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ORIGINAL ARTICLE
Year : 2021 | Volume
: 5
| Issue : 2 | Page : 90-96
Effects of the interval between ovulation induction using clomiphene citrate and frozen embryo transfer on pregnancy outcome
Yi-Ning Xu, Lu Li, Xiao-Xi Sun
Department of Reproductive Medicine, Shanghai Ji Ai Genetics and IVF Institute, Obstetrics and Gynecology Hospital of Fudan University, Shanghai 200011, China
Correspondence Address:
Xiao-Xi Sun
Shanghai Ji Ai Genetics and IVF Institute, Obstetrics and Gynecology Hospital of Fudan University, No. 352, Dalin Road, Shanghai 200011
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2096-2924.320883
Objective: To explore the best timing for frozen embryo transfer (FET) after ovarian stimulation and egg retrieval using the clomiphene citrate (CC) + human menopausal gonadotropin (hMG) ovulation induction regimen through a retrospective analysis.
Methods: Data of patients who underwent CC + hMG ovulation induction and FET from January 2014 to December 2019 were analyzed retrospectively. The patients were divided into three groups according to the interval from egg retrieval to FET: CC1 (within 1 menstrual cycle), CC2 (2 menstrual cycles), and CC3 (≥ 3 menstrual cycles). Indicators such as hormone levels and pregnancy outcomes were recorded to explore the effect of different intervals on pregnancy outcome.
Results: A total of 1,082 transfer cycles were included in this retrospective analysis. The implantation, clinical pregnancy, and live birth rates in the CC1 group were significantly lower than those in the CC2 and CC3 groups (P < 0.05). The E2/P4 ratio on progesterone injection day (3 days before thawed embryo transfer) was lower in the CC1 group than in the other groups (P < 0.05). After adjusting for all factors using multifactor regression analysis, the interval between egg retrieval and FET was found to be an independent predictor of the implantation, pregnancy, and live birth rates.
Conclusion: An interval of more than one menstrual cycle between the day of egg retrieval after ovarian stimulation with the CC + hMG ovulation induction regimen and the day of FET can result in high implantation, clinical pregnancy, and live birth rates, which can lead to an improved pregnancy outcome.
Keywords: Assisted Reproduction; Clomiphene Citrate; Endometrial Receptivity; Frozen Embryo Transfer; Ovulation Induction; Pregnancy Outcome
Clomiphene citrate (CC) has been widely used as an ovulatory stimulant in the treatment of infertility for decades. Although the antiestrogen effect of CC increases the ovulation rate and decreases the pregnancy rate, it has several advantages, such as ease of use, low cost, minimal invasiveness, and extremely low incidence of ovarian hyperstimulation syndrome. At present, CC is considered a first-line drug for ovulation induction.[1] Since the early 1980s, CC has been used to induce ovulation during in vitro fertilization and embryo transfer (IVF-ET), yielding favorable results. More recently, CC combined with gonadotropin (Gn) has been found to induce more eggs when reducing the use of Gn and has become a mainstream ovulation induction regimen in IVF. Because of the high affinity between CC and estrogen receptors, CC can inhibit endometrial proliferation, which inevitably leads to decreased endometrial receptivity, thus affecting the success rate of IVF. In this regimen, a luteinizing hormone (LH) surge occurs early due to the simultaneous development of multiple follicles, which is one of the primary reasons for the high cancellation rate of the CC ovulation cycle and subsequent criticism of CC.[2]
In recent years, with the development of frozen ET (FET) technology and popularization of vitrification technology, it has become possible to reduce the negative effects of the CC ovulation induction regimen on endometrial receptivity. After using the CC regimen for ovulation induction, a fresh ET can be avoided and embryos can be frozen and thawed before transfer during the endometrial recovery period. FET undoubtedly solves the problem of poor endometrial receptivity during the CC-induced ovulation cycle. However, at present, several issues associated with this assisted reproductive technology regimen still need to be addressed, and the best timing for FET remains unclear. Therefore, through a retrospective analysis, this study aimed to explore the endometrial conditions and outcome of FET within 30 days, 31–60 days, and >60 days after using the CC + human menopausal gonadotropin (hMG) regimen to induce ovulation. This study also aimed to determine the best timing for FET after CC application; provide a basis for the future clinical application of this regimen; and improve the embryo implantation, clinical pregnancy, and live birth rates of patients using CC to induce ovulation.
MethodsIn this study, we retrospectively analyzed the data of patients who underwent FET after CC + hMG ovulation induction at Shanghai Ji Ai Genetics and IVF Institute from January 2014 to December 2019. We discussed the timing of FET after CC + hMG ovulation induction by comparing endometrial thickness, hormone levels, and FET outcomes. This study was approved by the Ethics Committee of the Shanghai Ji Ai Genetics and IVF Institute. Written informed consent was obtained from each patient.
