Serum hormone levels in infertile women between conceived with and without hormone replacement therapy: Safety of estradiol and progesterone replacement therapy in frozen-thawed embryo transfer cycles for breast cancer survivors

Objective Recent advances in cancer treatment and reproductive medicine have made the post-treatment quality of life an important concern for cancer survivors. We aimed to evaluate the safety of sex hormone (estradiol and progesterone) replacement therapy (HRT) in women who conceived by assisted reproductive technology (ART) with hormone receptor-positive breast cancer. Methods We measured serum E2 and P4 levels at 4–10 weeks of gestation in women who conceived naturally or after timed intercourse or intrauterine insemination for infertility without HRT for luteal support (non-HR group; n=135). We conducted a retrospective comparison of the values from the non-HR group with those of women who conceived by ART with HRT for infertility (HR group; n=75). Results Serum E2 levels were significantly higher in the non-HR group than in the HR group at 5, 6, and 8 weeks of gestation. Similarly, serum P4 levels were significantly higher in the non-HR group than in the HR group at 4, 5, and 6 weeks of gestation. Conclusions This study suggests that in cancer reproductive medicine for hormone-dependent breast cancer survivors, HRT administered during the first trimester of a pregnancy after primary disease treatment may not increase the sex hormone levels to levels above those seen in spontaneous pregnancy.


Introduction
3 Recent advancement in cancer treatment and reproductive medicine has increased the importance 4 of post-treatment quality of life among childhood, adolescent, and young adulthood cancer survivors.
5 Fertility preservation is of major concern, and the use of assisted reproductive technology (ART), 6 such as cryopreservation of embryos, oocytes, or ovarian tissue, is important for conserving fertility.
7 For women with a history of breast cancer and other hormone-sensitive malignancies, hormone 8 replacement therapy (HRT), which is important for continued pregnancy using ART, risks the 9 exacerbation or recurrence of the primary disease. Hormonal exposure owing to pregnancy could be 10 also a risk factor; however, some studies have reported that the prognosis of breast cancer survivors 11 who underwent appropriate neoadjuvant therapy is not necessarily worsened by spontaneous  [5]. Although the rates of recurrence and mortality are lower for patients who 13 receive multidrug chemotherapy following breast cancer surgery than for those treated by surgery 14 alone [6], patients who receive multidrug chemotherapy show decreased fertility owing to 15 chemotherapy-induced ovarian failure and age-associated ovarian dysfunction resulting from 16 prolonged administration of hormone therapy. Furthermore, many patients encounter difficulty in 17 conceiving naturally. The levels of anti-Mullerian hormone (AMH), a parameter of ovarian reserve, 18 decrease to undetectable levels during chemotherapy and remain low even after completing of 19 chemotherapy [7]. Studies have reported low rates of pregnancy in breast cancer survivors [8] [9] 20 [10]; hence, before initiating treatment, patients must be provided with adequate information, 4 1 informed consent should be obtained, and patients should be offered consulting with a doctor 2 specializing in reproductive medicine. Under these circumstances, the standard practice is to offer 3 various options for preserving fertility such as cryopreservation of embryo, oocytes, or ovarian tissue 4 to women with cancer in the limited duration between their diagnosis and treatment. However, the 5 benefits and drawbacks of ovarian stimulation, embryo transfer, and HRT as part of ART are unclear 6 with respect to the effect on the primary disease and are currently a subject of debate. In particular, if 7 ovarian function decreased and ovulation is impaired after cancer treatment, HRT is essential for 8 embryo transfer using frozen embryos or oocytes once the patient is allowed to conceive. Even after 9 pregnancy is established, this support must be continued until the main site of hormone production 10 switches from the corpus luteum to the placenta (the luteo-placental shift).

11
In this study, we compared the hormone levels during the first trimester of pregnancy between 12 women who conceived naturally or after timed intercourse (TI) or intrauterine insemination (IUI), 13 which does not require HRT for infertility, and those who conceived with ART. We aimed to 14 evaluate the safety of estrogen and progesterone replacement therapy for frozen-thawed embryo 15 transfer in estrogen and progestogen replacement therapy for frozen-thawed embryo transfer in 16 women with hormone receptor-positive breast cancer who conceived using ART to compare with 17 those who conceived naturally or after TI or IUI.

