Understanding Implantation – Challenges & Management of Implantation Failure

Understanding implantation

Implantation in humans is an intricate but highly organized phenomenon that is dependent on multiple physiological and molecular processes.⁷   The event begins at the early stages of oocyte maturation, and specifically requires a mutual interaction between the blastocyst and the endometrium, eventually culminating in a small “window of opportunity” during which implantation can occur. This interaction involves the embryo, with its intrinsic molecular program of cell growth and differentiation, and the temporal differentiation of endometrial cells to attain uterine receptivity. By itself, the entire sequence is governed by an array of endocrine, paracrine and autocrine modulators, of embryonic and maternal origin.⁸

Histological changes in endometrium during normal ovulatory menstrual cycle

There exist three distinct histological stages in the monthly normal ovulatory endometrial cycle; menstrual, proliferative, and secretory phase (Figure 1).⁹   The proliferative phase – also referred to as the estrogen phase – begins at about 5 days after menstruation, and lasts for about 11 days. Estradiol (E) secreted by the ovary stimulates growth of the endometrium, and appears to trigger a series of events. The epithelial cells and stroma cells begin to proliferate rapidly, uterine glands starts growing and elongating, and spiral arteries begin to grow in order to supply the thickened endometrium. Rising levels of estradiol then trigger the midcycle luteinizing hormone (LH) surge, thereby inducing ovulation. At the time close to ovulation, the endometrium is about 3-4 mm thick. Now, endometrial glands secrete thin, stringy mucus, which acts to protect and lead the sperm into the uterus.

The ovulation event is followed by the secretory phase – referred to as the progesterone phase – that lasts for approximately 12 days. The change from proliferative to secretory endometrium is an essential part of achieving the receptive conditions required for implantation. The proliferative phase is associated with ovarian follicle growth and increased steroidal secretion, because of which, reconstruction and growth of the endometrium are achieved. The corpus luteum secretes large amounts of progesterone (P) and some estradiol. While estradiol leads to slight cellular proliferation in the endometrium, progesterone causes significant endometrial swelling, thereby converting the endometrium to an actively secreting tissue. Progesterone, in addition, inhibits myometrial (uterine smooth muscle) contractions, mostly by opposing the stimulatory actions of estradiol and prostaglandins. It thus augments vascularization, blocks estrogen activity, promotes decidua, enhances T-cell proliferation and evokes intense stromal edema. As such, the endometrium reaches a thickness of 7-8 mm about one week later the ovulation event. This entire process of endometrial changes is aimed at preparing the uterus for implantation of the ovum if fertilization occurs.

The process of implantation

The complex process of implantation is thought to involve a number of different stages: blastocyst apposition, adhesion, and invasion, which occur after the synchronous development of oocyte and maturation of the endometrium.⁸  Prior to implantation, blastocyst assumes a particular orientation as it approaches the endometrium, thus exhibiting an evidence of polarity. Then, the zona pellucida is shed once the blastocyst is oriented correctly (apposition). This is followed by blastocyst coming into contact with the epithelial layer and adhering to the endometrial surface (adhesion). In the adhesion phase, the polarized interaction between blastocyst and endometrium is established and becomes stronger, being mediated by adhesion molecules (integrins), immune cells, and cytokines.¹⁰   The final stage (of invasion) is characterized by the blastocyst penetrating the epithelial layer and invading the stroma. During trophoblast invasion, maternal tissue is degraded in a controlled manner to allow the blastocyst to enter the endometrial lining. The invasive capacities of trophoblasts are regulated, in both a positive and negative manner, by numerous cytokines including leukaemia inhibitory factor (LIF), interleukin-6, hepatocyte growth factor (HGF), granulocyte macrophage-colony stimulating factor, and others. These modulators interact via specific receptors with the trophoblast cells, in which they activate intracellular signalling cascades.^11,12

Pinopodes: A consistent morphological event observed during the short period of uterine receptivity is the formation of pinopodes, which are hormone-dependent protrusions on the apical cell membrane of the endometrial epithelium. Their presence may signify the nidation window on an individual basis.¹³   In normal fertile women, pinopodes are present on day 6-8 post-ovulation, whereas pinopode formation is observed 1-2 days earlier in patients undergoing controlled ovarian hyperstimulation.¹⁴

