Conditions for an Embryo at Oviduct and Uterus

In vitro fertilization (IVF) strives to ensure an in vitro environment that closely mimics the physiological environment of gametes. However, the providing of such conditions is limited by the lack of our knowledge on the actual natural levels of oxygen concentration, pH, and temperature within the reproductive tract.

The environments within the oviduct and uterus differ significantly in terms of nutrient availability, and the presence of a complex mixture of cytokines and growth factors in the uterus [1]. The oviduct is characterized by relatively high levels of pyruvate (0.14–0.32 mM) and lactate (5.4–10.5 mM), with relatively low levels of glucose (0.5–1.1 mM). In contrast, the uterus contains a relatively high level of glucose (3.15 mM) [2–3]. The relative availability of nutrients within the female reproductive tract precisely reflects the changing requirements of the preimplantation embryo at each stage of development.

The degree of tissue oxygenation is defined by partial pressure (pO₂). In contrast to pO₂, which represents partial pressure in mm Hg, dissolved oxygen (DO) is a measure of oxygen concentration, i.e., the number of DO molecules in the liquid (mg/l). Comparison of pO₂ in tissue with DO in a liquid is not equal; therefore, the environment of the embryo in culture differs from the environment in Fallopian tubes and uterus. DO is reliant on various key physical factors, such as temperature, pH, and salinity, in addition to external pressures. Historically, atmospheric oxygen tension (~21% or 750 mm Hg) has been used in the medium of tissue culture and then in IVF embryo culture. Nevertheless, the physiological oxygen concentration in the female reproductive tract has been found to be lower, ranging between 2% and 8% [4]. This pO₂ in the Fallopian tubes is dependent on the blood circulation in the subepithelial capillary network. Moreover, pO₂ within the uterus during the reproductive cycle, as found in humans, monkeys, rabbits, and hamsters, is less than ambient air pO₂ at sea level. In addition, the intrauterine pO₂ also depends on hormonal activity during estradiol (E₂) and progesterone (P₄) fluctuations [5]. The intrauterine pO₂ has been shown to vary considerably between individuals, with a range of 6.4–32 mm Hg [6]. The fluctuations in intrauterine pO₂ within the estrous/menstrual cycle are not consistent, and there are no clear data about uterine pO₂ around implantation and the early pregnancy states. The physiological significance of reduced pO₂ in the female reproductive tract is mostly unknown. However, it seems that this is a functional phenomenon as a means to protect the developing blastocyst from oxygen toxicity.

Various levels of pH are found in different parts of the female reproductive tract. Lower pH levels are measured within the cervix and vagina compared with the Fallopian tubes and uterus. During ovulation, pH in the uterus and endocervical mucus was shown to be increased [7]. Biological buffers have been introduced into embryo culture media to help stabilize pH [8]; however, the in vivo pH regulation mechanism within the reproductive tract is likely to be more comprehensive than these culture media.

The potential involvement of temperature in embryo development is studied within the IVF procedure. The temperature for embryo culture is accepted to be close to the natural body temperature. Basal body temperature is diurnal, with minimum body temperatures measured in the early morning. Female body temperature also tends to show a biphasic pattern with a phase of the cycle, increasing in the luteal phase by 0.31–0.46°C compared with the follicular phase [9], with the latter attributed to the thermogenic action of P₄ [10]. The regulation of temperature within the reproductive tract is multifarious. The region of isthmus of the Fallopian tube (sperm storage site) is 1–2°C cooler than the portion of ampulla (fertilization site) [11], and variation in temperature may affect fertilization or embryo development. Extreme changes in body temperature may trigger an immune response, orchestrated by molecular networks of cytokines and miRNAs [12], which may injure the embryo. The temperature of 37°C is accepted as a standard and practically used in embryo culture incubators.

