ABSTRACT
We explored the strategy of frozen-thawed embryo transfer (FET) in the women with advanced maternal age (AMA). We first determined the age cut-off point of AMA by retrospective analysis of pregnancy outcomes in the patients undergoing FET. The patients with AMA were divided into 3 groups including natural cycle (NC) group, controlled ovarian stimulation (COS) group, and hormone replacement therapy (HRT) group, and simultaneously were divided into 2 groups including cleavage-stage embryo transfer (CET) group and blastocyst-stage embryo transfer (BET) group. The clinical pregnancy, embryo implantation, abortion and live birth rates were compared between the 3 groups and the 2 groups, respectively. We found that in the women aged 38 years or over, the clinical pregnancy rate and live birth rate were all significantly decreased as compared with the younger than 38-year-old women (all P < 0.05), so the women aged 38 years or over were regarded as the patients with AMA in this study. In the patients with AMA, the clinical pregnancy rate and live birth rate were 22.95% and 18.03% in NC group, 23.68% and 15.79% % in COS group as well as 24.58% and 15.92% in HRT group, and there were no significant differences in the clinical pregnancy rate and live birth rate between the 3 groups. However, the clinical pregnancy rate (42.96% vs 15.87%) and embryo implantation rate (32.26% vs 9.67%) were all significantly higher in the BET group than in the CET group (all P < 0.01). We conclude that in the women aged 38 years or over, the choice of endometrial preparation protocols may depend on the individual specific conditions because the endometrial preparation protocols do not affect FET outcome, but BET can obtain better FET outcomes as compared with CET.
Abbreviations: AMA: advanced maternal age; FET: frozen-thawed embryo transfer; NC: natural cycle; COS: controlled ovarian stimulation; HRT: hormone replacement therapy; CET: cleavage-stage embryo transfer; BET: blastocyst-stage embryo transfer; LH: luteinizing hormone; HCG: human chorionic gonadotropin; HMG: human menopausal gonadotropin; FSH: follicle-stimulating hormone; BMI: body mass index
Introduction
After China’s two-child policy was gradually liberalized, more and more women with advanced maternal age (AMA) want to have the second child. The women with AMA commonly have underlying diseases due to physical function decline, so pregnancy may induce or stimulate the onset of these diseases. Because of diminished ovarian function in the women with AMA, the number of follicles decreases (Grondahl et al. Citation2010), oocyte aneuploidy rate increases (Vialard et al. Citation2007) and endometrial receptivity decreases (Soares et al. Citation2005), which all affects pregnancy outcome. Although modern assisted reproductive technology has solved some reproductive problems in the fertility of the patients with AMA, there still is a big challenge. Therefore, it is necessary to explore the strategy of embryo transfer in the patients with AMA. In this study, we first determined the age cut-off point of AMA by retrospective analysis of pregnancy outcome in the patients undergoing frozen-thawed embryo transfer (FET) in our reproductive center between January 2014 and December 2016, and then the patients with AMA were grouped according to different endometrial preparation protocols and different types of transferred embryos, respectively, to compare pregnancy outcome between groups and explore FET strategy in the patients with AMA.
Results and discussion
Relationship between age and pregnancy outcome
A total of 3775 FET cycles were included in this study. In this study, there were 1914 clinical pregnancies, 315 abortions and 38 ectopic pregnancies including 10 heterotopic pregnancies, 1562 live births and 9 with loss of follow-up. The ages were between 21 and 51 years in these patients. Our results showed that with the increase of age, the pregnancy rate and live birth rate gradually decreased, and the abortion rate gradually increased, and the clinical pregnancy rate and live birth rate were all significantly lower in the women aged 38 years or over than in the younger than 38-year-old patients (all P < 0.05) (). Therefore, 38 years of age was used as the age cut-off point of AMA women in this study, namely that the women aged 38 years or over were regarded as the patients with AMA.
Table 1. Comparisons of pregnancy outcome of frozen-thawed cycles between patients with different ages.
