Endometrial preparation and its impact on implantation

Introduction

Embryonic implantation is a dynamic process of paracrine interactions between the maternal compartment and the conceptus and involves a receptive endometrium and a developmentally competent blastocyst.

The process of implantation of a fertilized egg in the human uterus is governed by two crucial factors: endometrial receptivity and synchronization between embryonic and endometrial development. In a natural cycle, both these factors are related directly to cyclic ovarian hormone production and the timely occurrence of ovulation. In assisted reproduction, however, the events of ovarian stimulation of the donor and the artificial preparation of the endometrium of the recipient are completely separate. Therefore, it becomes essential to suitably manipulate endometrial growth with sequential administration of estrogen and progesterone in order to make the endometrium receptive to implantation.¹

Normal menstrual cycle

The menstrual cycle is controlled by the endocrine system. Normally, the endometrium proliferates under increasing levels of estradiol. This is known as the proliferative or follicular phase. Endometrial regeneration and proliferation are on-going by the time that menstrual sloughing is completed (days 5-6) and continues until day 14-15 of a normalized 28-day cycle. This is followed by the secretory or luteal phase, in which progesterone takes over estradiol as the main hormonal influence. The first rise in serum progesterone starts at the same time as the luteinizing hormone (LH) surge (day 13) and is appreciable by day 15 of the cycle.²   Day 16 is the earliest day on which secretory changes are consistently observed.³   These events are graphically depicted in Fig. 1.

Successful implantation requires the appropriately timed arrival of a viable blastocyst into a receptive endometrium.

The main function of the endometrium is to create a suitable environment for embryo implantation and pregnancy sustenance.

The endometrium is remodelled throughout the menstrual cycle. The proliferative stage prepares the endometrium for implantation of the blastocyst. Cells reach their maximum growth and sensitivity to the blastocyst at the time of implantation. This period of maximum sensitivity is referred to as the receptive phase and has a limited duration or window of time – the ‘implantation window’. Successful implantation and pregnancy depend upon the precise coordination of events that change the receptivity of the uterus.^4-6

In humans, during a natural cycle, the embryo enters the uterine cavity approximately 4 days after ovulation.⁷   The endometrium becomes receptive to blastocyst implantation 6-8 days after ovulation and remains receptive for about 4 days (cycle days 20-24).⁸

In order to improve implantation rates in stimulated cycles, it is important to pinpoint the implantation window, select the best embryo and synchronize embryo transfer with the time of optimal endometrial receptivity. In addition, ways of evaluating and enhancing endometrial receptivity and embryo quality without disrupting the process of implantation itself must be identified.⁹

Mimicking the natural cycle by hormonal manipulation of endometrial maturation

Assisted reproductive techniques (ART) have revolutionized the treatment of infertility. One of the well accepted methods of assisted reproduction is oocyte donation (Fig. 2). This technique is indicated in two types of infertility, namely:

• Infertility due to loss of ovarian function – It may result from premature menopause (spontaneous or subsequent to surgical removal of ovaries), chemotherapy/radiotherapy or gonadal dysgenesis (with or without chromosomal abnormalities)^10,11  
• ŠInfertility with functioning ovaries – It may be due to poor oocyte retrieval when various ovarian stimulation regimens prevent the development of follicles, genetic abnormalities or inaccessible ovaries^12-14  

Progesterone and estrogen are the major hormonal modulators of endometrial development. Ovarian estrogen and progesterone condition the uterus for implantation. Hence, in IVF programs for women with or without functioning ovaries, the endometrium is prepared artificially using exogenous hormones in a manner that mimics the natural cycle in preparation for embryo transfer (ET).¹⁵

Lutjen et al ¹⁶   were the first to devise such artificial endometrial cycles to induce pregnancy in a young woman with premature ovarian failure. For hormonal replacement therapy, estradiol valerate and progesterone were both given in varying concentrations so as to mimic the regular cyclic fluctuations of ovarian hormones in the normal cycle. Since then, several estrogen and progesterone protocols with different delivery routes, daily doses and active ingredients, have been used successfully.