The preimplantation embryo is known to be highly sensitive to the environment as it undergoes a process of dynamic changes and reprogramming in preparation for implantation. Conditions within the uterus and Fallopian tubes fluctuate throughout the estrous and menstrual cycles. They tend to show an oscillatory biorhythm, which may be important for the survival of the preimplantation embryo and may be influenced by hormones, blood supply, tissue integrity, and other external factors. The “natural” environment of the preimplantation embryo remains to be fully characterized. There is no simple single solution which can translate the dynamic physiological conditions to an IVF laboratory setting. Although the transfer of zygotes and cleavage-stage human embryos to the uterus can result in pregnancies, in all mammalian species studied to date, the transfer of cleavage-stage embryos resulted in significantly compromised transfer outcomes compared with transfer at the blastocyst stage. Therefore, the transfer of a cleavage-stage embryo to the uterus places it into an environment that is not ideal.

The relative conditions, such as the pH of commercially available culture media, the temperature, and concentration of oxygen in IVF incubators are based on results extracted from animal studies.

Selection of Embryo for Transfer

One of the most lottery aspects of assisted reproductive technology (ART) is the selection of embryos that will be able to yield a healthy pregnancy. A number of selection criteria for rationalization of the process have been suggested, including the zygote appearance score (pronuclei and nucleoli orientation), the rate of embryo development (cleavage rate), morphological assessments (equality of blastomeres, fragmentation), blastocyst development, and preimplantation genetic testing (PGT). Embryo scoring techniques have been developed to help assess fetal implantation potential.

Morphological embryo assessment has been employed for many years as a tool for determining pregnancy potential. Embryos are scored based on cell count, fragmentation pattern, cytoplasmic pitting, blastomere regularity, and vacuole presence [15–18].

Fragmentation is one of the typical morphological features used in assessing embryo quality. Grading systems evaluate embryo quality based on the cell (blastomeres) count and the percentage of fragmentation observed in the embryo. Low implantation rates were reported from embryos with higher than 15% fragmentation on day 2 of development [19]. While not all fragmentation appears to be detrimental to embryo development, its pattern has a profound effect on the embryo’s developmental potential. Large fragments appear to be more detrimental than small fragments. The correlation between fragmentation and apoptosis is not clear; however, fragmentation may be a sign of apoptosis if regulatory proteins are altered [20].

Embryo scoring based on cleavage rate and morphology is one of the major predicting factors in maximizing pregnancy rates [19; 21]. Embryos with eight cells (blastomeres) on day 3 have a lower percent of chromosomal abnormalities than embryos with altered (more than eight or less than eight) numbers of blastomeres at this stage of development [22]. Blastomere volume also has an essential impact on embryo quality. A positive correlation exists between blastomere volume and the number of mitochondrial DNA copies [23]. The cell stage at the time of transfer has become a significant factor in identifying the embryos with the greatest implantation potential. Embryonic genome activation occurs between the four-cell and eight-cell stages of preimplantation development [24]. ET on day 3 or day 5 of development allows for the selection of embryos undergoing embryonic activation. In fact, the embryo selection criterion should be based on the dynamics of all embryo development stages from zygote “Z”-scoring of pronuclei to embryo parameters scoring on day 3 or day 5 of development.