Timelines of “implantation window”

Successful implantation essentially requires appropriately timed arrival of a viable blastocyst into a receptive endometrium (see above). The endometrium is remodeled throughout the menstrual cycle, and exhibits only a short period of maximal uterine receptivity, better known as the ‘implantation window’. During a natural ovulatory cycle, the embryo enters the uterine cavity about 4 days after ovulation. The endometrium becomes receptive to blastocyst implantation 6-10 days after ovulation and remains receptive for about 4 days (cycle days 20-24) (Figure 2).¹⁵

Factors affecting implantation process

Several factors influencing this delicate process of implantation include: maternal age, endometrial receptivity, embryonic genetic abnormalities, immunological factors, and other uterine characteristics. A variety of congenital or acquired uterine pathologies (see below) could affect endometrial responsiveness to the circulating steroid hormones. Of note, endometrial abnormality at about the time of implantation may be a result of hormonal factors, or secondary to local, uterine pathology (Table 1).^16,17     These factors may affect pertinent characteristics of the endometrium and the embryo and their mutual interaction, thus having an impact on successful implantation.

The effect of advancing maternal age is evident in both normal and assisted reproduction. While there is a significant decline in human fecundity with advancing age, a significant decrement in success rate is also seen in older women undergoing assisted reproduction, including in-vitro fertilization (IVF).¹

Hormonal factors

The complex hormonal interplay plays a key role in entire pregnancy and begins early with implantation. Estrogen and progesterone are key hormonal modulators of endometrial development as they condition the uterus for implantation. Both the stromal and epithelial compartments express receptors for these hormones, wherein the response depends on the levels of these receptors and on the concentration of the hormones themselves. Over time, a better understanding has been gained concerning the types of receptors involved (ERa, ERb, PRA, PRB) and the dynamics of receptor expression.⁸   In effect, an appropriate cyclical pattern of receptor expression is crucial for achieving endometrial receptivity and successful implantation. Nonetheless, hormonal activity depends on not only the levels of these hormones and their receptors, but also on the rates of metabolism of these hormones. As well, their activity is also influenced by effects of co-activators and repressors, and their capacity to regulate the expression of several endometrial proteins.

Besides these dominant hormonal modulators of endometrial development – estrogen and progesterone, several other endocrinological factors may possibly play role in mediating endometrial function. Evidence from experimental models suggest that prostaglandins may facilitate increased vascular permeability during implantation, and enzymes involved in their production (COX-1 and COX-2) shown cyclical changes in expression. Human chorionic gonadotrophin (hCG) perhaps have direct effects on endometrium and also mediates interaction between the embryo and the endometrium, through chorionic gonadotrophin receptors present on epithelial cells.⁸   As well, androgen receptors are present on endometrial stromal and epithelial cells, and androgens (androstenedione and testosterone) induce changes in endometrial function that may impact implantation.

Intrinsic endometrial modulators of implantation

Intrinsic endometrial factors play important role in all phases of the implantation process. The epithelial cells of the endometrium are the first cells that an embryo encounters once it reaches the uterus. These cells secrete or express a variety of factors, such as LIF, which may affect not only the embryonic attachment but also further development of early placenta and embryo.^8,18

Effect of endometrial vascularity

Endometrial vascularity varies significantly during the menstrual cycle and is characterized by a pre-ovulatory peak and post-ovulatory nadir during the peri-implantation window. A dysregulation of endometrial blood flow may affect the implantation process.¹⁹   As such, endometrial vascularity appears to be an important determinant of endometrial receptivity during assisted reproduction. A decline in uterine artery resistance during the midluteal phase may reflect optimal vascularity for implantation of the blastocyst.²⁰

Effect of endometrial thickness and volume

There exists a positive relationship between endometrial thickness and pregnancy rates, which also affects the clinical outcome of IVF and embryo transfer.^21-23     It has been observed that no pregnancy does occur when the endometrium measures <7 mm.²⁴   As such, the target cross-section endometrial thickness is >7 mm, with a triple-line endometrial pattern, while endometrial thickness <6 mm is associated with a decreased probability of achieving a full-term pregnancy. Embryo implantation, clinical and ongoing pregnancy rates are significantly higher in patients with an endometrial thickness >9-10 mm.