Relationship between endometrial preparation protocols and pregnancy outcome
A total of 457 FET cycles were collected in the patients with AMA. In the 457 EFT cycles, there were 111 clinical pregnancies, 35 abortions and 3 ectopic pregnancies including one heterotopic pregnancy and 74 live births. According to different endometrial preparation protocols, the 457 patients with AMA were divided into NC group (61 cases), COS group (38 cases) and HRT group (358 cases). There were no significant differences in age, basal follicle-stimulating hormone (FSH), basal luteinizing hormone (LH), body mass index (BMI), times of embryo transfer, number of transferred embryos, D0 endometrial thickness, types of transferred embryos, embryonic quality and infertility factors between the three groups. Furthermore, the clinical pregnancy rate, implantation rate, abortion rate, ectopic pregnancy rate, and live birth rate also did not show significant differences between the three groups ( and ).
Table 2. Comparisons of clinical data between patients receiving different endometrial preparation protocols.
Table 3. Comparisons of clinical outcome between patients receiving different endometrial preparation protocols [n/n (%)].
Relationship between types of transferred embryos and pregnancy outcome
According to the types of transferred embryos, the patients with AMA were divided into cleavage-stage embryo transfer (CET) group and blastocyst-stage embryo transfer (BET) group. There were no significant differences in D0 endometrial thickness and endometrial preparation protocols between the CET group and BET group. The age was significantly older, the number of transferred embryos was significantly more, and the times of embryo transfer was significantly less in the CET group than in the BET group (all P < 0.01). Furthermore, the clinical pregnancy rate, implantation rate and live birth rate were significantly higher in the BET group than in the CET group (all P < 0.01), but there were no statistical differences in abortion rate and ectopic pregnancy rate between the CET group and BET group ( and ).
Table 4. Comparisons of clinical data between CET group and BET group.
Table 5. Comparisons of clinical outcome between CET group and BET group [n/n (%)].
In assisted reproductive technology, female age is one of the important factors affecting clinical pregnancy outcome (Wang et al. Citation2008; Veleva et al. Citation2013), and has gradually become a main predictor of pregnancy outcome (Kim et al. Citation2014). With the increase in age, fertility decreases in women, especially in the over 35-year-old women. In patients with AMA, assisted pregnancy faces a huge challenge because diminished ovarian function and oocyte quality affect embryo quality (Munne et al. Citation2007) and decreased endometrial receptivity affects embryo implantation. It has been reported that the age cut-off point of AMA is 37 years because the physiological index shows a downward trend in the over 37-year-old women (Setti et al. Citation2013; Lin-Yan et al. Citation2016). With the increase of age, pregnancy rate markedly decreases, especially in the over 40-year-old women. Increasing the number of transferred embryos does not improve the clinical outcome in the patients with AMA, but increases multiple birth rate and reproductive risk (Wang et al. Citation2001). Therefore, the number of transferred embryos was not more than 2 in all patients of this study. In this study, using retrospective analysis we compared a large number of clinical pregnancy outcomes between different age groups. Both the clinical pregnancy rate and the live birth rate were significantly lower in the patients aged 38 years or over than in the younger than the 38-year-old patients, so 38 years of age was used as the age cut-off point of AMA in our reproductive center.
In FET cycles, endometrial preparation protocols commonly include NC, COS, and HRT. The three methods have their own advantages and disadvantages, and there is considerable debate on the effect of endometrial preparation protocols on clinical outcomes. Hill et al. (Citation2010) reported that the live birth rate was significantly higher in HRT than in NC. Levron et al. (Citation2014) described that the clinical pregnancy rate and embryo implantation rate were significantly higher in CN than in HRT and Candido et al. (Citation2012) indicated that the pregnancy loss rate was significantly lower in CN than in HRT. However, Ermanno et al. (Citation2016) found that clinical outcomes were similar between CN and HRT, and it has been reported that endometrial preparation protocols were not related to clinical outcome (Hui et al. Citation2008; Gui-Jia and Tan Citation2013). This study also showed that there were no significant differences in endometrial thickness and clinical pregnancy rate between the three groups, demonstrating that the three endometrial preparation protocols (NC, COS and HRT) all obtained ideal endometrial thickness and similar clinical outcome. Therefore, we should consider the most suitable, simple, economical, and effective endometrial preparation protocol based on the patient’s specific condition. In addition, this study also indicated that the abortion rate markedly decreased and the live birth rate markedly increased in NC group as compared with COS and HRT groups, but there were no statistical differences in the abortion rate and live birth rate between the three groups. This may indicate that the sample size in this study is not large enough, and that it is necessary to increase the sample size in future study.