Extended embryo culture to the blastocyst stage was suggested as a possible selection criterion of the most vital embryo for transfer (only half of all zygotes have the potential to develop to the blastocyst stage) as a means of reducing the risks of aneuploidy. Nevertheless, extended culture to the blastocyst stage does not eliminate embryos displaying chromosome abnormalities; ~40% of embryos exhibiting normal morphological development to blastocyst are aneuploid. Selection of blastocyst-stage embryos at this time may allow for the transfer of a single blastocyst (day 5). At this stage, the transfer is associated with a significant increase in implantation rate compared with cleavage-stage transfer (day 3) [25]. A scoring system for blastocyst development, based on cavity formation, inner cell mass (ICM) definition, and trophectoderm (TE) distinction, was first described by Dokras et al. [26] and later extended by Gardner et al. [27]. The two grading systems, compared by a randomized study, illustrated the superiority of the Gardner system over the Dokras system in predicting blastocysts with a higher chance of implantation and clinical pregnancy. Although the advantages of blastocyst transfer have been presented, efficacy of this time-point procedure is still in dispute. A subsequent analysis of the various studies involving blastocyst transfer revealed that there were striking differences in culture conditions. In particular, studies that did not find a benefit in blastocyst transfer had used atmospheric oxygen for embryo culture, whereas the studies reporting significant benefits from day 5 transfer had used 5% O₂ for embryo culture. This latter point highlights the difficulties of comparing studies assumed to have performed IVF in the same way. There is proof that blastocyst transfer increased implantation rates, decreased pregnancy losses, and slightly reduced the time to pregnancy.

Both meiotic and mitotic types of chromosome errors have been observed during preimplantation development. Meiotic errors primarily arise during oogenesis and become more frequent with advancing female age. Mitotic errors arise after fertilization and most commonly occur during the first three cleavage divisions. Mitotic errors lead to a phenomenon known as mosaicism (chromosomally distinct cells within the same embryo). PGT is a more reliable and useful technique to eliminate chromosomally abnormal embryos. Regardless of the many criteria proposed to assist in the selection process of the normal embryo, no single criterion offers a significant advantage over the others. Most embryo selection systems are based on a combination of criteria, including morphology, cleavage rate, and embryonic genomic activation.

Older patients (more than 35 years old) are more likely to have “slow” blastocysts, and this asynchrony, which increases with maternal age, should be taken into account when synchronizing the embryo to endometrium receptivity [28].

Despite the intensive research performed on the various aspects of human reproduction (genomics, proteomics, and metabolomics), most embryologists still daily use routine assessments using standard variables, which include developmental rate, based on cell counts on day 2 and day 3 and development of a blastocyst on day 5, and morphological features such as fragmentation, degree of symmetry in cleavage-stage embryos, and ICM or TE quality in blastocysts (Figures 11.1 and 11.2).

Figure 11.1 Embryo at different developmental stages.

Figure 11.2 Low embryo quality. (A) Cleavage-stage embryo; (B) blastocyst.

Assisted Hatching

Manipulation of the zona pellucida (ZP), termed “assisted hatching” (AH), has been introduced to favor embryo hatching and ultimately improve ART outcomes. Assisted zona hatching is a part of ART in which a small hole is made in the ZP, using a micromanipulation or laser, thereby facilitating ZP hatching. The AH procedure was first described by Jacques Cohen, who, in 1988, reported the first pregnancy after AH [29]. Various AH procedures have been developed to help embryos escape from their ZP during blastocyst expansion. These include mechanical incision of the ZP, chemical ZP drilling with acidic Tyrode’s solution (pH = 2.5), enzymatic zona removal with pronase treatment, laser-assisted AH, and piezo (Figure 11.3) [30].

Figure 11.3 Assisted hatching of cleavage embryo and blastocyst. (A–B) Partial dissection of ZP in cleavage-stage embryo; (C–D) ZP-drilled embryos; (E–F) ZP-drilled blastocysts.

During mechanical incision, the embryo is held firmly in position by the holding pipette, and an opening is made by introducing a dissecting pipette through the ZP, followed by rubbing the embryo gently against the holding pipette until the embryo is released [31].

When drilling with Tyrode’s solution, the solution is expelled with a micropipette until one-third of the ZP is dissolved to perform the thinning. A disadvantage of this procedure is the possibility of an embryo damaged by acidic pH.

Bathing embryos in pronase solution and thinning of the ZP by enzyme treatment before transfer yields similar effects to other AH procedures.