While routine sonographic detection of average endometrial thickness is no guarantee for pregnancy, as it cannot quantify function, advances in three-dimensional ultrasound, together with automated software for endometrial measurements, provide more accurate and reproducible measures of endometrial volume.²⁵   However, endometrial volume was not found to be a better predictor of IVF outcome when compared to endometrial thickness measured on the day of hCG administration.

Embryonic factors in implantation

The embryonic factors affecting implantation include intrinsic features of the embryo, such as its genetic constitution, morphology and hatching. Abnormal genetic constitutions or unsuccessful transitions from maternal to embryonic transcription could account for many failures of early embryonic growth and implantation. Besides, a number of signals are there that needs to be exchanged between the embryo and the mother to ensure satisfactory implantation, such as platelet activating factor (PAF), prostaglandins, histamine related factors, steroids, proteins, metabolic products and immune-active factors. ²⁶

Improving implantation rates

Despite many advances in assisted reproductive technologies (ART), implantation rates are still low. Increasing embryo implantation rates to improve the overall efficacy of the IVF cycle is, therefore, the need of the hour. This goal may, in part, be achieved by adopting strategies aimed at improving the endometrial receptivity without disrupting the delicate process of implantation.²⁷   Essentially, efforts should be made to develop ovarian stimulation protocols that enhance the endometrial receptivity, to improve uterine vascularity, and to treat the underlying pathology.^1,28     As well, ensuring best embryo selection, and synchronizing embryo transfer with the time of optimal endometrial receptivity are also important. Several modalities of treatment that have a potential to improve implantation rates are discussed below.

Treatment of underlying gynecological conditions

As endometrial receptivity is a major limiting factor in establishment of pregnancy in a number of gynecological diseases, treatments to optimize implantation, in such cases, should be directed towards the underlying uterine disorder (Table 2).²⁸

The treatment of endometriosis is highly individualized and dependent on the desire for fertility or requirements for contraception, though there is little evidence to support the use of medical treatment in women with endometriosis who wish to conceive. The other option for treatment of endometriosis in such women is to remove the lesions with laparoscopic excision or laser/diathermy ablation.²⁸

The treatment of myomas for fertility preservation or enhancement is surgical removal (myomectomy), either by laparotomy, laparoscopy or hysteroscopy. The treatment goals include restoration of uterine morphology, return of normal menstrual function and enhancement of fertility.

The preferred treatment method of endometrial polyps is polypectomy. Herein, the operative technique may be dependent on the size of the polyp or their location.²⁸

Patients with hydrosalpinges have low implantation and pregnancy rates. The condition has been associated with altered endometrial histology and a lack of expression of endometrial adhesion molecules, which may play important roles in the implantation process.²⁹   Hence, treatment is advised in such patients prior to IVF treatment. The treatment options for hydrosalpinges include drainage, salpingostomy, proximal tubal occlusion and salpingectomy. Laparoscopic salpingectomy is advised for all women with bilateral hydrosalpinges prior to IVF treatment, and for a unilateral hydrosalpinx.²⁸   In cases where salpingectomy is technically difficult or not feasible, proximal tubal occlusion is a valid alternative.

In cases where inflammatory or infectious conditions alter endometrial receptivity, treatment should be directed toward eliminating the source of inflammation or infection.