Study population
Female patients (1) aged < 40 years; (2) whose infertility was caused by problems with the uterine tube, severe oligospermia, ovulation disorder, unexplained cause, or mixed factors; (3) who underwent FET; (4) who underwent third day (D3) ET; (5) who underwent IVF/intracytoplasmic sperm injection (ICSI) as the fertilization mode of assisted reproduction; or (6) who underwent hormone replacement therapy for endometrial preparation before transfer were included in the study.
Female patients who had (1) uterine malformation and uterine cavity adhesion, (2) endometriosis, (3) repeated implantation failure, or (4) genetic disease, serious physical disease, or mental or psychological disorders were excluded.
The patients were divided into the following three groups: (1) the CC1 group, the interval between egg retrieval using the CC + hMG regimen and FET was within one menstrual cycle; (2) the CC2 group, the interval between egg retrieval using the CC + hMG regimen and FET was two menstrual cycles; and (3) the CC3 group, the interval between egg retrieval using the CC + hMG regimen and FET was ≥3 menstrual cycles.
Ovulation induction regimen
A recombinant follicle-stimulating hormone (rFSH; Gonal-F®; Merck Serono, Fenil-sur-Corsier, Switzerland) or hMG (Zhuhai Lizhu Pharmaceutical Group, Zhuhai, China) was used from the second to the 3rd day of the menstrual cycle. The initial dose depended on the patient's age, body mass index (BMI), antral follicle count, FSH level, estradiol (E2) level, anti-Müllerian hormone level, and ovarian response in the previous ovulation induction cycle and was generally 150–225 IU/day. Approximately 100 mg/day of clomiphene (Cyprus Codal Synto Ltd., Limassol, Republic of Cyprus) was used simultaneously until the human chorionic gonadotropin (hCG) injection day. From the 6th day of Gn injection, the follicles were monitored by transvaginal ultrasound, and the serum E2, progesterone (P4), and LH levels were tested. Gn dosage was adjusted based on the results until the hCG injection day.
Egg retrieval, embryo culture, freezing, and thawing
When the diameter of at least three follicles was ≥18 mm, 10,000 IU of hCG (Livzon Pharmaceutical Group Inc., China) was injected into each patient's body. After 36 h, the eggs were collected through the vagina under B-mode ultrasound guidance.
For IVF, the eggs were incubated for 3 h and then individually transferred into a live sperm droplet with a density of approximately 100,000/mL. For ICSI, a single sperm was injected within 4 h after egg retrieval.
The embryos were cultured in 5% CO2 sequential medium (Vitrolife, Goeteborg, Sweden), and fertilization (fertilization rate) was observed 16–18 h after insemination. The formation of blastomeres (cleavage rate) was observed 72 h after fertilization, and the embryos were graded. The Edward RG standard was used for embryo grading: the blastomeres were equal in size, transparent, and free of fragments (Grade I); the blastomeres had an uneven size with a fragment rate of < 10% (Grade II); and the blastomeres had an uneven size with a fragment rate of 10%–50% (Grade III), and the blastomeres had an uneven size with a fragment rate of > 50% (Grade IV). The number of blastomeres was ≥6, and the fragment rate was ≤ 50%. Cleavage-stage embryos on D3 were frozen.
The embryos were frozen using vitrification technology, and the specific process was as follows: the embryos were rinsed twice in Dulbecco's phosphate buffer (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), transferred to equilibrium solution (MediCult, Fornebu, Norway) for 5 min, transferred to vitrification solution (MediCult), dripped into a Cryotop straw (AOLIJING, China) within 60 s, sleeved, and placed in liquid nitrogen for preservation.
After thawing, the straw was removed from the liquid nitrogen, placed in the air for 30 s, placed in thawing solution (1.0 mol/L sucrose) at 37°C for 1 min, and then placed in 0.5 mol/L sucrose, 0.25 mol/L sucrose, and G2 culture medium (Vitrolife AB, Västra Frölunda, Sweden) for 3 min at room temperature. The survival of embryos was observed after the culture medium was rinsed off. Embryos in which more than half of the blastomeres survived after thawing were regarded as viable embryos. After 16–18 h of culture after thawing, embryos were selected for transfer based on their condition. The embryos were graded based on the number of embryonic cells and morphological evaluation results.
