to an anteverted position.

2.8 Full bladder

Just like many other factors in embryo transfer, the benefit of a full bladder at the time of embryo transfer has been a subject of controversy. The anatomical portion of the uterus in most cases is that of anteversion with slight angulation at the internal os of the cervix. A full bladder is thought to passively straighten the uterine curvature. Straightening of cervico-uterine angle allows easy entry of the catheter into the uterine cavity [46, 47]. Bladder distension could create some level of discomfort to the patient and in the presence of retroverted uterus catheter visualization may be difficult.

Many clinicians would prefer to perform embryo transfer under ultrasound guidance with a partially filled bladder and some empty bladder. Their main reason is to avoid immediate post embryo transfer micturition. Lorusso et al. [48] in RCT concluded that bladder distension has not shown any possible impact on the in vitro fertilization success rate. Abou- Setta in a systematic review and meta-analysis, observed significantly higher clinical pregnancy and ongoing pregnancy rates with a full bladder [49]. The study observed that the need for instrumental assistance and incidence of difficulty was significantly greater with an empty bladder.

2.9 Flushing or removal of cervical mucus

Controversy exists about whether or not to remove cervical mucus from the ectocervix before embryo transfer. The presence of cervical mucus has been thought to interfere with the embryo transfer process by obstructing the passage of the embryo through the catheter tip. It may also pull the embryo back from the expulsion site. There is also the possibility of contaminating the uterine cavity with cervical microbes. Conversely removing the cervical mucus could be detrimental as it may cause bleeding in the cervix and may stimulate uterine contraction.

There has not been extensive research on this subject matter. However, of the few studies, published, majority favor removing cervical mucus before embryo transfer as this has been associated with a higher success rate at embryo transfer [50, 51].

The ASRM practice committee, therefore, concluded that there is fair evidence of the beneficial effect of cervical mucus removal at the time of embryo transfer in improving clinical pregnancy rate and live birth rate [22].

2.10 Bed rest after embryo transfer

Traditional practice in In Vitro Fertilization programs established bed rest for a variable duration following embryo transfer [up to 2 weeks in some instances]. It is presumed that the supine position and bringing physical activity to the barest minimum is likely to reduce the chances of embryo expulsion from the uterine cavity post embryo transfer. However, over the years the role of bed rest became questionable. This prompted a lot of research to determine the value of bed rest in the successful outcome of embryo transfer. The etiology of implantation failure in most cases has no relationship with physical activity. It can therefore be inferred that bed rest may not have a positive impact on pregnancy rate following embryo transfer.

One study followed via an ultrasound scan of the air bubble within the endometrial cavity in a patient who stood immediately after embryo transfer and discovered that the position of the air bubble before and after standing did not change [52]. Hence it was concluded that standing immediately after embryo transfer has no significant impact on the eventful position of embryo-associated air, consequently not associated with embryo expulsion.

The association between pregnancy rate following embryo transfer and physical activity level was assessed in a prospective observational cohort study which demonstrated that ambulation following embryo transfer has no adverse effect on pregnancy rate [53]. They, therefore, recommended immediate resumption of regular activity following embryo transfer. In the same light a meta-analysis by Cozzolino et al. [54], concluded that immediate mobilization after embryo transfer does not have any negative influence on the success of in vitro fertilization.

Some studies have shown a harmful impact of bed rest on the success of embryo transfer following in vitro fertilization [55, 56]. Garkwad et al. in a randomized controlled trial (among oocyte recipients in IVF donor oocyte cycle) showed a statistically significant live birth rate in the non-bed rest group and recommended a further study to evaluate the anatomical/physiological or psychological reason for the positive effect of physical activity on success rates post embryo transfer [56]. A study was abandoned in clinical trial for ethical reasons due to poor results in the group observing bed rest [57]. Waterstone et al. noted that the supine position makes the anteverted uterus, become vertical therefore predisposing its content to the action of gravity. As such bed rest could be seen as detrimental to the success of embryo transfer [55]. A series of systematic reviews and meta-analysis on randomized controlled trials concluded that bed rest following embryo transfer did not improve clinical pregnancy and live birth rates, instead, there was a reduction in implantation rate [58].

A Cochrane review did not provide any sufficient evidence to support the usefulness of bed rest following embryo transfer [59]. The ASRM practice committee summarized that there is good evidence not to recommend bed rest after embryo transfer [22].