Multiple other alternative treatment modalities have been studied. Systemic treatment with heparin and aspirin was performed to improve endometrial perfusion, intravenous immunoglobulins to modulate endometrial immune cell function, prednisone to suppress fetal rejection, recombinant LIF and progesterone to improve endometrial receptivity. However, with the exception of luteal phase support by progesterone administration in IVF, none of these treatments was shown to be efficient in increasing implantation or pregnancy rates.²⁸

Proper preparation of endometrium by using steroids [estradiol valerate (EV) + progesterone (P)]

As estrogen and progesterone play key role in modulating endometrial changes that occur during pregnancy, a unique way to modify the endometrium could be through external administration of these hormones in a closely regulated manner. Essentially all reported regimens of exogenous steroid administration for the induction of endometrial receptivity use E2 in combination with P administration.³⁰   Estrogen preparations used effectively promote endometrial growth and proliferation. In addition, progesterone helps in induction of receptive secretory endometrium.²⁷

EV for thin endometrium

A regimen designed to mimic the natural pattern of Estradiol (E2) levels in the circulation would consist of E2 administration in increasing doses – endometrial receptivity can be induced by exogenously administered E2 and P in a variety of regimens. However, the optimal duration of estrogen stimulation before the start of progesterone has not been established. In majority of normal responders, a short (5-7 days) stimulation phase seems sufficient, though most replacement protocols favor duration of 10-14 days. Duration may be extended if the endometrium fails to respond to a short course of estrogen. An artificial follicular phase length of 5 weeks has been evaluated, and found to result in optimal subsequent luteal-phase endometrial histology.³⁰

Role of EV supplementation in clomifene citrate induced cycles

Clomifene citrate (CC) is successful at inducing ovulation in 50%-75% of cases, but only 30-40% becomes pregnant, a difference that has been attributed to a negative action of this drug in form of prolonged anti-estrogenic effects on endometrial receptivity, which ultimately results in low pregnancy rates. Hence, estradiol valerate is used as an adjunct to counter these effects; addition of EV stimulates E2 receptors. The effect of addition of EV to CC on endometrial morphology and pinopodes was investigated by Favaedi et al in a randomized clinical trial. The results of this trial showed that adding EV to clomiphene can positively affect endometrial morphology and increases the pinopodes on implantation window during induction ovulation.³¹

A meta-analysis was conducted by Torres et al to determine the effectiveness of adding estrogen during CC-induced ovulation cycles among infertile women in improving endometrial thickness, endometrial echogenicity and pregnancy rates. This meta-analysis demonstrated that estrogen supplementation in clomiphene citrate ovulation induction increases endometrial thickness and serum estradiol levels with a trend towards increased chances of pregnancy among infertile women.³²   When reduced endometrial thickness is observed in CC-induced cycles, addition of 6 mg/day EV following the CC treatment can prevent the endometrial thinning without perturbing folliculogenesis and ovulation.³³

Controlled endometrial development

In ART, controlled formation of the endometrium is necessary.³⁴   The unique and key features of donor oocyte, in-vitro maturation (IVM), Frozen embryo transfer IVF cycles relate to the need for embryo/endometrial synchronization and exogenous hormonal support of early pregnancy until the luteal-placental shift.

Careful synchronization between endometrial developments with the embryos to be transferred must be orchestrated.³⁵   In order to synchronize, recipients with functioning ovaries are first down regulated with a long-acting Gonadotrophin-releasing hormone (GnRH) agonist, (women with ovarian failure do not require). GnRH-agonists render the patients functionally agonadal in order to evade cycle asynchrony between the donor and the recipient. A programmed regimen of sequential estrogen and progesterone replacement is used to simulate the natural cycle and to promote normal endometrial growth and maturation. The rationale is that GnRH-analogues have an indirect, central mechanism of action of inhibition, through pituitary desensitization, of the synthesis of gonadotropins, thus inducing profound hypoestrogenism.³⁶   Therefore, EV may be added to the regimen to balance the counterintuitive actions of GnRH-agonists.³⁷

A wide variety of treatment regimens have been used successfully to achieve controlled endometrial development and maturation. Micronized EV (2-6 mg) can be administered orally designed to achieve serum levels that approximate those observed in the late follicular phase in natural cycles (200-400 pg/mL). EV administration is aimed at mimicking the regular cyclic fluctuations throughout the cycle.³⁸   The duration is quite flexible and can range from as little as 7 days to as long as 3 weeks or more. Progesterone can be administered intra-muscularly (i.m), in doses designed to approximate20 ng/mL (50-100 mg daily) or vaginally in the form of suppositories, tablets (180-600 mg daily).³⁵