19
Materials and Methods

20
We measured the serum E2 and P4 levels at 4-10 weeks of gestation in non-HR group participants.

1
The study subjects were women treated in the Department of Obstetrics and Gynecology or 2 Reproduction Center, Toho University Omori Medical Center, between November 2018 and April 3 2019, who conceived naturally or after TI or IUI for infertility. The non-HR group participants did not 4 undergo HRT for luteal support (Fig 1). TI and IUI cycles were performed naturally or using 5 medication for ovulation induction with follicle growth monitoring by ultrasonography (Fig 2).
6 We retrospectively compared the serum E2 and P4 levels at 4-10 weeks of gestation between the 7 non-HR and HR groups. The HR group included women who conceived after frozen-thawed embryo 8 transfer and HRT with estrogen and progesterone in our reproduction center between January and 9 December 2018 (Fig 1). The members of both groups continued their pregnancies until at least 12 10 weeks of gestation.
11 Estrogen replacement therapy was administered in the form of transdermal estrogen patch applied 12 every alternate day from day 3 of menstruation. The initial dose was 2.16 mg, and this was increased 13 after a few days to 2.88 mg and then to 3.60 mg. Transvaginal natural progesterone at a dose of 90-800 14 mg/day was administered when the thickness of the endometrium was ≥8 mm; depending on the stage 15 of the frozen embryo to be transferred, the embryo transfer was performed on day 2 (P+2), day 3 (P+3),  19 The serum E2 and P4 levels in the non-HR and HR groups increased over time during the first 20 trimester of pregnancy (Figs 4a, 4b, 5a, and 5b). There was no significant difference in the pregnancy 7 1 continuation rate between the two groups. The formulas for the approximate curves of the serum E2 2 and P4 levels for the non-HR group are y=234 x+ 500 (R 2 =0.17) and y=0.8x + 23.3 (R 2 =0.027) and 3 those for the HR group are y=258x−112 (R 2 =0.63) and y=2.5x + 9.3 (R 2 =0.27). 4 ng/mL at 9 weeks, and 28.5±8.8 and 27.6±9.3 ng/mL at 10 weeks of gestation, respectively. The 5 serum P4 level was significantly higher in the non-HR group than in the HR group at 4, 5, and 6 6 weeks of gestation (4 weeks, p <0.01; 5 weeks, p <0.01; 6 weeks, p <0.01). There was no significant 7 difference in the levels at 7, 8, 9, or 10 weeks of gestation between the groups ( 4 Hence, careful consideration is necessary before offering this option to patients with cancer. As serum 5 estrogen levels increase with COS, the risk of recurrence is of particular concern in patients with 10 whose fertility has declined are likely to require HRT, including the administration of estrogen and 11 progesterone. This plays a major role in embryo implantation and the continuation of pregnancy.
12 Similar to COS, estrogen and progesterone replacement therapies are administered to patients 13 without an ovulatory cycle who are undergoing thawed embryo transfer. The effect of these therapies 14 on breast cancer is concerning. Currently, the safety of these replacements is not known. In this study, 15 we measured the levels of serum E2 and P4 levels in the first trimester of pregnancy in patients who 16 did not receive HRT.

17
Our results suggest that in the first trimester, the serum E2 and P4 levels in the HR group were not 18 higher than those in the non-HR group. Thus, the amount of HRT required to establish a pregnancy 19 may not increase the cancer risk associated with hormonal exposure in patients with hormone receptor-20 positive breast cancer compared to that in those with spontaneous pregnancy.

1
We found that the serum E2 levels were significantly lower in the HR group than in the non-HR group 2 at 5, 6, and 8 weeks of gestation. Differences at 4 and 7 weeks of gestation may not have been 3 statistically significant owing to the small sample size. In the non-HR group, the levels tended to 4 increase over time (Table 1). At approximately 6-7 weeks of gestation, the main site of hormone 5 production switches from the corpus luteum to the placenta (luteo-placental shift) [34], and the levels 6 rapidly increased during this period (Fig 4a). There was no significant difference in the serum E2 levels 7 at 9 and 10 weeks of gestation between the two groups; this may be because estrogen replacement 8 therapy was discontinued in the HR group at 8 weeks of gestation, and in both groups, the main site of 9 hormone production was now the placenta.

10
The serum P4 levels were significantly lower in the HR group than in the non-HR group at 4, 5, and 11 6 weeks of gestation (Table 2). In the non-HR group, the serum P4 levels gradually increased between 12 4 and 10 weeks of gestation to 20-30 ng/mL (Fig 4b), and in the HR group, the level remained at 13 approximately 15 ng/ml, which was significantly lower than that in the non-HR group. As these 1 concentration in the endometrial tissue is maintained at a higher level [37]. In this study, because few 2 samples were collected from patients in the non-HR group at 7 weeks of gestation, we could not 3 evaluate this aspect. However, as the placenta started to produce hormones by approximately 6-7 4 weeks of gestation, the hormone levels increased after 8 weeks of gestation in the HR group, and the 5 significant difference between the groups disappeared.
6 Our results suggest that increased serum E2 and P4 levels in the first trimester of pregnancy owing 7 to HRT are lower than the serum E2 and P4 levels noted during the first trimester of spontaneous 8 pregnancy or pregnancy following regular infertility treatment. This suggests that the risk of breast 9 cancer associated with thawed embryo transfer with HRT of estrogen and progesterone may not be 10 greater than that associated with spontaneous pregnancy.
11 This study has several limitations. First, only a few samples were collected from the patients in the 12 non-HR group. Therefore, we contained patients undergoing medication for ovulation induction in non-13 HR group. This was unavoidable because the study design involved the use of left-over blood from 14 blood drawn during scheduled hospital visits by women who conceived naturally or after IUI. Second, 15 because this study did not include breast cancer survivors, we were unable to assess the prognosis or 16 risk of recurrence in breast cancer survivors who underwent thawed embryo transfer with HRT of 17 estrogen and progesterone. It is an ethical dilemma not to offer HR to breast cancer survivors when they 18 have a rare opportunity to conceive using ART. Hence, we opted for a study design in which we 19 compared the non-HR and HR groups.

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In conclusion, our results showed that thawed embryo transfer with HRT did not increase the serum 13 1 hormone levels beyond those observed in spontaneous pregnancy. This suggests that in patients with 2 hormone receptor-positive breast cancer, HRT administered during the first trimester of a pregnancy, 3 established as a result of ART, after treatment of the primary disease may not increase the sex hormones 4 levels beyond those observed in spontaneous pregnancy.