Transfer timing, luteal support after transfer, and pregnancy monitoring
The endometrium was prepared by the hormone replacement method. Beginning on the 3rd day of the replacement cycle, 4 mg of estradiol hydrochloride tablets (Progynova®, Bayer Healthcare, Leverkusen, Germany) was administered every day. When the endometrial thickness was ≥ 8 mm, 60 mg of progesterone was administered once a day. ET was carried out on the 4th day after progesterone injection. After transfer, 60 mg of progesterone was injected once a day for 14 days.
The serum hCG level was measured 14 days after ET, and pregnancy was confirmed if the hCG test result was positive (hCG > 5 IU/L). The gestational sac and fetal heart were examined 28 days after ET and then re-examined every 4 weeks until the 12th week.
Outcome evaluation
The primary outcomes were as follows: (1) implantation rate = number of gestational sacs/number of transferred embryos × 100%; (2) clinical pregnancy rate = number of clinical pregnancy cycles/number of transfer cycles × 100%; and (3) live birth rate = number of live birth cycles/number of transfer cycles × 100%.
The secondary outcomes were as follows: (1) abortion rate = number of abortion cycles/number of transfer cycles × 100%.
Statistical analysis
The following population characteristics were obtained: age; BMI; cause of infertility (tubal factors, ovulation factors, male factors, unexplained cause, or mixed factors), endometrial thickness; and serum E2, P4, and LH levels on the progesterone injection day during endometrial preparation before ET (3 days before ET). Hormone levels were measured using a Beckman Coulter Access 2 immunoassay system (Brea, CA, USA) using the chemiluminescence method. The preparation, setting, dilution, adjustment, assay, and quality control of the test were carried out in accordance with the manufacturer's instructions. In all hormone tests, the coefficients of variation within run and between runs for each hormone were < 5%. The number of transferred embryos and the average embryo score using the Edward RG standard (the morphological score of each embryo multiplied by the number of blastomeres) were recorded.[3] The cumulative embryo score was obtained by multiplying the number of implanted embryos by the embryo score. In this study, the average ET score (the cumulative embryo score/number of transferred embryos) was used. The specific scores were as follows: Grade I, 4; Grade II, 3; Grade III, 2; and Grade IV, 1.
All statistical analyses were performed using SPSS Statistics 19 software (IBM SPSS, Armonk, NY, USA). The implantation, clinical pregnancy, live birth, and abortion rates of each group were calculated using Chi-square tests. The population characteristics, hormone levels, endometrial thickness, and average embryo score of each group were compared according to the mean values to detect any significant differences. P < 0.05 was considered statistically significant. Multifactor regression analysis was performed to further verify the influence of each factor on the implantation, clinical pregnancy, and live birth rates.
ResultsAmong the patients who underwent CC (CC + hMG) ovulation induction and FET at our reproductive center from January 2014 to December 2019, based on the inclusion and exclusion criteria, 1,082 transfer cycles were included in the final study. Based on the interval between the date of egg retrieval and the date of ET, the patients were divided into three groups: the CC1 group (interval within 1 menstrual cycle), the CC2 group (2 menstrual cycles), and the CC3 group (≥ 3 menstrual cycles).
Comparison of general characteristics of the study population
As shown in [Table 1], there was no significant difference in age, BMI, or cause of infertility among the three groups (P > 0.05).
Comparison of pregnancy outcome statistics in the study population
The pregnancy outcome statistics in each group are shown in [Table 2]. Results of the statistical analysis showed that the implantation, clinical pregnancy, and live birth rates of the CC1 group were significantly lower than those of the other groups (P < 0.05), but there was no significant difference between the CC2 and CC3 groups. There was no significant difference in the abortion rate of each group (P > 0.05). [Table 2] shows specific statistical analyses.
Comparison of endometrial thickness, embryo score, and hormone levels in each group
According to the data analysis in [Table 3], there was no significant difference in the embryo score between groups (P > 0.05). The mean endometrial thickness on progesterone injection day (3 days before ET) in the FET cycle of the CC1 group was higher than that of the CC3 group (P < 0.05) but not significantly different from that of the CC2 group (P > 0.05). The mean endometrial thickness on progesterone injection day of the CC2 group was significantly higher than that of the CC3 group (P < 0.05). On the day of progesterone injection, the serum E2 level of the CC1 group (656.05 ± 589.02 pg/mL) was lower than that of the other groups (P < 0.05), while the serum P4 level of the CC1 group (0.89 ± 0.72 pg/mL) was significantly higher than that of the other groups (P < 0.05). There was a significant difference in the serum LH level on the day of progesterone injection in each group (P < 0.05). The E2/P4 ratio on progesterone injection day of the CC1 group (1,096.70 ± 1,283.75) was significantly lower than that of the CC2 and CC3 groups (2,047.51 ± 2,620.73 and 2,210.99 ± 2,979.42, respectively; P < 0.05). There was no significant difference in the E2/P4 ratio between the CC2 and CC3 groups.