The methods and extent of monitoring vary among programs. Many use transvaginal ultrasonographic measurements of endometrial thickness, aiming to achieve a thickness greater than 6-7 mm, at a minimum. In the occasional woman not achieving desired endometrial thickness in response to the standard replacement regimen, vaginal estrogen administration can help to promote additional proliferation. To maximize the probability for successful implantation, embryo transfer must be carefully timed. In order to achieve the same coordination of embryo and endometrial development that occurs in natural conception cycles, progesterone treatment should begin on the day the donor undergoes retrieval. Estrogen therapy in general begins at or near the time that stimulation begins in the donor, allowing ample time to achieve the desired degree of endometrial proliferation before the donor’s retrieval.³⁵

Controlled endometrial development for frozen-embryo transfer cycle

In cases where a fresh embryo, ART procedure cycle is unsuccessful and frozen embryos are available, a frozen-thawed embryo transfer can be performed. Frozen-thawed embryo transfer enables surplus embryos derived from IVF treatment to be stored and transferred at a later date.³⁹   In some specific cases women may undergo oocyte donation treatment, a frequent treatment option for women who have ovarian insufficiency (premature menopause), poor ovarian capacity to respond to stimulation, or have had several unsuccessful IVF cycles; particularly for older women. In both such instances of egg donor and frozen embryo replacement cycles, adequate hormonal preparation of the endometrium is of importance in order to provide optimal chances of pregnancy.

Frozen-thawed embryos can be transferred in a monitored natural cycle in women with normal ovulatory function.³⁵   Alternatively, transfer can occur in an artificial cycle wherein endometrial development is carefully controlled using programmed sequential treatment with exogenous estrogen (oral micronized estradiol valerate 4-6 mg daily) and progesterone (50-100 mg daily i.m), beginning with or shortly before the onset of menses.³⁵   Preliminary down regulation with GnRH agonist also can be used, as is typical in donor oocyte recipients. A 2010 systemic review, which included 22 randomized trials comparing different endometrial preparation regimens, found no evidence regarding one method of endometrial preparation being superior to others. In both natural (days after ovulation) and artificial cycles (days of progesterone treatment), time of transfer is synchronized with the stage of embryo development, as in donor oocyte recipients.³⁵

Controlled endometrial development for IVM

Technically, the term in-vitro maturation (IVM) describes the maturation of immature oocytes in culture after their retrieval from follicles not exposed to exogenous LH or hCG in-vivo. In efforts to improve the relatively low efficiency of classical IVM, new methods involving preliminary “follicular priming” have been developed. One method involves FSH treatment for 3-6 days, followed by retrieval on cycle day 9-10. Another involves a single injection of hCG (10,000 IU), administered when the largest follicle reaches 10-12 mm in size and 36 hours before retrieval. A third method combines the two techniques, involving sequential treatment with follicle stimulating hormone (FSH) and hCG before oocyte retrieval.

In IVM, a dominant follicle or a corpus luteum is not routinely formed which compromises the follicular and luteal sex steroid contribution to the development of the endometrium, Therefore, adequate endometrial development is paramount in IVM. Barnes et al. suggested that the dominant follicle be spared at the time of retrieval and the luteal phase supplemented with hCG.⁴⁰   Most clinical protocols today rely on the use of exogenous E and P supplementation. EV is started on the day of oocyte retrieval for endometrial preparation. If the endometrial thickness is 6-8 mm a dose of 8-10 mg daily is given, and if it is 8 mm, a 6 mg dose is administered in divided doses. If endometrial thickness is less than 6 mm a dose of 12 mg is advocated. Timing of estrogen replacement has been debated with some authors advocating an earlier (mid-follicular phase) start.

