Optimal means of Preparing the Endometrium & Luteal Support in Frozen-thawed Embryo Transfer

Cryopreservation and frozen embryo transfer: An overview

Rapid advancement in the field of assisted reproductive technologies (ART) has boosted a parallel increase in the use of cryopreservation techniques for infertile individuals.¹   Cryopreservation, in reproductive medicine, is defined as the preservation of structurally intact sperms, oocytes or embryos at ultra-low temperatures.^2,3   The most well-established method of fertility preservation, which has received considerable importance in the ART setups, is embryo cryopreservation.⁴   This method serves as an attractive option for infertile individuals contemplating frozen embryo transfer (FET) for conception as it enables the excess embryos generated by ART methods to be stored and utilized for future requirement.⁵   In addition to preventing wastage of embryos, and increasing cumulative pregnancy rates, FET also mitigates the risks of multiple pregnancies and ovarian hyperstimulation. In light of these data, FET is considered both successful and safe for use, and has become an integral part of ART setups. In the recent years, the number of FET cycles performed has increased dramatically due to the trend towards transferring fewer embryos after a fresh IVF cycle, and as a result of advanced laboratory procedures.^5,6

The history of the first clinical pregnancy from FET stretches back to 1983, a year after which, the first successful birth through this method was reported.⁶   Looking back to those years, dimethyl sulphoxide (DMSO) was the main, traditionally accepted agent employed in the slow-cooling of multicellular embryos. Gradually, emergence of 1,2-propanediol and sucrose as cryoprotectants, and slow-cooling to -30℃ prior to plunging into liquid nitrogen, led to cryopreservation becoming the standard protocol in IVF programs. Today, slow freezing is widely accepted as a safe technique as it requires low concentrations of cryoprotectants leading to a negligible risk of toxic and osmotic cell damage. However, low concentrations of cryoprotectants may be inadequate for preventing ice crystal formation within the cells, which is a major cause of cell death.^1,7   Moreover, although this technique potentially helps freeze all kinds of human embryos, success rates obtained have been highly variable across clinical settings, casting doubts on its reliability.⁸   Slow-freezing is also time-consuming and demands costly equipments. Due to these drawbacks, cryopreservation by slow freezing is gradually phasing out, and novel protocols such as vitrification are receiving attention. In vitrification, both intra- and extracellular matrices apparently vitrify after cellular dehydration has already occurred. Compared to slow-freezing, vitrification involves the use of greater concentrations of cryoprotectants. Thus, ice cannot form in any part of the sample. As no ice forms, cooling does not have to be slow. The freezing solutions that are commonly used for vitrification are made up of permeating and nonpermeating agents such as DMSO and sucrose, respectively. In some protocols, the vitrification medium is also supplemented with macromolecules such as polyethylene glycol, or ficoll. Vitrification is believed to be a desirable method for the freezing of a small number of embryos at a time. Scores of research papers in literature validate its superiority over slow-freezing in terms of post-thaw survival rate, implantation rate, and clinical pregnancy rate. Moreover, unlike slow-freezing, vitrification offers the ability to control the solute penetration and dehydration rate, and maintain the physiological temperature during the Unlike equilibrium procedure. It is also more time efficient and less costly as it does not require expensive programmable freezing machine.^1,7,9-12

Influence of clinical factors on success rate in FET

The conception rate in frozen-thawed embryo transfer cycles is generally believed to be lower than that of fresh transferred embryos. Some authors have recorded biochemical pregnancy and clinical abortion in 15-20% and 20-25% patients undergoing FET, respectively. The factors responsible for impaired conception and increased spontaneous abortion rates after FET are far from completely known and remain an area of great interest and research. Recent research reveals that age of the woman, technique of oocyte fertilization (IVF or intra-cytoplasmic sperm injection (ICSI)), duration of infertility, FSH serum concentration, type of infertility (primary or secondary), embryo selection/quality, and endometrial parameters on the day of embryo transfer are some of the important factors influencing FET success (figure 1).¹³   Some of these factors are discussed here in brief.