The introduction of laser has substituted acidic and enzymatic removal of ZP. Lasers represent an ideal tool for microsurgical procedures, as the energy is easily focused on the targeted area, producing a controlled and precisely positioned hole, with high interoperator consistency. Two methods of laser-assisted AH can be used. In the first method, the contact mode, the laser is guided through optical fibers touching the embryo and ultraviolet light is delivered by a glass pipette, or infrared light is delivered with a quartz fiber. In the second method, the noncontact mode, the laser beam is directed through the ZP using an optical lens tangential to the embryo. The noncontact mode using various laser wavelengths is preferred for gamete micromanipulation. The 1.48 μm laser can be focused through the conventional optics of an inverted microscope, to illuminate a polystyrene culture dish filled with culture medium, providing for easy non-touch and objective-driven targeting of the laser light to specific cellular subcomponents, such as the embryo ZP. The location, duration, and increment of the delivered energy can be precisely defined with the aid of computer programs. A laser can be used to thin or to make actual holes in the ZP. ZP thinning can be performed on the outer or inner side of the ZP. Both light electron microscopy and scanning electron microscopy have revealed no degenerative ultrastructural alterations of oocyte and embryo ZP following laser-mediated zona drilling [32]. In various protocols the time of AH varies, from immediately to 24 hours before ET, as well as irradiation time and size of the hole (10–40 μm). It should be emphasized that the live birth rate is not affected.

The clinical relevance of AH on fresh or frozen embryos within an ART program remains controversial [33–34]. There is no significant evidence that ZP drilling in selective embryos helps in poor-prognosis cases [35] or in cases of older patients [36]. The implantation rate of ZP-drilled embryos and control embryos was 25% and 18%, respectively [37], and selective AH appeared most effective in women over 38 years of age and in those with elevated basal FSH levels [37]. However, the benefit of AH in the older age group remains unclear [38]. An unselected group of ET patients showed similarity between the outcomes of AH performed using partial ZP dissection [39] or acidic Tyrode ZP drilling [40] versus non-treated. However, it has to be noted that AH might effectively increase embryo implantation rates in humans, but only in selected cases. Recommendation of the European Society for Human Reproduction and Embryology (ESHRE) and the American Society for Reproductive Medicine (ASRM) for this procedure remains unclear and has become even fainter over the years.

Embryo Transfer Procedure

The ET procedure is one of the most critical steps in the process of IVF (Figure 11.4). The success rate of ART directly depends on embryo quality and uterine receptivity.

Figure 11.4 Embryo transfer procedure.

The first step in ET is the “time-out” process, during which identification and matching of patients and embryos are performed. A double identity check of the patient, the patient’s file, and the culture dish(es) is mandatory immediately before the transfer. To perform ET the preparation of patient is required that includes positioning the patient, introducing the speculum, cleaning the cervix, and manipulating the uterus. There is no compelling evidence that patient position affects the outcome of ET [41], and it is recommended to choose a position most comfortable for both patient and therapist. A bivalve speculum is then gently introduced into the vagina to expose the cervix. Maneuvering the speculum can improve cervical–uterine alignment to allow easier access by the catheter. Removing mucus from the endocervical canal using sterile cotton swabs, or aspiration with a catheter improves clinical outcomes [42]. Also, it has been reported that passive bladder distension results in significantly higher pregnancy rates in contrast to patients with an empty bladder [43]. The ET procedure requires abdominal ultrasound for monitoring of catheter placement. Limited centers have utilized transvaginal ultrasound for ET, which has been associated with improved patient comfort relative to transabdominal ultrasound due to the lack of bladder filling [44]. Cervical dilatation can be performed in patients with cervical stenosis, where the passage of the catheter is challenging. The cervix can be cleaned by swabbing, vigorous rinsing, or aspiration to remove excess mucus; there is no clear agreement on the best method. To gain a better understanding of the patient’s anatomy, a “mock” ET (trial run that allows determining the best route and the ideal location to place the embryo in the uterus) can be performed [45]. Mock ET has been shown to minimize the problems associated with the actual ET procedure and to improve pregnancy and implantation rates. Usually, the ET procedure is performed without any anesthesia. Local or general anesthesia is sometimes required if the ET procedure is complicated.