In clinical practice, HRT is more acceptable in endometrial preparation due to its flexibility (Gui-Jia and Tan Citation2013). However, in patients with AMA, oral large-dose estrogen may cause some side effects such as nausea, vomiting, and dizziness because of their body function decline, and large-dose estrogen may increase the risk of clinical complications such as venous thrombosis (Bagot et al. Citation2010). Stevenson et al. (Stevenson Citation2009) found that oral estrogen can increase the triglyceride level and its agglutinating activity, so estrogen should not be used for the patients with elevated level of triglycerides. In addition, its metabolite, estrone, can activate oncogenes which are associated with breast tumors, ovarian cyst and uterine fibroids (Xian et al. Citation2012). Therefore, for endometrial preparation, estrogen should be carefully considered before use in the patients with AMA. Furthermore, estrogens are designated as American Food and Drug Administration category X and in the USA, it is contraindicated in the women who are pregnant, although it is not clear whether estrogens affect offspring. To reduce potential harm to offspring, the dosage of estrogen must be controlled in HRT. The lowest dose of estrogen that permits endometrial growth to reach above 8 mm should be selected in order to reduce the side effects and possible long-term harm caused by estrogen.
Compared with young patients, the numbers of retrieved oocytes and high-quality embryos are less in patients with AMA because of diminished ovarian reserve, so prolonging incubation time may lead to no transferable embryos in some patients. Therefore, the patients with AMA have the risk of no transferable embryos if they receive blastocyst transfer. However, it has been confirmed that the embryos with genetic defects or no developmental potential fail to develop into blastocysts, so the transferable embryos may be screened out by blastocyst culture (Gardner et al. Citation1998). Blastocyst transfer allows embryo development to synchronize with the endometrial implantation window, which is in line with the physiological implantation time and is conducive to embryo implantation. Moreover, uterine contraction is gradually reduced following blastocyst transfer, which reduces the possibility of abortion (Bazer et al. Citation2011). Our previous study indicated that blastocyst culture could significantly improve pregnancy outcome in patients with repeated embryo implantation failure (Jie et al. Citation2017). The results of this study further showed that blastocyst transfer could significantly increase the clinical pregnancy rate in the patients with AMA. This study also displayed that the age was significantly older and the time of embryo transfer was significantly less in the CET group than in the BET group. This may be that due to older age, the number of transferable embryos was less, so FET had to be carried out only after repeated oocyte retrievals. This likely led to a reduced number of embryo transfers in the CET group than in the BET group; and the patients in the BET group chose blastocyst transfer due to repeated embryo transfer failures, so the times of embryo transfer was higher but the clinical pregnancy rate was better as compared with the patients of CET group.
In summary, for the patients with AMA, the choice of endometrial preparation protocols depends on the individual specific conditions. In HRT, large dose estrogen should be carefully considered for AMA patients as it is associated with potential diseases. For patients with AMA, one could consider to choose blastocyst transfer if conditions permit.
Materials and methods
All study methods were approved by the Ethics Committee of Hubei Maternal and Child Health Hospital. All the subjects enrolled into the study gave written informed consent to participate.
Materials
Inclusion criteria included (1) no intrauterine lesions; (2) no severe uterine malformation; (3) no hysteromyoma; (4) no endometriosis; (5) no hydrosalpinx; (6) no abnormal hysteroscopy; and (7) no chromosomal abnormalities in male and/or female.
The patients who were in line with the inclusion criteria and underwent FET in our reproductive center between January 2014 and December 2016, were grouped according to their ages, and then the clinical pregnancy rate, abortion rate, ectopic pregnancy rate, and live birth rate were compared between groups to determine the age cut-off point of AMA. The patients with AMA were further grouped as follows.
Grouping and outcome measures
All the patients with AMA were divided into 3 groups including natural cycle (NC) group, controlled ovarian stimulation (COS) group, and hormone replacement therapy (HRT) group according to different endometrial preparation protocols, and simultaneously were divided into 2 groups including CET group and BET group according to the types of transferred embryos. And then, the clinical pregnancy rate, embryo implantation rate, abortion rate, ectopic pregnancy rate, and live birth rate were compared between the 3 groups and the 2 groups, respectively.
Endometrial preparation protocols
NC: NC was suitable for the patients with regular menstruation and ovulation.
The follicle development was monitored by transvaginal B mode ultrasound from the 10th day of menstruation; and when the dominant follicle diameter was 16mm or more, the urine luteinizing hormone (LH) level was monitored every day until the occurrence of urinary LH peak or ovulation showed by B ultrasonic examination. If urinary LH peak still did not appear when the dominant follicle diameter was more than 18 mm, human chorionic gonadotropin (HCG, 10000IU, Livzon Pharmaceutical Group Inc, Zhuhai, China) was intramuscularly injected in order to induce ovulation.