Female age

Among women aged 35 years, diminished ovarian reserve, poor quality of remaining oocytes, decreasing endometrial receptivity and abnormal genetic factors are commonly seen. Reports have emerged demonstrating the pregnancy rate after FET to drop with advancing age. A group of investigators demonstrated the overall conception rate among women aged 30-34 years to be notably higher than that in women aged 35-44 years. The implantation rate was also remarkably higher in the younger women than the older ones.¹⁴   These findings were corroborated in another study which showed deteriorating clinical outcomes after FET cycles with advancing female age (figure 2).¹⁵

Serum FSH levels

Correlation between basal serum FSH level and the clinical outcome of FET has been extensively studied. Basal serum FSH level before IVF/ICSI cycle is believed to be inversely proportionate to pregnancy rates. A retrospective observational study¹⁶   demonstrated that the basal serum FSH in 25.2% women who had a clinical pregnancy was considerably lower compared to that in those who did not have a clinical pregnancy. This was in congruity with another report¹⁷   which suggested the implantation, biochemical, and clinical pregnancy rates to be more favorable in patients with low age-specific basal FSH concentrations.

Embryo selection

To enhance success rates in ART, selection of the most viable embryo(s) for transfer is deemed important. The view that cryopreservation of embryos increases the probability of implantation failure is widely held. However, recent developments in FET challenge this view. Transfer of a cryopreserved top quality embryo has been demonstrated to boost success outcomes in many patients after FET cycles. Reports have emerged which reveal that embryos of good quality before freezing have a reasonably good cryopreservation survival rate and are associated with a high conception rate. Some authors have further suggested that the use of frozen-thawed top quality embryos may lead to live birth rates comparable to those seen in fresh cycles.¹⁸

To achieve the best possible live birth rates after IVF while diminishing the possibility of multiple pregnancy, one or two embryos which seem to have the highest probability of implanting are chosen for transfer. Following this, supernumerary embryos with a reasonably good chance of implanting are selected for cryopreservation and possible transfer at a future date, and rest of the embryos are discarded. Among the available methods for embryo selection, morphological evaluation stands out as the most preferred. Based on multiple morphological characteristics at one or several stages of preimplantation development, embryos are selected for transfer. An alternative selection method is preimplantation genetic screening (PGS).¹⁹

Endometrial parameters

It is known that successful implantation of embryo depends on the degree of interaction between the embryo and receptive uterus (endometrium lining). FET cycles need to be synchronized so that the age of the embryos after thawing corresponds to age of the endometrium on the day of embryo transfer. The parameters that serve to predict endometrial receptivity, include endometrial volume, thickness, and artery blood flow.^20-22      A triple layered endometrial pattern and a thickness >7 mm indicate appropriate endometrial receptivity. A thin endometrium has a suboptimal vascular supply which leads to compromised endometrial receptivity, thereby jeopardizing success outcomes. In a recent study, the subendometrial-endometrial blood flow of 165 patients undergoing FET was evaluated. The results revealed that the pregnancy rates were much higher in the presence of subendometrial-endometrial blood flow than in its absence. In light of these data, endometrial thickness seems to have a critical bearing on the success of FET.²³   The importance of a sufficiently thick endometrium and the treatment protocols that may help circumvent thin endometrium in patients contemplating FET are discussed in the following sections.

Endometrial thickness: A success determining factor

Among the factors affecting implantation and conception in ARTs, endometrial thickness is deemed critically important.²⁴   It is utilized as an indicator of endometrial receptivity, as previously mentioned, and has been shown to be a valuable prognostic parameter for successful pregnancy in IVF.²⁵   A myriad of investigators believe that an endometrial thickness of less than a specific value is capable of reducing the likelihood of pregnancy to almost zero.²⁶   In an endometrial line of ²⁷

Accumulating evidences suggest that a thin endometrium reduces the chances of conception in relatively young patients (²⁸

The effect of endometrial thickness on clinical outcomes in FET cycles was specifically examined in a retrospective analysis²⁹   which recruited 35 patients with endometrial thickness of

Based on these data, adequate endometrial preparation logically becomes an important part of FET cycles.

Importance of endometrial preparation and luteal support

In many cases of infertility where there is no identifiable pathology, treatment to improve the poorly prepared endometrium has proved successful. However, achieving the optimal endometrial thickness remains a challenge in clinical practice.³⁰   The two main methods discussed in literature are:

• A natural cycle (NC) which is based on endogenous sex steroid production from developing follicles in individuals with regular ovulatory cycles; and
• An artificial cycle (AC) which is based on the sequential administration of estrogens and progestogens to imitate a normal cycle. It is more preferred in women with irregular ovulatory cycles.

