Ovarian reserve and stimulation response before chemotherapy treatment in breast cancer patients undergoing fertility preservation: a historical cohort study

In this single-center historical cohort study, the data of 78 breast cancer patients undergoing COS for fertility preservation were compared with those of 66 healthy oocyte donors between April 2014 and March 2022 at the Infertility Center of Royan Institute, Tehran, Iran. The data for this study were collected from medical case records of (files) of patients. The Institutional Review Board approved this study and the ethical clearance was issued by the Royan Institute Ethics Committee (IR.ACECR.ROYAN.REC.1398.206), Tehran, Iran, in compliance with the Helsinki Declaration. All patients met the following inclusion criteria: women with a breast cancer diagnosis confirmed by histopathology with an indication for chemotherapy if they visited our infertility clinic before the beginning of their cancer treatment; if they desired for reproduction after cancer treatment; and if they aged 35 years and less. Exclusion criteria were women with poor ovarian response (according to the Bologna criteria [ 21 ]), with polycystic ovary syndrome (according to the Rotterdam criteria [ 22 ]), severe male factor infertility (azoospermia), endometriosis, and those who used oral contraceptives. According to our center’s practice, when young patients with a cancer diagnosis were referred by an oncologist for follicle puncture, the patients were counseled by a gynecologist about different fertility preservation strategies and were assessed by pelvic ultrasound and hormonal profile including Follicle-Stimulating Hormone (FSH) mIU/mL, Luteinizing Hormone (LH) mIU/mL, Estradiol (E2) pg/mL, Progesterone (P) ng/mL and AMH ng/mL. In both groups, ovarian stimulation was initiated with a gonadotropin-releasing hormone antagonist (GnRH-anta) protocol. Only the first cycle of the patients in both groups was studied. For all breast cancer patients presenting around the start of menses, ovarian stimulation was initiated using the conventional-Start COS method, with gonadotropin administration starting on day 2 or 3 of their menstrual cycle accompanied by the use of GnRH-anta. For breast cancer patients who did not present during menstruation, ovarian stimulation was initiated in the random-start manner as described in a previous publication from our center [ 23 ]. Briefly, the initial dose of gonadotropins was determined based on the patient’s age, body mass index (BMI), and ovarian reserve, according to AFC and AMH levels, and was adjusted based on the patient’s ovarian response (follicle number/size) as visualized by transvaginal ultrasound (performed every 24 to 48 h). Letrozole (Femati ® , Atipharmed company, Iran) was administered at a dose of 5 mg for all patients regardless of estrogen receptor status from the beginning of ovarian stimulation and was increased to 10 mg per day with the aim of maintaining estradiol levels below 500 pg/ml and continued until the trigger day. If the estradiol level on the trigger day was more than 500 pg/ml, the estradiol level was checked every other day and letrozole administration was continued until the estradiol level fell below 500 pg/ml, and then it was discontinued. In control group, the patients were received similar COS protocol (standard GnRH antagonist), except that these patients were not given letrozole. Final oocyte maturation was triggered with GnRh agonist (decapeptyl ® , Ferring, Saint-Prex, Switzerland) or recombinant HCG (rhCG) (Ovitrelle@ Choriogonadotropin alfa, Merck Serono, Germany) or both administration of GnRH agonist and rhCG under vaginal ultrasound guidance. Oocyte retrieval was performed 34–36 h after ovulation trigger and the oocytes in each woman were assessed by an embryologist for the number of oocytes retrieved and the number of Metaphase II (MII) oocytes. ICSI was performed using sperm cells of ejaculated with MII oocytes for patients who desired to have frozen embryos. Cryopreservation was performed by vitrification. The primary outcome measure was total number of MII oocytes retrieved, which was defined as oocytes that were retrieved at the metaphase II. Secondary outcome measures include: LH (mIU/mL), FSH (mIU/mL), AMH (ng/mL), starting dose of gonadotropins, total dose of gonadotropins administered, duration of ovarian stimulation, days of GnRH antagonist regimen, estradiol level on trigger day, number of follicles ≥ 13 mm on trigger day, number of retrieved oocytes, oocyte maturation rate (was defined as the ratio between the MII number and total number of retrieved oocytes) and ovarian hyperstimulation syndrome (OHSS). The statistical package for the social sciences (SPSS) software (version 22.0 for Windows; SPSS Inc., IBM, USA) was used for all statistical analyses. Data were initially tested for normal distribution using the Kolmogorove-Smirnov test. Data were normally distributed. Continuous variables were expressed as mean ± SD and categorical variables as number (percentage). Data were analyzed using the 2-tailed Student t-test for independent data, Fisher exact test, and a two-by-two table between groups, where appropriate. Pearson correlation analysis was conducted to examine the relationship between MII and AMH. Multiple linear regression analysis (multivariate analysis) was performed with the independent variables that were significant in univariate analyses at a level of 0.10. P  < 0.05 was considered statistically significant in multivariate analysis.