Luteal phase support

In ART, the hCG or progesterone levels, or both, are low, making the natural process insufficient; hence, the luteal phase is supported with either progesterone, or hCG. Luteal phase support improves the implantation rate, and therefore pregnancy rates. However, the addition of GnRH agonist to the treatment regimen in IVF cycles is, by itself, thought to create a luteal phase defect⁴¹  , favouring hormone (estrogen and progesterone) supplementation. In this context, a benefit has been observed from addition of progesterone to GnRH agonist for the outcomes of live birth, clinical pregnancy and ongoing pregnancy.⁴²

Generally, controlled ovarian hyperstimulation with exogenous gonadotropins yields multiple corpora lutea that might well be expected to sustain supraphysiologic serum concentrations of estradiol and progesterone during the luteal phase of IVF cycles. Co-treatment with GnRH analogs for prevention of premature LH surge and luteinisation effectively suppresses endogenous LH secretion. This suppression of endogenous LH even continues after treatment with GnRH analogs, and can remain suppressed for as long as 10 days after treatment with a GnRH agonist ends. As such, luteal function is frequently inadequate in amount or duration. The abnormally low levels of LH during the luteal phase may be insufficient to stimulate and maintain the level of luteal function required to promote timely endometrial maturation in preparation for implantation, or to support an early pregnancy once established.³⁵   Per se, a defective corpus luteum function resulting from relatively short endogenous LH surge may be detrimental to endometrial receptivity.⁴³   Aggressive luteal phase support and monitoring is therefore essential considering the overwhelming evidence suggestive of abnormal luteal phase steroid profile. This may be achieved by use of adequate estradiol and progesterone supplementation in the luteal phase and the first trimester. Integrated estradiol and progesterone levels are abnormally low, accompanied with grossly short luteal phase duration, in GnRH antagonist treatment cycles, particularly when a GnRH agonist rather than hCG is used to stimulate the final stages of ooycyte maturation.

As it is difficult to predict who may or may not require luteal support in any given cycle, some form of treatment should be provided for all. Progesterone supplementation generally begins on the day of oocyte retrieval, or at the time of embryo transfer. Progesterone can be administered orally (300-800 mg daily), vaginally as cream or tablet (100-600 mg daily), or i.m (25-50 mg daily). Supplemental doses of hCG generally have been administered every 3 days (1500-2500 IU).³⁵   As well, there is no evidence to support that any one-treatment regimen is superior. Treatment regimens vary widely, from discontinuation of supplementation at the time of pregnancy test to continuation throughout the first trimester.³⁵

Since luteal phase endometrium also requires estradiol support, the potential benefit of estradiol treatment seems to be promising. A combined estradiol and progesterone substitution of the luteal phase of ovulation induction cycles was observed to increase the overall pregnancy rate. Furthermore, it was conceivable that a higher improvement in pregnancy rates could be possible if earlier estradiol substitution of the luteal phase is initiated.⁴⁴

Clinical evidence to support the addition of estradiol to progesterone substitution has been provided in a number of clinical trials. Lukaszuk et al studied 166 women younger than 40 years who were undergoing IVF with long protocol controlled ovarian hyperstimulation (COH). The researchers concluded that for women treated with a long GnRH analog protocol for COH, addition of a high dose of E2 to daily progesterone supplementation significantly improved the IVF embryo transfer results.⁴⁵   Later, Drakakis and colleagues also evaluated the effect of estradiol addition to progesterone supplementation during the luteal phase on pregnancy and implantation rates in patients undergoing IVF/ICSI-ET cycles, and noted higher pregnancy and implantation rates with estradiol supplementation without any adverse effects.⁴⁶   More recently, Chang and colleagues conducted a retrospective analysis of 155 poor responder patients subjected to IVF/ICSI to find out if administering E2 during the luteal phase of the preceding cycle and during ovarian stimulation in GnRH antagonist cycle could enhance follicular response and hence improve outcomes in poor responders. The investigators concluded that estrogen priming through luteal phase and stimulation phase improved ovarian responsiveness and this could lead to an increase in pregnancy rate in poor responders with failed cycle.⁴⁷