In in vitro maturation (IVM) cycles, estrogen therapy for endometrial preparation usually begins on the day of oocyte retrieval. This practice was questioned by Martins and colleagues,¹⁷   who hypothesized that estradiol used since the beginning of the menstrual flow would be able to improve endometrial thickness and volume without loss of ovarian follicle count. In order to compare this alternative estrogen therapy with the standard one, they assigned 20 women with polycystic ovary syndrome to two estrogen therapy schedules:

• Standard schedule, in which estradiol valerate was begun on the day of planned egg retrieval (DPER), i.e., 14 days after the beginning of the menstrual flow. Dosage was based on endometrial thickness – 6 mg/day if ≥5 mm and 10 mg/day if <5 mm.
• ŠLong schedule, in which estradiol valerate was started on the first day of menstruation, and the dose was sequentially increased – 2 mg/day for 4 days, 4 mg/day for 4 days and 6 mg/day until DPER, and then as per the standard schedule

In both groups, dehydrogesterone, 30 mg/day, was begun 2 days after DPER (day of planned intracytoplasmic sperm injections). All the patients stopped the use of estradiol valerate and of dehydrogesterone one week after DPER (Fig. 3). Natural micronized progesterone can also be used instead of dehydrogesterone.

No oocyte retrieval or embryo transfer was performed. Endometrial thickness was evaluated by three-dimensional ultrasound on the day of planned egg retrieval and one week later, i.e., on days 14 and 21 after the beginning of menstruation.

The long schedule was found to result in a higher endometrial thickness and volume on both evaluations, indicating that early use of estrogen improves endometrial tissue and requires lower daily dosage.

The significance of endometrial thickness with respect to the outcome of medicated frozen-thawed embryo replacement (FER) cycles was evaluated in a retrospective observational study.¹⁸   Patients were started on a daily dose of 6 mg of oral estradiol valerate on menstrual day 1, and vaginal progesterone (800 mg daily) was administrated when the endometrial thickness reached 7 mm or more, with embryo transfer (ET) taking place 2-3 days later. The FER cycles were categorized according to endometrial thickness on the day of progesterone supplementation.

Analysis of the 768 consecutive medicated FER cycles revealed the following facts:

• The lowest pregnancy rates were associated with endometrial thickness <7 mm and >14 mm
• ŠSignificantly higher implantation (19% vs. 12%), clinical pregnancy (30% vs. 18%), ongoing pregnancy (27% vs. 16%), and live birth (25% vs. 14%) rates were achieved in cycles where endometrial thickness was 9-14 mm, compared with those in which endometrial thickness was 7-8 mm (Fig. 4).

The above results led to the conclusion that in medicated FER cycles, an endometrial thickness of 9-14 mm measured on the day of progesterone supplementation is associated with higher implantation and pregnancy rates compared with an endometrial thickness of 7-8 mm.

Preparation of endometrium in patients with ovarian failure

Estrogen replacement protocols

Estradiol valerate and micronized estradiol are the most extensively used hormones for estrogen replacement therapy. Most workers^19-23      have used estradiol valerate in various concentrations throughout the cycle, as follows:^16,24,25

• Days 1-5: 1 mg
• Days 6-9: 2 mg
• Days 10-13: 6 mg
• Days 14-17: 2 mg
• Days 18-26: 4 mg
• ŠDays 27-28: 1 mg

According to Norfolk’s protocol, micronized estradiol is given as follows:¹³

• Days 1-5: 2 mg
• Days 10-13: 4 mg
• ŠDays 14-28: 1 mg

The oral estradiol regimen²⁶   has several advantages:

• It can produce consistent serum values, well within the normal physiological estradiol range
• Estradiol is relatively inexpensive
• ŠGiven in the physiological estradiol range, estradiol is virtually free from side effects

Progesterone replacement protocols

Progesterone administration serves to replace corpus luteum function in women with hypergonadotrophic hypogonadism. Progesterone can be administered as oral tablets, intravaginal suppositories and intramuscular (IM) injections. Orally administered progesterone is poorly absorbed, but vaginal or IM administration can achieve sufficient concentrations.