2.11 The use of embryo glue

There is no robust evidence in favor or against the universal use of embryo glue in improving clinical outcome of embryo transfer. Most of the studies on this subject are limited by sample size and study design. Most studies have failed to demonstrate the universal benefit of embryo glue hence only showed that it could be beneficial in selected groups of patients particularly patients with repeated implantation failures [60, 61]. Yung et al. in a randomized, double blind controlled trial involving 550 women concluded embryo glue use does not improve live birth rate following frozen embryo transfer [62]. In a review article Atkinson and Woodland concluded that the use of embryo glue may be justified for selected group of patients since there is no evidence to show any harmful effect [63]. However, higher pregnancy and live birth rates using embryo glue (hyaluronic acid) were observed in a Cochrane review of 16 randomized controlled trial [64]. In this review, except for a higher rate of multiple pregnancy, there was no other complication associated with the use of embryo glue.

2.12 Acupuncture

The role of acupuncture in IVF-ET outcome remains controversial. Wang et al. in 2021 [65] took a critical review of the role acupuncture in IVF-ET outcome. In their review, 312 original randomized control trials and 65,388 participants were included. They concluded that even though acupuncture seem to provide beneficial effect in increasing clinical pregnancy rates, there were significant limitation to the studies, which weaken the power of the observed outcome. They further suggest that primary studies of high quality are in high need in this subject area.

While some meta-analysis showed positive efficacy of acupuncture in improving ET outcome [66], some have shown no benefit in outcome, particularly live birth rate [67, 68, 69, 70]. Despite the observation that the problem with the efficacy of acupuncture could be with the failure of actual protocol tested rather than acupuncture itself, the ASRM practice committee [22] concluded that there is fair evidence that acupuncture performed around the time of the embryo transfer does not improve live birth rate in IVF.

2.13 Other factors

Transcutaneous Electrical Acupoint Stimulation (TEAS): This involves placing electrodes over the acupoints instead of needles. There are limited data in literature on the role of TEAS in IVF outcome. A randomized controlled trial showed a significant increase in clinical pregnancy and live birth rates with TEAS performed on embryo transfer date [71]. However due to paucity studies on this subject the ASRM practice committee concluded that no recommendation for or against TEAS can be made with regards to improving ET outcome [22].

Use of sterile Latex-free gloves: The nature of the gloves used during embryo transfer has been thought to affect the outcome of the embryo transfer. Powdered latex gloves particularly have been taught to be detrimental to outcome of ET as the [power is thought to escape into the air or be in direct contact with the embryo, which is toxic to the embryo. However, in a randomize control trial involving 712 women comparing effect of powdered versus non-powdered gloves on ET outcome, the pregnancy rate was similar in both groups. The author therefore pointed that the key factors was avoidance of direct contact of the gloves or powder with the embryo [72]. ASRM practice committee [22] suggested no recommendation for any specific gloves to be used, as more research is needed to make any definitive inference.

Massage: Body massage is a non-invasive therapeutic modality employed to alleviate both psychological and physical stress and discomfort. It is thought to be of benefit in IVF ET outcome. There is however paucity of research to elucidate its actual effect on IVF ET [22]. One observational retrospective analysis however showed its benefit increased pregnancy and live birth rate [73]. More robust clinical studies is required in this area to ascertain the usefulness of massage in IVF outcome hence the ASRM practice committee submitted that there is insufficient evidence to recommend for or against massage therapy to improve IVF—ET outcome [22].

ASRM practice committee [22] looked at some other factors in patient preparation before and at embryo transfer. These factors include: the use of Analgesics, Anesthesia, Prophylactic antibiotics and Whole-System Traditional Chinese Medicine (WS-TCM). It was concluded that there is insufficient evidence to recommend for or against analgesics and WS-TCM to improve In Vitro Fertilization embryo transfer (IVF-ET). In the case of anesthesia, there is insufficient evidence that it improves outcomes. Therefore, knowing the possible risk of anesthesia and unconfirmed benefit, routine anesthesia is therefore not recommended to improve outcomes. Prophylactic antibiotics has shown no clear benefit and as such not recommended.

3. Conclusions

Embryo transfer is critical in the overall process of IVF. Consequently, adequate measures must be put in place to ensure its efficacy. Some factors have been associated with improvement in the outcome of embryo transfer procedures while other factors may have no value at all in outcome determination. It is also reported that some of these factors could have a negative impact on the outcome. Intervention supported by literature for improving pregnancy rates are [22]

• Abdominal ultrasound-guided embryo transfer.

• Removal of cervical mucus.

• Use of soft embryo transfer catheters.

• Placement of embryo transfer tip in the upper or middle central area of the uterine cavity greater than 1 cm firm the fundal endometrium, for embryo transfer procedure is completed.

• Immediate ambulation after embryo transfer procedure is completed

While some other interventions would require further research to authenticate their usefulness, others have been proven to be ineffective.

Acknowledgments

My thanks to Chioma Tojue who did the secretariate work and Dr. Farida Adeyemo who was of great assistance in completing the work’.

Special appreciation to my lovely wife Julie and children- Destiny, David, Derwin and Daniella, who tolerated my absence and gave all the moral support in the course of writing this text.