In this study, the treatment cycles of 144 patients (78 women in the cancer group and 66 women in the control group) were examined. The most common receptor type in the group of women with breast cancer was E positive and P positive receptors (62.5%), and the least common receptor types were P positive and E negative receptors (1.6%) and E positive and P negative receptors (3.1%). In addition, the majority of subjects were in grades A2 (41.8%) and A1 (25.5%). Comparisons of demographic data between groups revealed a significant difference between the two groups by mean age and BMI. Compare to controls, mean age was significantly higher and BMI was significantly lower in the breast cancer group ( p  < 0.001 and p  = 0.034, respectively). In most of patients with breast cancer, early proliferative and luteal phase were common days of starting the ovulation stimulation protocol ( P  < 0.001). According to hormonal examines, LH was statistically higher in breast cancer patient than controls ( p  = 0.001). On the contrary, AMH was statistically lower in breast cancer patient than controls ( p  = 0.005). The starting dose of gonadotropin and total dose of gonadotropin used were significantly higher in patients with breast cancer group compared to donors ( p  < 0.001and p  = 0.005, respectively). There was a statistically significant difference between the two groups by type of trigger used. GnRH agonist was frequent trigger in each group ( P  = 0.001). FSH, duration of ovarian stimulation, days of GnRH antagonist regimen, number of follicles ≥ 13 mm on trigger day, number of retrieved oocytes and number of MI oocytes were comparable between groups. Although the number of MII oocytes was not statistically different between groups, mean of oocyte maturation rate was significantly lower in breast cancer patients compared than controls. (Table  1 ). In contrast, the mean number of GV oocytes was significantly higher in breast cancer patients than controls. In our study, 30 out of 78 breast cancer patients had embryo cryopreservation. According to our analysis, the mean obtained embryos were insignificantly lower in breast cancer patients than oocyte donors (Table  1 ). Only one person in the donor group reported mild OHSS. Table 1 Demographic characteristics and cycle outcome of breast cancer and control groups Breast cancer ( n  = 78) Control ( n  = 66) P -value* Age (years) 30.62 ± 3.12 28.3 ± 3.10 < 0.001 BMI (kg/m2) 24.32 ± 2.90 25.67 ± 4.32 0.034 Day of starting ovulation stimulation Early proliferative 32 (41%) 66 (100%) < 0.001 Late proliferative 13 (16.7%) 0 (0%) Luteal 33 (42.3%) 0 (0%) LH (mIU/mL) 7.98 ± 1.02 4.22 ± 0.28 0.001 FSH (mIU/mL) 4.99 ± 0.29 5.23 ± 0.17 0.494 AMH (ng/mL) 3.51 ± 0.33 5.06 ± 0.43 0.005 E2 (pg/mL) level on trigger day 436.14 ± 40.79 - - Starting dose of gonadotropins (IU) 179.33 ± 49.34 156.82 ± 21.73 < 0.001 Total dose of gonadotropins used (IU) 2031.73 ± 103.03 1680.68 ± 67.43 0.005 Duration of ovarian stimulation 9.85 ± 2.03 9.65 ± 1.79 0.547 Days of GnRH antagonist regimen 4.71 ± 1.50 4.95 ± 1.27 0.289 Number of follicles ≥ 13 mm on trigger day 9.37 ± 5.19 10.31 ± 4.57 0.252 Trigger types GnRh agonist 52 (66.7%) 65 (98.5%) 0.001 rhCG 23 (29.5%) 1 (1.5%) GnRh agonist + rhCG 3 (3.8%) 0 (0%) Number of retrieved oocytes 11.38 ± 7.50 11.71 ± 5.89 0.770 Number of MII 8.35 ± 5.64 9.59 ± 4.79 0.160 Number of MI 1.00 ± 0.18 0.89 ± 0.13 0.651 Number of GV 1.62 ± 0.25 0.89 ± 0.22 0.033 Oocyte maturation rate 75.81 ± 20.07 83.57 ± 18.18 0.017 Number of obtained embryos ⁑ 7.03 ± 1.04 9.17 ± 0.83 0.124 Values are presented as the mean ± Standard deviation (SD) and number (percent). *Obtained by independent t test and chi square test. Statistically significant level was 0.05. BMI: body mass index. LH: luteinizing hormone. FSH: follicle-stimulating hormone. AMH: anti-Mullerian. hormone. E2: estradiol; MII: metaphase II. MI: metaphase I. GV: germinal vesicle. Oocyte maturation rate: MII oocyte/ total number of oocytes retrieved× 100. ⁑ 30 out of 78 breast cancer patients had embryo cryopreservation Demographic characteristics and cycle outcome of breast cancer and control groups Values are presented as the mean ± Standard deviation (SD) and number (percent). *Obtained by independent t test and chi square test. Statistically significant level was 0.05. BMI: body mass index. LH: luteinizing hormone. FSH: follicle-stimulating hormone. AMH: anti-Mullerian. hormone. E2: estradiol; MII: metaphase II. MI: metaphase I. GV: germinal vesicle. Oocyte maturation rate: MII oocyte/ total number of oocytes retrieved× 100. ⁑ 30 out of 78 breast cancer patients had embryo cryopreservation Based on the Pearson correlation test, there was a statistically significant relationship between AMH level and MII number in women with breast cancer ( r  = 