Devroey and Pados¹   have described the use of two different progesterone preparations during the replacement cycle:

• Natural progesterone IM (50 mg on day 14, 100 mg on days 15-26)
• Micronized progesterone vaginally (100-200 mg on day 14, 300-600 mg on days 15-26)

The vaginal route was found to induce an endometrial secretory pattern similar to that of a natural cycle.²⁷   It also caused less local irritation or general intolerance, leading to better patient acceptability and compliance.

Micronized progesterone releases a stable amount of progesterone (60-100 nmol/l) in the vagina for at least 3 months without any adverse systemic effects, and enables the embryo to be implanted and pregnancy to be maintained.¹

Preparation of endometrium in patients with intact ovarian function

In patients with functioning ovaries, regular hormonal cyclicity makes cycle control and synchronization between the donor and recipient more difficult. This problem can be mitigated by synchronizing the luteinizing hormone (LH) between the donor’s and the recipient’s LH surge. Other ways to overcome the problem are cryopreservation of the donated embryos^11,14      and administration of GnRH-a to donor and recipient.^28,29

Clinical studies

Study: Using the model of ovum donation in women without ovaries, Navot et al ¹⁹   showed that:

• Sequential estradiol and progesterone supplementation can produce physiological serum levels of estradiol and progesterone (Fig. 5).
• Sequential administration of estradiol and progesterone is sufficient to produce normal endometrial maturation, as proved by both light and electron microscopy.
• The normal morphological changes are accompanied by the ability to promote and sustain embryo implantation.
• Only sequential estradiol and progesterone, and no other ovarian secretagogues, are essential for embryo implantation and pregnancy maintenance.

Ben-Nun and colleagues³⁰   conducted a review of studies that examined the relative role of endometrial development in in vitro fertilization (IVF) and embryo transfer (ET) treatment in relation to the treatment outcome. They found that human chorionic gonadotropin or natural progesterone supplementation given after controlled ovarian stimulation with gonadotropin-releasing hormone and hMG effectively corrected the luteal phase defect and resulted in an improved conception rate. Since the endometrium of agonadal women is highly conducive to hormonal manipulation, all estrogen preparations effectively promoted endometrial growth and proliferation. Natural progesterone was found to be superior to synthetic progestins for induction of receptive secretory endometrium.

Conclusion: The study authors concluded that the outcome of IVF and ET treatment largely depends on the presence of an adequately receptive endometrium.

Study: Navot and coworkers²⁶   devised three experimental protocols to induce endometrial maturation in 12 women with ovarian failure.

• A short follicular phase protocol consisted of only 6 days of estrogen administration before addition of progesterone (13 cycles).
• In the long follicular phase protocol (5 cycles), estradiol was given for 3-5 weeks, and progesterone administration was accordingly postponed.
• In 6 accelerated secretory transformation cycles, 150 mg/day progesterone were administered intramuscularly from day 15 onward.
• A fourth protocol comprising 18 cycles of sequential estradiol (2-6 mg/day) and progesterone (progesterone; 25-50 mg/day, intramuscularly) simulating the 28-day natural cycle, served as a control protocol.

The adequacy of the induced endometrial cycles was evaluated by hormonal, morphological, and histochemical criteria relevant to endometrial normalcy and receptivity. Serum estradiol levels and the areas under the estradiol curves for the long and short follicular phase protocols differed significantly from those during the control cycles. Areas under the estradiol curves in the accelerated secretory transformation protocol yielded significantly higher progesterone values than those in all other protocols. All biopsies in the 3 experimental protocols compared favorably with those of the control protocol.

Conclusion:Based on the above findings, the investigators concluded that a very short exposure of the human endometrium to estradiol or, conversely, prolonged estradiol stimulation, will allow normal endometrial maturation with the addition of progesterone. Supraphysiological doses of progesterone in the accelerated secretory transformation protocol significantly enhanced endometrial maturational processes.