ET pregnancy rates depend on the physician performing the procedure [46–47]. Examples of difficult transfers, retained embryos, or other mishaps can be recounted, many with happy endings [46].

Microbial contamination of the ET catheter tip is correlated with a significant reduction in pregnancy rate [48]. Prophylactic antibiotics administered during oocyte retrieval and on the day of ET significantly reduce the incidence of positive microbial cultures from ET catheter tips 48 hours after antibiotic administration [49], but have not been shown to improve clinical pregnancy rates.

There is insufficient evidence regarding the effect of acupuncture, analgesics, traditional Chinese medicine, or massage therapy on ET pregnancy rates.

Catheters

Many different catheters are commercially available for ET. Catheters are classified according to their tip structure, flexibility, and presence of a separate outer sheath, location of the distal port (end- or side-loading), degree of stiffness, thickness, length, diameter, and echogenic visibility. However, they are generally grossly classified as soft or stiff (or firm). There is little difference in concept and technology between ET catheters and so there is no distinctly superior catheter. The main characteristic required of a transfer catheter is the ability to maneuver it into the uterine cavity without causing trauma to the embryos and endometrium. Some advantage was shown with an increased chance of clinical pregnancy when soft ET catheters are used [50]. Soft catheters can more easily follow the contour of the endometrial cavity, reducing the risk of endometrial trauma or of plugging the catheter tip. However, at the same time, soft catheters are more difficult to insert and pass. In some cases, the insertion of the catheter is difficult due to cervical stenosis. An operable stylet device can be used with some soft catheters to negotiate a difficult internal os. In technically difficult ETs, mainly where difficulties are encountered in negotiating the internal cervical os, there is often a need for the stiffer firm catheters. The switch from soft to firm catheters for these transfers may account for help in ET and pregnancy rate. There is sufficient evidence demonstrating that soft ET catheters improve IVF-ET pregnancy rates.

Catheter Loading

The critical part of the ET process is embryo loading into the transfer catheter. The type of syringes and catheters selected for ET and how they are handled are very important. Three main options exist for transfer syringes: 1 ml glass Hamilton syringes, 1 ml rubber-free syringes, and 1 ml syringes with rubber stoppers. No clear advantage of one syringe type over another has been shown. Critical factors considered for selection of a particular device include the preferred amount of resistance when depressing the plunger and the type of plunger with regard to chemical toxicity concerns. Glass Hamilton syringes require sterilization between cases and can wear out over time.

Rinsing of the catheter, to remove any substances or debris that may be present inside the catheter before loading embryos for transfer, is recommended by some specialists. However, such rinsing can form air bubbles inside the catheter wall and may affect the smoothing of the medium flow during aspiration and embryo loading.

Once the catheter and syringe are selected, and catheters are prepped, various embryo loading methods can be used (Figure 11.5). Wide debate exists over the impact of air loading in the ET catheter. Alternation of medium–air loading can help with ultrasound visualization during the transfer. Various studies failed to show a clear advantage of the medium–air catheter loading method compared with medium only, in terms of pregnancy, implantation, miscarriage, or live birth rates [51]. Furthermore, the embryo can be stuck to an air bubble in the uterine cavity after ET and degenerate. An additional disadvantage of air in the catheter/uterus is the possible pH change of the expelled medium. Moreover, air is not generally found in the uterus and should not be present in the uterine cavity.

Figure 11.5 Embryo loading options. (A) medium loading; (B–F) variants in medium/air proportion.

The volume of media in the catheter may be an important factor impacting pregnancy and implantation rates. More fluid (50 µl) yielded superior pregnancy and implantation rates than smaller fluid volumes (20 µl) [52].