0.626, P  < 0.001). We performed a multiple linear regression analysis to adjust the possible effects of variables that were significantly different between the two groups (Table  1 ) on the primary outcome. In the multiple linear regression analysis for MII, only AMH was statistically significantly associated with the number of MII (Table  2 ). Table 2 Multiple linear regression analysis of factors related to MII in breast cancer patients Independent factors β SE p -value B 95% CI Lower Power Age (years) -0.056 0.197 0.616 -0.100 -0.495 0.296 BMI (kg/m2) -0.150 0.199 0.164 -0.281 -0.681 0.118 LH (mIU/mL) 0.065 0.072 0.535 0.045 -0.100 0.190 AMH (ng/mL) 0.633 0.228 < 0.001 1.195 0.737 1.652 Starting dose of gonadotropins (IU) 0.039 0.015 0.758 0.005 -0.025 0.034 Total dose of gonadotropins used (IU) 0.010 0.001 0.951 5.677 -0.002 0.002 Days of starting ovulation stimulation -0.042 1.228 0.710 -0.458 -2.921 2.004 Trigger types 0.019 0.747 0.882 0.111 -1.386 1.608 CI: confidence interval; B, unstandardized coefficient; SE, standard error; β, standardized coefficient. BMI: body mass index. LH: luteinizing hormone. AMH: anti-Mullerian hormone Multiple linear regression analysis of factors related to MII in breast cancer patients CI: confidence interval; B, unstandardized coefficient; SE, standard error; β, standardized coefficient. BMI: body mass index. LH: luteinizing hormone. AMH: anti-Mullerian hormone

It appears that patients with cancer experience diminished ovarian reserve prior to cancer treatment. Further prospective studies with larger sample sizes are recommended.

Based on studies conducted on female cancer patients, it has been hypothesized that malignancy may affect ovarian reserves, follicular growth, and oocyte quality through increased catabolic states, malnutrition, or hypothalamic dysfunction caused by stress [ 24 ]. The aim of this study was to evaluate the relationship between breast cancer and ovarian function including ovarian reserve and ovarian stimulation response in patients undergoing fertility preservation programs. Previous research findings in this area have been inconsistent and contradictory. In a 2017 cohort study by Quinn et al. [ 25 ] on 191 newly diagnosed breast cancer patients, it was shown that there was no evidence of impaired ovarian reserves before gonadotoxic treatment. Cancer grade did not affect ovarian stimulation outcomes in this study. In contrast, data from several sources have identified impaired ovarian stimulation responses for cancer patients undergoing fertility preservation than the control group [ 15 – 17 , 26 , 27 ]. In preliminary studies on cancer patients, baseline ovarian reserve assessments were not reported. Without this information, it is difficult to determine whether differences in outcomes were due to ovarian reserves or stimulation responses. Based on our results, it seems that patients with cancer face reduced ovarian reserves and diminished ovarian stimulation responses before cancer treatment. Similar to another study [ 28 ], our results showed that despite higher starting doses and total doses of gonadotropins used in the breast cancer group and similar duration of stimulation days in both groups, the number of dominant follicles and retrieved oocytes were comparable. Furthermore, the levels of AMH and oocyte maturation rate was significantly lower in our breast cancer patients. Similarly, data from a prospective, non-interventional study [ 26 ], on cancer patients, recruited before starting chemotherapy, showed that the numbers of total oocytes and MII oocytes and oocyte maturation rate were significantly lower in cancer patients compared than age-matched healthy controls. Results from another large multicenter study [ 15 ] on three patient groups showed that women with hormone-dependent tumors had lower responses to COS compared to those with hormone-independent cancers and control groups. Alvarez et al. in a large study [ 29 ] on 306 female cancer patients underwent ovarian stimulation for oocyte or embryo cryopreservation, analyzed the records of each type of cancer separately. The number of mature oocytes retrieved was significantly lower in patients with gynecological malignancy (7.73 ± 6.33) and breast cancer patients (9.64 ± 6.31) compared with hematological (13.33 ± 9.01) ( P  = 0.005 and P  = 0.016, respectively). In another investigation comparing IVM outcomes between women with malignancies and age-matched control group [ 30 ], it was shown that women diagnosed with breast cancer obtained fewer oocytes compared to the infertile control group. Nevertheless, ovarian reserves and oocyte maturation were found to be comparable between other malignancies and controls. Another research group [ 31 ] also reported a 2.4-fold reduction in oocytes among women with breast cancer