Luteal phase and early pregnancy support in donor oocyte cycle

In naturally conceived early pregnancies, progesterone helps in preparing the endometrium for pregnancy by stimulating proliferation in response to hCG that is produced by the corpus luteum. This occurs in the luteal phase of the menstrual cycle. The rapidly rising levels of hCG first “rescue” and then stimulate the corpus luteum to maintain high levels of estrogen and progesterone secretion necessary to ensure endometrial stability in support of early embryonic growth and development until the emerging placenta achieves the capacity to take that responsibility.³⁵   The donor oocyte recipient has no corpus luteum. Therefore, exogenous luteal support must be provided for the requisite interval. Normally, luteal-placental transition is completed between 7-9 weeks of gestation (menstrual dates), between 5-7 weeks after embryo transfer. Administration of exogenous estrogen and progesterone treatment therefore must continue until at least 7 weeks, though many recommended treatment until approximately 10 -12 weeks gestation, for added safety. Some prefer monitoring of serum concentrations of estradiol and progesterone during early weeks of pregnancy, decreasing the dose of exogenous hormone treatment by half after observing a sharp increase in levels, and discontinuing treatment after an additional week if concentrations continue to rise normally.³⁵

Luteal phase support in FET cycle

Many different protocols for Frozen-embroyo transfer (ET) (FET) cycles exists, and this applies to both “natural cycle” FETs and “hormone replacement cycle” frozen-thawed ETs. The latter is often the standard in many fertility centers. Hormonal replacement with only estrogen and progesterone is a frequently used protocol in women with and without functioning ovaries.⁴⁸

Protocols for luteal phase support also vary wildly, sometimes starting before transfer, and sometimes the day of transfer.^49,50     Box 1 depicts a typical hormone replacement cycle, controlled FET cycle with luteal phase support.⁵⁰

Luteal phase support in IVM cycle

A difference in implantation rate described for normal IVF and IVM, with no evident difference between oocytes retrieved, fertilization rate, and number or quality of the embryos transferred, except for endometrial thickness, may, however, be attributable to the fact that gonadotropin ovarian stimulation provides higher plasma levels of estrogen and consequently better endometrial preparation for embryo implantation.

In this context, Martins et al questioned the efficacy of the estrogen therapy used in IVM cycles for endometrial preparation that usually begins on the day of oocyte retrieval. The authors hypothesized that estradiol used since the beginning of the menstrual flow would be able to improve endometrial thickness and volume without loss on ovarian follicle count. Twenty women with PCOS were submitted to two estrogen therapy schedules: standard schedule; estrogen (estradiol valerate) began on day of planned egg retrieval (dosage based on endometrial thickness); and long schedule; estrogen began on first day of menstruation. Three-dimensional ultrasound was performed on the day of planned egg retrieval and one week later for endometrial evaluation. Results showed a higher endometrial thickness and volume in long schedule on both evaluations, thus clearly showing that early use of estrogen improves endometrial tissue. Interestingly, estrogens used since the beginning of menstrual flow did not reduce the ovarian follicle count or ovarian volume, events that would be deleterious for oocyte retrieval. The finding of long schedule may be important since the estrogen-progesterone therapy in IVM is generally continued until the 12th week of pregnancy.^51,52

Embryo characteristics

Morphology of the embryo

Significant morphological variations exist among oocytes that may affect the developmental competence and implantation potential of the derived embryo. These morphological variations may result from intrinsic factors, such as age and genetic defects, or extrinsic factors, such as stimulation protocols, culture conditions and nutrition.⁵³

The quality of the oocyte and embryo scoring can help to improve the implantation rates. It is therefore the vision and experience of the embryologist to select a good embryo after grading for transfer. In this context, pronuclear morphology and early cleavage rates are good indicators of healthy embryos.⁵⁴   In a cohort of morphologically good embryos, assessment for alignment/polarization of nucleolus precursor bodies (NPB) and/or early cleavage can, alongside conventional morphological criteria, serve as a simple non-invasive method for selection of embryos with high implantation potential.⁵⁵     Polarization of NPB in both pronuclei is as reliable marker of implantation as embryo morphology on day 3. However, pronuclear morphology assessment improves embryo selection only when it is combined with embryo morphology evaluation on day 3.^56,57