Time Frame of an Embryo in the Catheter

The timing of each ET step may also be involved in success rates. The interval between catheter loading and embryo discharge into the uterine cavity may affect IVF outcomes. At intervals of more than 120 seconds, both implantation and pregnancy rates were lower [53]. This phenomenon may be related to environmental stress caused by exposure of the embryo outside the incubator. Thus, minimizing the time between loading and transfer of embryos is highly recommended.

Depth of Catheter Placement in the Uterus

There is no consensus to the depth of placement of the ET catheter within the uterus. It is widely accepted that contact with the uterine fundus can stimulate contractions that may be responsible for ET failure or ectopic gestation [54–55]. High-frequency uterine contractions on the day of ET have been found to decrease implantation and clinical pregnancy rates, possibly by expelling embryos from the uterine cavity [56].

Embryos placed too high in the uterine space may increase the probability of endometrial trauma and may induce uterine contractions with potentially adverse effects. Significantly higher pregnancy rates were obtained when the selected location was approximately 2 cm from the uterine fundus compared with 1 cm from the fundus [57]. Results of others show no difference in implantation and pregnancy rates when embryos were placed in the upper versus lower half of the endometrial cavity [58–59].

Uterine movements must be distinguished from uterine contractions. Ultrasound assessments have shown that endometrial wave movements flowing from the cervix to fundus and from fundus to cervix begin at both ends simultaneously. The intensity of the endometrial movements differs in every patient at different times. The uterine wave movements help the embryo implant into the “right place.” We can conclude that expulsion of the embryo during ET to the exact point is not really necessary.

Complications during ET may be related to inability or difficult ability to insert the catheter into the uterine cavity leading to trauma of the embryo and/or endometrium. That is one of the critical determinants of success. Easy or intermediate transfers resulted in approximately two-fold higher pregnancy rate than complicated transfers. In order to assist more accurate catheter placement within the uterus, ultrasound-guided ET is highly recommended instead of relying on clinician “feel” [60]. Improvements in both implantation and clinical pregnancy rates can be achieved by using ultrasound-guided ET [61]. Transabdominal and transvaginal ultrasound appear to be similarly effective in terms of pregnancy outcomes [62].

Embryo Expulsion Speed

The exact speed of embryo–fluid expulsion into the uterus is unknown and may also impact ET outcome. Rapid expulsion increases the rates of shrunken and/or collapsed embryos and the apoptotic index of rapidly injected embryos is higher than those injected slower [63]. A pump-regulated ET device provided reliable and reproducible injection speeds, whereas manual injection showed considerable variation in speed even with a standardized protocol [64]. Great caution is required with expulsion speed to avoid embryo trauma. To prevent embryonic damage inject the fluid as slow as possible. During the ET procedure, the syringe plunger should be gently pushed to the end of a syringe without depression; negative pressure built up by depression may suck the embryo back.

To minimize potential embryo expulsion from the uterus following ET, biological glue (hyaluronic acid [HA]- or hyaluronan-enriched medium) has been used to attach the embryo to the endometrium at the site of embryo deposition. However, the hatched blastocyst continues to move within the uterine cavity and routine use of embryo glue, as ET medium, does not improve the ART outcomes [65]. From another side, HA improves clinical pregnancy rates in patients with previous IVF failures [66], and the use of HA-enriched ET medium is beneficial [67], increasing the live birth rate by 8% [68]. The potential positive complimentary effect of HA on IVF outcome cannot be denied. HA is a member of the glycosaminoglycan family, secreted into the extracellular matrix from the cell surface. HA has a wide variety of physiological functions in the body, including maintenance of a viscoelastic cushion to protect tissues, control of tissue hydration and water transport, lubrication of bio-interfaces, the formation of large protein and proteoglycan assemblies, and receptor-mediated signaling roles in cell detachment, mitosis, migration, and inflammation. HA secretion is regulated by interleukins and gonadal steroids [69]. HA presents in follicular, oviductal, and uterine fluids. Human embryos at all stages of development possess HA receptors [70], and HA and its receptor are present in the uterine endometrium with the most abundant expression at the time of implantation. HA may directly stimulate the embryo growth or provides the nourishing environment. Cell–cell adhesion and cell–matrix adhesion have been shown to be increased by HA, which may facilitate the apposition and attachment of the embryo. HA and its receptor CD44 have both been implicated in the angiogenesis of endometrium. HA can promote angiogenesis by both degradation products and interaction with epidermal growth factor. On the day of implantation, uterine HA levels increase significantly and appear to be associated with regions that contain stromal cells that are proliferating in preparation for embryo implantation.