in comparison with women of a similar age stimulated by conventional COS protocols. Comparing AMH levels between breast cancer patients and individuals undergoing elective fertility preservation further supports this claim [ 25 ]. Studies of ovarian reserves in individuals with measured AMH levels indicate that women with cancer have lower AMH levels before beginning chemotherapy than expected for their age group [ 32 , 33 ]. Similarly, in our study, the mean AMH was significantly lower in the breast cancer group compared to the control group. A recent systematic review and meta-analysis [ 20 ] investigating the association between cancer and ovarian function prior to gonadotoxic treatment and demonstrated that cancer patients had lower serum AMH levels ( P  = 0.001) and AFC ( P  = 0.033) compared to healthy controls. According to a recent cohort study [ 34 ] on 101 stimulation cycles of breast cancer women indicated that higher grade of breast cancer is associated with lower AFC at baseline and need for higher doses of gonadotropin during ovarian stimulation. It is worth noting that many reproductive-age women use oral contraceptives to prevent pregnancy or manage menstrual irregularities [ 35 ]. There is a correlation between recent long-term oral contraceptives use and reduced AFC and AMH [ 36 ]. One effective way to overcome any suppression caused by long-term use of hormonal contraceptives for patients undergoing elective cryopreservation is to delay ovarian stimulation for several months, whereas breast cancer patients do not have this opportunity before cancer treatment [ 25 ]. In our study, women who had used oral contraceptives were not included. According to our analysis, ovarian stimulation began during the luteal phase in 42.3% of breast cancer patients, which may explain the higher mean LH levels at the start of the treatment cycle in the breast cancer group compared to the control group. A number of studies have linked the use of letrozole to lower numbers of mature oocytes in breast cancer patients. In a retrospective cohort analysis [ 37 ], the benefits and risks of using letrozole for COS in young estrogen receptor-positive breast cancer patients was evaluated and reported that the number of mature oocytes was significantly (approximately 40%) lower when letrozole was used compared to gonadotropin alone. One possible explanation for this finding may be that in this study, both the starting dose and total dose of gonadotropins were significantly lower in letrozole group than the group that did not use letrozole. Conversely, another study [ 10 ] showed that breast cancer patients undergoing COS with letrozole and gonadotropins had more mature oocytes compared with patients treated with gonadotropins alone for selective oocyte cryopreservation. The authors concluded that higher yield of mature oocytes in the breast cancer group may be attributed to variations in hCG trigger criteria and triggering at follicle sizes larger than 18 mm in letrozole-based protocols. In this regard, some studies have suggested that triggering at follicle size between 17 and 18 mm as standard follicle size in letrozole stimulation protocols results in a higher number of immature oocytes [ 38 , 39 ]. Some other studies reported similar results in patients with or without aromatase inhibitors [ 40 , 41 ]. In an in vitro mice study, it was reported that when pre-antral follicles were cultured with an aromatase inhibitor, the antral space formed earlier compared to the control group, although oocyte quality did not change [ 42 ]. In our center, during the time interval of our study, all breast cancer patients who were elected for ovarian stimulation for fertility preservation received aromatase inhibitor regardless of estrogen receptor status and after 2022, only estrogen receptor positive patients received aromatase inhibitors. In present study, the starting and total doses of gonadotropins were significantly higher in the breast cancer group compared to the control group. Similarly, in Almog et al. study [ 43 ], the FSH dose in the upper quartile of the study group was non-significantly higher than the one in control group (225 IU). A possible explanation for these observations could be the physicians’ inclination towards achieving the maximum number of oocytes in patients who are about to undergo chemotherapy. Obviously, since all oocytes and embryos in the study group are frozen, the concern for OHSS is reduced. Notably, in our study, no cases of moderate to severe OHSS were observed among the COS cycles conducted for fertility preservation. The conflicting results between studies can partly be explained by differences in the control group populations. Infertile patients struggling with unsuccessful pregnancies clearly represent a different population