The proper identification of ovulation and determination of the most probable subsequent period of adequate endometrial receptivity has critical bearing on the success of the NC-FET cycle. For correct identification of ovulation, detection of spontaneous LH surge is important; hence, monitoring of LH concentrations in blood or urine at appropriate time intervals is emphasized, as it is presumed that ovulation will occur 36-40 hours after LH peak. Unfortunately, the LH surges in urine lag up to 21 hours behind the appearance of the blood surge, making accurate evaluation both challenging and confusing. To address these issues, a modified-NC (mNC) has been proposed, which comprises hCG triggering when dominant follicle is of appropriate size (>17 mm), following ultrasonographic assessment to ensure the right timing of hCG administration.

The AC-FET is however different from NC-FET, as the timing of embryo thawing and transfer is decided as per the moment of progesterone supplementation in AC-FET. However, the sequential administration of exogenous sex steroids does not assure desired pituitary suppression, thereby increasing the probability of the endometrium being exposed early to progesterone. Just like the NC-FET, a modified cycle (mAC) has also been proposed for AC-FET which comprises co-treatment with GnRH agonist for down-regulating the pituitary and avoiding early follicular luteinizations. A metaanalysis that established a comparison between many of these endometrial preparation methods in FET revealed no noteworthy advantage of one specific approach for FET with regards to clinical pregnancy or live birth rates. When true NC-FET and modified NC-FET were specifically compared, no notable difference in terms of clinical conception, ongoing pregnancy or live birth rate was seen. Also luteal phase support was evaluated as potential influencing factor on pregnancy rate but the meta-analysis failed to provide strong evidence in support of a favorable effect of luteal phase support in patients undergoing NC-FET. However, other studies have contradicted this finding, and demonstrated luteal phase supplementation to reduce miscarriage rate and increase live birth rate in NC-FET cycles.³¹

There is voluminous evidence to show that endocrinal insufficiency during the luteal phase of the ART cycle signals an increased risk of abnormal or unsuccessful implantation.³²   Luteal phase defect is a clinical entity per se and a robust indicator of implantation failure in assisted conception. Luteal phase defect is regarded as a major problem in ART cycles due to very low luteinizing hormone (LH) concentrations during the luteal phase in agonist and antagonist cycles; and in artificial cycle FET (AC-FET), due to the lack of corpus luteum. Due to this support of corpus luteum function with aggressive luteal phase supplementation and monitoring is mandatory. The rationale of using luteal phase support is to recover the deficient endocrine secretion resulting from inhibition of LH release via the negative feedback mechanism at the hypothalamopitutary axis level caused by supraphysiological levels of during the early luteal phase.^33-35

Assimilating the aforementioned evidences, we can surmise that both endometrial preparation and luteal phase support are prudent for a successful FET cycle.

Estrogen and progesterone for endometrial preparation and luteal support in FET

Data on priming and preparation of the endometrium prior to and after embryo transfer has remained sparse. However, it is worthwile to examine the current evidences for the rationale and timing of hormone supplementation in embryo transfer cycles. Investigators have accumulated a large body of data showing estrogen and progesterone to play pivotal roles in endometrial receptivity, blastocyst implantation, and sustenance of pregnancy. It is also apparent from assisted conception studies that the ratio of progesterone to estrogen in serum is important for the secretory maturation of the endometrial glands during the midluteal phase. These findings have been corroborated by both experimental and human studies.

Clinical reports delineating the effects of these hormones report that they function to mobilize several molecular modulators in a spatiotemporal manner thereby facilitating embryo attachment. Further, they regulate the cascade of growth factors and cytokines which are the prime paracrine mediators of embryoendometrial dialogue. Progesterone during the luteal phase, particularly orchestrates the synthesis and secretion of numerous proteins.³⁶