Once the embryo is discharged from the embryo catheter, the physician can immediately withdraw the transfer catheter or pause before doing so. Immediate withdrawal and a 30-second delay were found to have no difference in pregnancy rates [71].

It was noted that the presence of mucus on the ET catheter, once it is withdrawn, is not associated with a lower clinical pregnancy rate or live birth rate [72]. The presence of blood on the catheter following ET was also not adversely associated with pregnancy rates [73]. However, others have reported that the presence of blood on the catheter has been related to poorer results [74–75]. Intrauterine blood coagulating with the embryo inside the uterus may lead to the adverse effects. To summarize, the success rate of IVF depends on factors such as patient preparation, medication, mock ET, type of catheter, and optimal transfer technique.

After ET, the catheter should be carefully examined to rule out embryo retention; this should be done by gently rinsing the catheter, avoiding bubbles.

Bed Rest

Historically, in the past, bed rest following ET has been advised. During the early years of IVF, there were significant variations in the time recommended to remain in a supine position, practiced in hope of avoiding uterine contractions and “premature expulsion” of embryos from the uterus. Anecdotal reports have included durations of bed rest for 24 hours to as long as 2 weeks. To date, there is no significant evidence that bed rest improves the implantation rates [76–77]. Moreover, some works have even indicated a possible detrimental effect of bed rest [78]. Thus, although recommendations should be individualized to patient preference and anxiety levels, bed rest after ET has no proven benefit.

When Should the Embryo Be Transferred?

Embryos can be transferred on day 2, day 3, day 4, or day 5 postfertilization. The highest pregnancy and implantation rates were reported when embryos were transferred on day 3 rather than on day 2 [79]. Another report showed no changes in implantation or live birth rates when delaying transfer from day 2 to day 3 [80]. Delaying ET until day 4 resulted in similar implantation and pregnancy rates to those achieved following ET on day 2 or day 3 [81]. No significant difference in pregnancy and implantation rates was determined when ET was performed on either day 3 or day 5 [82]. However, delay of ET to day 5 enabled the identification of embryos with very high implantation potential. Clinical pregnancy rates were almost double those recorded for day 3 transfers when exclusively cavitating embryos were transferred on day 5 [25; 83–84]. The number of ET cycles necessary until achieving the first live birth was significantly lower for embryos transferred on day 5 compared with day 3. However, the cumulative live birth rates were the same for cleavage-stage and blastocyst-stage transfers.

One of the disadvantages of delaying ET to day 5 is that ET may be canceled if the cleavage-stage embryo will not develop to blastocyst in culture. It should be noted that in patients with a high number of embryos, blastocyst transfer is desirable. Overall, blastocyst transfer has advantages over cleavage ET, in both higher implantation rates and lower miscarriage rates, but with an increased risk of monozygotic and monochorionic twins.

Transfer of the Frozen-Thawed Embryo

Frozen–thawed ET (FET) is a procedure used for the transfer of cryopreserved embryos. FET prevents embryo waste and increases the chance of pregnancy in a single stimulated cycle. FET in a “more physiologic,” non-stimulated endometrium may not only result in higher pregnancy rates [85–86], but may also decrease maternal and neonatal morbidity [87–88]. It is suggested that the natural cycle (NC) is superior to hormonal replacement treatment because the NC is close to the physiology of a natural process. In terms of ET timing, initiation of P₄ intake (luteal support) should preferably be on the theoretical day of oocyte retrieval, and blastocyst transfer should be at human chorionic gonadotropin (trigger) + 7 days or luteinizing hormone (surge) + 6 days in modified or true NC, respectively.