than oocyte donors or patients undergoing fertility preservation. In our study, the control group consisted of oocyte donors with no previous history of infertility. Similarly, in the study by Garcia-Velasco et al., [ 44 ] where the control group consisted of women undergoing elective fertility preservation for “non-medical” reasons, fewer mature oocytes were observed in cancer patients compared to the control group. The strengths of our study were the inclusion of recently diagnosed breast cancer patients with no prior history of infertility and an appropriate control group comprising oocyte donors undergoing fertility cycles referred to a referral infertility center. Additionally, only data from the first ovarian stimulation cycle were analyzed, reducing the potential for bias from repeated cycles. Despite the importance of the present findings, this study has some limitations. An inevitable limitation is that aromatase inhibitor prescribed only in case group which may affect the result. Another limitation of our study lies in its retrospective feature. Because of the retrospective design of our study, conclusions may be influenced by the limited sample size and individual variations which may limit the generalizability of our findings. To overcome the limitation of a small patient population, it is recommended to conduct multicenter studies to determine the number and quality, and fertilization rates of oocytes in breast cancer patients. This would allow more effective counseling for patients wishing to preserve their fertility potential.

Breast cancer is the most common malignancy in adult women. The worldwide incidence of breast cancer has been increasing during the past decades, among young reproductive-aged women [ 1 , 2 ]. Recent advancements in early diagnosis and oncological treatments have led to improved 5-year survival rates [ 3 ]. This survival rate validates worries about gonadotoxicity related to chemotherapy in women with fertility goals. Chemotherapy can have direct (through impact on primordial follicles or the population of growing follicles) [ 4 , 5 ] or indirect (through age-related ovarian aging due to time required to attempt pregnancy after healing) detrimental effects on ovarian reserves [ 3 ]. Thus, it is crucial to consider fertility specialists’ counselling before cancer treatment for patients who have not completed family planning [ 4 ]. In light of recent advances in cryopreservation techniques, different fertility preservation options have been proposed, among which two main strategies are promising: ovarian tissue cryopreservation, and oocyte or embryo cryopreservation [ 6 ]. For oocyte or embryo cryopreservation as the most suitable and efficient strategy [ 5 ], the first step is controlled ovarian stimulation (COS), which should be considered. In breast cancer patients, concerns about COS include delays in initiating chemotherapy and exposure to high estradiol levels resulting from multiple follicle growth. Random-start ovarian stimulation, which allows women to start COS immediately instead of waiting for menstrual cycle phase, has become a standard approach, enabling oocyte retrieval in less than two weeks in most cases [ 7 , 8 ]. The possibility of estradiol levels rising above physiological levels is another concern among women undergoing COS which potentially may have negative affect on estrogen receptor-positive cancers, such as breast cancer [ 9 ]. Adjuvant treatment with aromatase inhibitors such as letrozole are regarded as standard approaches to maintain estradiol concentrations at lower levels [ 10 ]. Previous research on the clinical outcomes of ovarian stimulation for fertility preservation in women with cancer have yielded conflicting results. Contrary to some investigators who have shown that ovarian reserve and response in cancer patients are similar to normal population [ 11 – 14 ], some studies suggest that breast cancer patients respond less effectively to ovarian stimulation compared to control groups [ 15 – 18 ]. This is while some investigators believe that cancer patients do not have a lower response to ovarian stimulation but have diminished ovarian reserves, as indicated by anti-Müllerian hormone (AMH) levels or antral follicle counts (AFC) [ 19 , 20 ]. In this study, the result of ovulation stimulation cycles including ovarian reserve and ovarian stimulation response was investigated in breast cancer patients and oocyte donors as healthy women. This study was conducted to evaluate the ovarian performance including ovarian reserve and ovarian stimulation response in breast cancer patients undergoing COS for fertility preservation before chemotherapy, compared with oocyte donors as healthy women undergoing COS for in vitro fertilization/intracytoplasmic sperm injection (IVF-ICSI).