COS: COS was suitable for the patients with anovulation or irregular ovulation. Intramuscular injection of human menopausal gonadotropin (HMG, 75 U/d, Livzon Pharmaceutical Group Inc, Zhuhai, China) was given from the fifth day of menstruation to promote follicular development. At the same time, the follicular development was monitored by B-ultrasound. When the dominant follicle diameter was more than 18 mm, HCG (10000IU, Livzon Pharmaceutical Group Inc, Zhuhai, China) was intramuscularly injected in order to induce ovulation. In NC and COS, the endometrial thickness on the ovulatory day should be more than 8 mm, the ovulatory day was set as D0, cleavage-stage embryos were thawed and transferred on D3 and blastocyst-stage embryos on D5.
HRT: HRT was suitable for the patients with COS failure, irregular menstrual cycle, or endometrial thickness <7 mm during previous ovulation period. From the 3rd day of menstruation, patients took progynova (2 mg/d, Bayer Company, Germany) which was gradually increased every 4 days according to endometrial thickness until 12 mg/d. When transvaginal B-mode ultrasound showed that the endometrial thickness was more than 8 mm, progesterone (40–60 mg/d, Guangzhou Baiyunshan Pharmaceutical Industry, Guangzhou, China) was intramuscularly injected in order to induce endometrial transformation to secretory phase. The day of progesterone administration was set as D0. Cleavage-stage embryos were thawed and transferred on D3 and blastocyst-stage embryos on D5.
Freezing, thawing and grading of cleavage-stage embryos and blastocyst-stage embryos
Freezing and thawing of cleavage-stage embryos and blastocyst-stage embryos were performed according to the routine procedures of cryopreservation in our center (Jie et al. Citation2017). The cleavage-stage embryos with grade III or above (fragment rate < 50% and the cleavage-stage embryo with more than 4 cells on D3) were frozen. After thawing, the cleavage-stage embryo in which intact cells were more than 50% was regarded as survival. The cleavage-stage embryos with grade I was regarded as high-quality cleavage-stage embryos, and those with grade Ⅲ and below were regarded as low-quality cleavage-stage embryos. D5 or D6 blastocysts-stage embryos with above grade 3CC were frozen after laser drilling. After thawing, the blastocysts-stage embryos with blastocyst cavity expansion were regarded as survival. The blastocysts-stage embryos with grade 4AA or above were regarded as high-quality blastocysts-stage embryos, and those with grade 3AC below were regarded as low-quality blastocysts-stage embryos. The ovulatory day was set as D0 in NC and COS cycles, and the day of progesterone administration was set as D0 in HRT cycle. Cleavage-stage embryos were thawed and transferred on D3 and blastocysts-stage embryos on D5. Cleavage-stage embryos or blastocysts-stage embryos were thawed 2 h before transfer. Cleavage-stage embryos subjected to laser drilling before transfer. The number of transferred embryos was not more than 2 in all the patients.
Luteal supporting therapy
In all patients, after embryo transfer, progesterone (60 mg/d) was injected intramuscularly for 14 days, and dydrogesterone (duphaston, Netherlands) was given orally for 14 days. For HRT cycle, patients continuously took progynova (2 mg/d) until 8 and 10 gestational weeks.
Pregnancy outcome
Serum β-HCG was detected 14 days after embryo transfer and β-HCG > 5 mIU/ml was defined as biochemical pregnancy. D28 B mode ultrasound showed gestational sac, which was regarded as clinical pregnancy. Live birth referred to live birth with 28 gestational weeks or more. Abortion refers to the abortion occurring before gestational weeks.
Statistical analysis
Statistical treatment was performed using SPSS18.0 software. Measurement data were expressed as mean ± standard deviation and were compared with t test. Chi-square test was used in the comparison of rates and Fish exact probability was suitable to the theoretical frequency less than 5. Statistical significance was established at P < 0.05.
Disclosure statement
No potential conflict of interest was reported by the authors.
Additional information
Notes on contributors
Jie Zheng
Conceived and designed the study: JZ; Performed the study: JL, Y-LL; Analyzed the data: X-FW; Wrote the manuscript: JL.
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