Hence, FET should be performed at optimal window time when the embryo seeking implantation matches to the receptive/selective endometrial stage. Although FET is increasingly used for multiple indications, the optimal protocol has not yet been determined.

FET and fresh ET appear to result in similar pregnancy outcomes [89]. Singletons born after cryopreservation of embryos had a significantly higher birth weight (macrosomia) than those conceived after fresh ET [90]. This increased risk of higher than gestational age weight cannot be explained by intrinsic maternal factors or birth order [91]. The increase in birth weight ranged from 50 g to 250 g [92]. This phenomenon may also be the result of the epigenetic effect of freezing–thawing procedures, manifested by abnormal methylation of the H19 gene in both maternal and paternal alleles.

Whether fresh or frozen ETs yield the best clinical outcomes is still under discussion. Elective freeze-all embryo cycles are growing in popularity due to increasing evidence of success rates of FET [93]. However, FETs have added benefits only for patients who produced large numbers of oocytes (15 or more at collection). Such patients had slightly higher birth rates for frozen compared with fresh embryos. Patients who produced low (1 to 5) or intermediate (6 to 14) numbers of oocytes had higher birth rates with fresh compared with frozen embryos [94]. In addition, freezing can also lead to another 1 or 2 months of waiting without knowing if the procedure will work, which can be emotionally draining for patients.

How Many Embryos Should Be Transferred?

When considering how many embryos to transfer, several variables must be taken into account, i.e., prognosis for live birth, the health risks for the children, the costs to the couples and society. A few decades ago, the implantation rate was low, but was considered acceptable for an IVF clinic. Taking into account the low implantation rate, transferring two or more embryos was considered necessary to achieve an acceptable implantation rate or pregnancy rate. Although high success with multiple blastocyst transfer leads to a high multiple pregnancy rate (MPR), the high MPR has been a problem for ART (pregnancy complications, premature delivery, and health of newborns) over the past two decades. Multiple pregnancy is associated with well-documented increases in maternal morbidity and mortality as a result of gestational diabetes, hypertension, cesarean delivery, pulmonary emboli, and postpartum bleeding. It also increases the risk of premature birth, resulting in fetal, neonatal, and childhood complications as a result of neurological insults, ocular and pulmonary damage, learning disabilities, retardation, and congenital malformations [95–96]. Reducing the MPR has become a crucial public health requirement in IVF practices. The most effective method to avoid multiple pregnancies (aside from monozygotic twinning [MZT]) is the transfer of a single embryo [84].

MZT is a rare phenomenon in humans, occurring in 0.42% of spontaneous pregnancies. MZT occurs more frequently after single blastocyst transfer compared with cleavage-stage ET [97]. Several studies also reported monozygotic twins and triplets following single blastocyst transfer [98–99].

A cumulative live birth rate following single ET (SET) is not substantially lower than that of double ET (DET) [100], but the multiple birth rate is markedly reduced. If no live birth occurs in the fresh SET cycle, a SET of a cryopreserved–thawed embryo is performed. The SET strategy is better than the DET strategy when considering a number of deliveries with at least one live-born child, the incremental cost-effectiveness ratio, and maternal and pediatric complications [101].

After SET, the prevalence of multiple pregnancies with splitting is 1.36% [102]. Splitting pregnancies are associated with frozen–warmed ET cycles, blastocyst culture, or AH. In fresh SET cycles, the prevalence of splitting pregnancy after single blastocyst transfer is significantly higher than after SET cycles with cleavage embryos [102]. However, there is no evidence that splitting is a result of ovarian stimulation and fertilization methods.

In summary, the single embryo transfer is more beneficial than multiple embryo transfer.