Table 3: Comparison of endometrial thickness, embryo score, and hormone levelsLogistic regression analysis
As shown in [Table 4], [Table 5], [Table 6], single-factor regression analysis showed that age, LH level on progesterone injection day, and the interval between egg retrieval and FET had statistically significant effects on the implantation, pregnancy, and live birth rates (P < 0.05). Multifactor regression analysis was carried out after removing the confounding factors, and the results showed that the interval between egg retrieval and FET was an independent predictor of the implantation, pregnancy, and live birth rates (implantation rate: odds ratio [OR], 0.577 and 95% confidence interval [CI], 0.436–0.763; pregnancy rate: OR, 0.561 and 95% CI, 0.424–0.741; and live birth rate: OR, 0.578 and 95% CI, 0.432–0.774).
DiscussionDrug-induced ovulation is a common treatment for patients with infertility. As a first-line drug for ovulation induction, CC has been widely used in clinical practice for many years. The administration of CC in the early follicular phase inhibits the negative feedback effect of estrogen on the hypothalamic pituitary gland to increase the secretion of FSH by the pituitary gland and promote the growth of follicles through the antagonistic effect of estrogen to occupy the estrogen receptors of the hypothalamus.[4] However, because of its antiestrogen effect, CC occupies the estrogen receptors in the endometrium, which leads to thinning of the endometrium in the fresh cycle and thus restricts its clinical application. In recent years, with the development of embryo cryopreservation technology, a regimen using FET has been proposed as a replacement for the regimen using fresh ET after CC-induced ovulation. This regimen allows the endometrium to recover, thus preventing endometrial thinning. At the same time, a better uterine environment also improves the success rate of FET compared to fresh ET.[5]
In this retrospective study, there was no significant difference in age, BMI, or cause of infertility among the groups. Based on the above results, we presumed that the pregnancy outcome statistics among the groups were not affected by age, BMI, or infertility factors and thus were comparable.
The statistical analyses of various pregnancy outcomes showed that the implantation, clinical pregnancy, and live birth rates of the transfer group within one menstrual cycle after CC + hMG ovulation induction and egg retrieval were significantly lower than those of other groups, but there was no significant difference in the abortion rate among the groups. The above results showed that FET after two or more menstrual cycles from CC + hMG ovulation induction and egg retrieval could provide better pregnancy outcomes. At present, there is no experimental evidence to support this finding. This finding may be related to the mechanism of CC, that is, it affects endometrial receptivity. CC can inhibit endometrial proliferation, promote endometrial cell apoptosis, and affect endometrial receptivity through various channels.
The biological half-life of CC is 5–6 days; however, its metabolites have been found in feces for up to 6 weeks.[6] The long half-life of CC may also be related to the results of this study. Because of the long metabolic half-life of CC and the prolonged use of CC during ovulation induction, it may require a longer time to complete metabolism clearance in vivo. At present, no study has reported the specific time required to completely eliminate CC from the blood after ovulation induction treatment with CC + hMG.
The results of this retrospective study showed that it may take more than one menstrual cycle for the endometrium to recover and create an implantation environment conducive to pregnancy after discontinuing the use of CC, which may be the time required for the estrogen and receptor levels to gradually return to normal,[7] prostaglandin synthesis,[8] steroid receptor coactivator 1 recruitment inhibition[9] to return to normal, or metabolic clearance of CC. Hence, further studies are needed to determine the specific mechanism.
To further explore the effect of CC on endometrial receptivity, the endometrial thickness, embryo score, and hormone levels of each group were recorded in this study. Endometrial thickness on progesterone injection day during endometrial preparation before ET (3 days before ET) was compared. Hormone levels were also measured on the day of progesterone injection. The study showed that there was no significant difference in the average embryo score of each group, which essentially excluded the influence of embryo quality on the pregnancy outcome.
The mean endometrial thickness of the CC1 group on the day of progesterone injection was higher than that of the CC3 group. However, the CC1 group had a poor pregnancy outcome and a lower E2 level before transfer, which seemed contradictory. In general, a higher E2 level can lead to a thicker endometrium, which is more likely to achieve a better pregnancy outcome. Therefore, the increase in mean endometrial thickness in the CC1 group may be due to the influence of CC on endometrial receptivity, which is not only reflected in endometrial thickness.