Assisted hatching

Hatching of the blastocyst from the zona pellucida is a natural process wherein the embryo expands and emerges before implantation.³⁵   Assisted hatching describes a variety of techniques for artificially thinning or opening the zona. The procedure is intended primarily to improve implantation potential, though varying results of clinical trials employing different techniques do not allow confident conclusions regarding its clinical value.^58,59     An assortment of methods has been used for assisted hatching, including zona drilling with acidified Tyrode’s solution, partial zona dissection with a glass microneedle, laser photoablation, enzymatic thinning, and the use of a piezo-micomanipulator. Routine or universal hatching is not warranted, as it may have benefit for selected individuals. Women aged more than 38 years appear to derive the most benefit from the procedure.²⁹

Blastocyst culture and transfer

Advances in cell culture media have led to a shift in IVF practice from early cleavage embryo transfer to blastocyst stage transfer. Culture of human embryos to the blastocyst stage (day 5 of culture) is repeatedly explored to identify embryos with higher implantation potential. The rationale for blastocyst culture is to improve both uterine and embryonic synchronicity and enable self-selection of viable embryos, thus resulting in higher implantation rates. Activation of embryonic genome occurs at the 8-10 cell stage (day 3 of culture). Embryos which cleave after day 3 in culture are therefore no longer dependent on maternal RNA transcripts and have made to successful transition from maternal to embryonic genomic control. Embryos that progress to blastocyst stage thus represent embryos with higher implantation potential, especially useful for patients with history of repeated implantation failures.²⁹

A recent Cochrane Database Systematic Review provided evidence for a small significant difference in live birth rates in favour of blastocyst transfer (Day 5 to 6) compared to cleavage stage transfer (Day 2 to 3).⁶⁰   However, cumulative clinical pregnancy rates from cleavage stage (derived from fresh and thaw cycles) resulted in higher clinical pregnancy rates than from blastocyst cycles. The most likely explanation for this is the higher rates of frozen embryos and lower failure to transfer rates per couple obtained from cleavage stage protocols. Further randomized controlled trials are required to enable ART consumers and service providers to make well-informed decisions on the best treatment option available.

Preimplantation genetic diagnosis

There is significant evidence identifying a close link between implantation failures in advanced maternal age with embryonic aneuploidy. Preimplantation genetic testing is used to identify genetic defects evaluated by removal of one or more nuclei from polar bodies (oocytes) or cells (blastomeres, trophoectoderm) from embryos. Preimplantation genetic diagnosis (PGD) determines the embryo’s genotype to identify whether the specific mutation or an unbalanced chromosomal complement has been transmitted to the oocyte or embryo. Preimplantation genetic screening (PGS) assesses an embryo’s karyotype, and has been used in screening chromosomal aneuploidy.⁶¹

Conclusion

In the pursuit of optimizing outcome with ART, the clinician has a profound responsibility to make every effort at enhancing the environment for implantation. In doing so, the ART provider will not only maximize the chance of pregnancy occurring following IVF, but also provide a healthy lifeline for the developing conceptus and fetus.

Implantation in humans is a very intricate process dependent on multiple factors, alongside the cross talk between the embryo and the endometrium. It therefore requires a better comprehension of the process of implantation and the relative importance of the factors involved to increase implantation rates further, given that implantation failure can occur because of impairment of embryo developmental potential and/or impairment of uterine receptivity and the embryo-uterine dialogue. Importantly, endometrial environment is a critical element in implantation process, particularly in form of the narrow “window of endometrial receptivity” or “window of implantation”. Hence, it requires developing better strategies to enhance endometrial receptivity, and, thus, improving clinical outcomes in form of successful pregnancies.

Medications play a crucial role in ART in stimulating ovaries to produce several oocytes and prepare the endometrium to be receptive after replacing one or more embryos into the uterine cavity. Their availability has in fact allowed the tailoring of several stimulation schemes, which have enhanced the pregnancy outcome after ART treatment. In particular, orally bioactive therapeutic preparations may play significant role in IVF treatment in the near future. Currently, GnRH-agonist and hormone supplementation with estrogen and progesterone is a key element of different ART protocols with a common intent to enhance the endometrial receptivity. As well, varying technological advances are being evaluated to modulate both maternal and embryo characteristics in order to enhance implantation process and pregnancy rates.

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