In the past few years, artificial endometrial preparation, comprising induction of endometrial proliferation with estrogens and secretory transformation with progesterone, has gained importance.³⁷   In AC-FET, estrogen and progesterone are administered in a sequential regimen in order to mimic the endocrine exposure of the endometrium in the normal cycle. In the beginning, estradiol is administered to trigger the proliferation of the endometrium, while inhibiting the development of the dominant follicle. This is continued until the endometrium is observed to be 7-9 mm thick on ultrasound, at which time progesterone is added to initiate secretory changes.⁵   In this context, a study²¹   assessed the effect of sequential hormone therapy in FET. Oral estradiol valerate was administrated at 2 mg/d from cycle day 1-4, 4 mg/d from day 5-8, 6 mg/d from day 9-12. Administration with progesterone was done when the endometrium reached a thickness of 8 mm or maximum. The results demonstrated that this regimen increased the possibility of pregnancy in patients undergoing FET. In addition to improving success outcomes, this procedure also has the advantage of requiring minimum ultrasonographic monitoring and therefore can be scheduled more conveniently placing less burden on both patients and clinicians.⁵   Moreover, these hormone preparations are considered safe for use in these patients. A group of researchers³⁸   showed oral estradiol valerate 2 mg to significantly improve ultrasonographic thickness of endometrium and enhance endometrial response by producing adequate plasma levels of estradiol without any adverse effects.

Besides endometrial preparation, the role of estradiol and progesterone for luteal supplementation is FET is also well-recognized. The findings of a retrospective cohort study¹⁸   revealed that luteal phase progesterone administration during NC-FET resulted in significantly higher live birth rates when compared with natural cycles without progesterone. A similarly designed study³¹   evaluating 228 patients also showed that luteal support with progesterone led to a fall in miscarriage rate and an increase in the live birth rate in NC-FET cycles.

To take this notion a step further, an investigatory team³⁹   studied the effect of micronized form of progesterone in 435 women undergoing FET. The results suggested the live birth rates to be significantly higher in those who received micronized progesterone daily starting on the day of embryo transfer than those who did not receive progesterone (30% vs. 20%) (figure 3). Micronization leads to enhanced bioavailability of progesterone and increases its clinical potential.⁴⁰   Similar advantages are also seen with micronized formulations of estradiol valerate. According to Martindale, micronized preparation of estradiol is the only natural unconjugated estrogen which is orally active.⁴¹

In addition to the benefits of micronization, the clinical potential of the micronized, sustained release (SR) form of progesterone is also highlighted in the literature. It is suggested that the oral SR natural micronized form enables consistent tissue concentrations for endometrial support allowing once daily dosage convenience, thereby enhancing patient compliance. The pharmacokinetic profile of SR preparation is reasonably good as it reduces drug exposure to hepatic metabolic enzymes at individual time points, thereby offering sustained activity with negligible central adverse effects. Research papers confirm the success of ART procedures using oral SR natural micronized progesterone 400 mg administered once a day at bedtime with good tolerability and no significant undesirable effects such as drowsiness.^42,43

Conclusion

Cryopreservation is increasingly becoming a crucial component of successful ART cycles. It prevents the wastage of supernumerary embryos. It not only reduces the number of transferred embryos but also minimizes the risk of multiple pregnancies. Recent research reveals that age of the woman, technique of oocyte fertilization (IVF or intra-cytoplasmic sperm injection (ICSI)), duration of infertility, FSH serum concentration, type of infertility (primary or secondary), embryo selection/ quality, and endometrial parameters on the day of embryo transfer are some of the important factors influencing FET success.

In order to optimize pregnancy rates, it is of utmost importance that the development of embryo and endometrium is synchronized. This can be achieved by different methods. The simplest method of endometrium preparation is by employing natural cycle FET (NC-FET), which is based on endogenous sex steroid production from a developing follicle. An artificial cycle (AC) is based on the sequential administration of estrogens and progestogens to imitate a normal cycle. It is more preferred in women with irregular ovulatory cycles. Both endometrial preparation and luteal phase support are prudent for a successful FET cycle. A triple layered endometrial pattern and a thickness >7 mm indicate appropriate endometrial receptivity. A thin endometrium has a suboptimal vascular supply which leads to compromised endometrial receptivity, thereby jeopardizing success outcomes. Investigators have accumulated a large body of data showing estrogen and progesterone to play pivotal roles in endometrial receptivity, blastocyst implantation, and sustenance of pregnancy. Moreover, these hormone preparations are considered safe for use in these patients.

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