In the morphological evaluation of endometrial receptivity, the pinopode is a critical marker. The lack of a pinopode may lead to repeat embryo implantation failure. An endometrium with abundant pinopodes often results in a better pregnancy outcome.[10] However, the use of CC may reduce the number of endometrial pinopodes.[11]
In addition, CC may affect endometrial receptivity and embryo implantation by affecting the expression of integrin, homeobox gene 10 (HOXA10), and leukemia inhibitory factor (LIF). Unexplained infertility, endometriosis, hydrosalpinx, and luteal phase deficiency (LPD) are related to the abnormal expression of integrin.[12] The physiological downregulation of progesterone receptors (PRs) and the expression of αVβ3 integrin are considered key steps in the implantation process. The downregulation of PRs is the premise of the expression of endometrial epithelial proteins (such as αVβ3 integrin); hence, the failure of PR downregulation is closely related to the abnormal expression of αVβ3 integrin, which leads to infertile conditions like LPD.[13] In women treated with CC, low expression of β3 integrin and the absence of PR expression downregulation were observed in glandular epithelial cells using endometrial biopsy in the middle luteal phase,[12] which may have an impact on embryo implantation. HOXA10, which can regulate endometrial proliferation, differentiation, and decidualization, plays an important role in endometrial receptivity and embryo implantation. HOXA10 expression increased significantly in the middle luteal phase, which was consistent with the implantation time.[14] Some scholars studied 36 female Kunming mice and found that the use of CC can significantly reduce HOXA10 expression in the endometrium compared to the natural cycle.[15] LIF is a cytokine involved in the implantation process and an important marker of endometrial function.[16] LIF is involved in many aspects of implantation and related processes, including uterine transition to the receptive state, decidualization, blastocyst growth and development, embryo–endometrium interaction, trophoblast invasion, and immune regulation.[17] CC can significantly reduce the expression of LIF in patients with unexplained infertility.[18]
In this study, the endometrial thickness of the CC1 group measured by B-mode ultrasound was higher than that of the other groups, but the pregnancy outcome was poor. This finding may indicate that although endometrial thickness was already high when CC was discontinued for one menstrual cycle, the number of endometrial pinopodes was reduced or the expression levels of integrin, HOXA10, and LIF were decreased due to the use of CC, thus affecting endometrial receptivity and embryo implantation, which further led to reductions in the implantation, clinical pregnancy, and live birth rates.
In terms of hormone levels, on the day of progesterone injection, the serum E2 level of the CC1 group was lower than that of the other groups, while the P4 level was higher than that of other groups. This result demonstrates that the impact of CC persists even after CC has been discontinued for one menstrual cycle. After CC was discontinued for one menstrual cycle, the E2 level in the peripheral blood remained low, while the P4 level was high, which may also have contributed to the low implantation, clinical pregnancy, and live birth rates. Some animal studies have suggested that CC can increase the apoptosis of ovarian follicular cells (possibly granulosa cells), thus reducing the E2 value in the ovaries and peripheral blood circulation and affecting the growth and maturation of follicles.[19] In addition, the E2/P4 ratio on progesterone injection day in the CC1 group was significantly lower than that of the other groups, indicating that the low E2 levels and high P4 levels before transfer had adverse effects on embryo implantation and pregnancy. Some scholars have made similar arguments that a high P4/E2 ratio may indicate that the endometrium accepts the effect of progesterone prematurely, which leads to asynchronous development of the embryo and thereby affects endometrial receptivity.[20]
In the single-factor regression analyses, age, LH level on progesterone injection day, and the interval between egg retrieval and FET had statistically significant effects on the implantation, pregnancy, and live birth rates. In the comparison of hormone levels, the LH level of the CC1 group on progesterone injection day was high, which may have also contributed to the increased progesterone level, thus affecting embryo implantation and pregnancy. Further multiple-factor regression analyses showed that the interval between egg retrieval and FET was an independent predictor of the implantation, pregnancy, and live birth rates. This finding further confirmed that a better pregnancy outcome could be achieved if the interval between egg retrieval using the CC + hMG ovulation induction regimen and FET is two or more menstrual cycles.
This retrospective study showed that the endometrium recovered better and that endometrial receptivity was higher when the interval between egg retrieval using the CC + hMG ovulation induction regimen and FET was two or more menstrual cycles, thus providing a better pregnancy outcome. A total of 1,082 transfer cycles were included in the study. There were a sufficient number of cases in each group, and the statistical results were reliable. However, one limitation of the study is that there were cases of single ET in a retrospective study, which may have had certain impacts on the pregnancy outcome statistics. In addition, as this was a single-center retrospective study, the results may be biased and need to be further verified by prospective studies.
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Conflicts of interest
There are no conflicts of interest.
References
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