Study: Various regimens are recommended for replacing sex steroids in ovarian failure patients attempting donor embryo transfer. Sauer et al ³¹   histologically assessed endometrial biopsies obtained on simulated cycle day 26 from 19 functionally agonadal patients receiving hormone replacement according to three different regimens:

• Regimen 1: oral micronized estradiol 2 mg days 1-5, 4 mg days 6-9, 6 mg days 10-13, 4 mg days 14-28, with progesterone vaginal suppositories, 100 mg day 15 followed by 200 mg days 16-28
• Regimen 2: oral micronized estradiol 1 mg days 1-5, 2 mg days 6-9, 6 mg days 10-13, 2 mg days 14-28, with progesterone vaginal suppositories, 100 mg day 15 followed by 200 mg days 16-28
• Regimen 3: oral micronized estradiol 1 mg days 1-5, 2 mg days 6-9, 6 mg days 10-13, 2 mg days 14-28, progesterone 50 mg intramuscularly delivered day 15 followed by 100 mg intramuscularly days 16-28

While all regimens resulted in variable degrees of stromal pseudodecidualization, Regimen 1 biopsies uniformly demonstrated glandular abnormalities consistent with excessive estrogen stimulation. This included aberrant maturation, intraluminal papillary excrescences and variations in epithelium size and stratification. Regimen 2 biopsies were morphologically normal in most patients, yet many manifested minor variations in gland maturity. Only Regimen 3 biopsies were consistently normal on day 26 or slightly advanced in maturation.

Conclusion: These results show that endometrial morphology differs according to the hormone replacement preparation and route of administration. A combination of oral estradiol and intramuscular progesterone, given according to Regimen 3 design, reliably produces in-phase secretory endometrium that morphologically mimics a natural spontaneous cycle.

Study: A retrospective study³²   compared two groups of women with regular menstrual cycles in whom oral estradiol valerate and vaginal progesterone were used to prepare the endometrium:

• Group A (n = 212) had FET in a natural cycle after spontaneous ovulation
• ŠGroup B (n = 205) had FET in a pituitary-desensitized hormonally controlled cycle

In group B, GnRH agonist was started in the midluteal phase of the previous cycle and stopped 3 days before embryo transfer. Embryo transfer (ET) was carried out 3 days after detection of the endogenous LH surge in group A and on day 3 of progesterone administration in group B.

In Group A, 172 ETs were performed and 247 embryos replaced, while in Group B, 173 embryo transfers were performed and 243 embryos replaced. The outcomes are shown in Table 1.

Conclusion:From the above results, it is clear that both FET protocols are equally effective in terms of implantation rate and pregnancy outcome in women with regular menstrual cycles.

Study: Simon and colleagues³³   described a flexible and convenient protocol for artificial endometrial preparation without prior GnRH agonist suppression in patients with functioning ovaries undergoing frozen ET. It involved the use of a fixed dose of 6 mg/d of micronized estradiol started on day 1 of the cycle, followed by concomitant administration of micronized progesterone given intravaginally.

In the 175 ET cycles found evaluable, the pregnancy rate was found to be 21.7% and implantation rate, 9%. The mean (± SD) duration of the proliferative phase was 15 ± 1.9 days.

Conclusion: Thus, for patients with functioning ovaries, controlled endometrial preparation for the transfer of frozen-thawed embryos can be done successfully using oral estradiol from day 1 of the cycle followed by progesterone preparation. Prior suppression with GnRH agonist is not necessary.

Conclusion

The human endometrium is highly compliant to a wide range of estradiol and progesterone stimulation. Various doses of estrogen are capable of producing morphologically and ultrastructurally normal endometrium, thus making it suitable for embryo implantation and its sustenance. Estradiol is the most potent naturally-occurring estrogen. Hence, estradiol valerate and micronized estradiol are most commonly used for estrogen replacement therapy.

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