Using Letrozole Cotreatment With Progestin-Primed Ovarian Stimulation In Women With Polycystic Ovary Syndrome Treated For IVF

Background: Women with polycystic ovary syndrome (PCOS) often experience poor oocyte quality and a high risk of ovarian hyperstimulation syndrome (OHSS) when treated with controlled ovarian stimulation (COS) in vitro fertilization (IVF). Progestin-primed ovarian stimulation (PPOS) shows good potential to compete with conventional protocols in women with PCOS. However, it always accompanied by increased pituitary suppression and gonadotropin consumption. Letrozole (LE) has the ability to increase luteinizing hormone (LH) levels and appears to have the potential to alleviate pituitary inhibition during COS in women with PCOS. A retrospective cohort trial was performed to evaluate the ecacy of PPOS with or without letrozole in infertile women with PCOS. Methods: This retrospective cohort study included 448 women with PCOS who underwent COS with human menopausal gonadotropin (hMG) and medroxyprogesterone acetate (MPA) (n=224) or hMG and MPA cotreatment with LE (n=224) from January 2018 to March 2021. Baseline characteristics of the two groups were balanced with propensity score matching using the nearest neighbour random matching algorithm at a ratio of 1:1. The primary outcome measure was the implantation rate. The secondary outcomes were the endocrinological proles, gonadotropin dose and duration, number of oocytes retrieved and viable embryos, clinical pregnancy rate, miscarriage rate and ectopic pregnancy rates. Result(s): The implantation rate was signicantly higher in the study group than that in the control group (42.22% vs. 34.69%, P < 0.05). Compared with the control group(cid:0)the study group had a higher LH concentration on the trigger day (3.85±3.6 mIU/ml vs. 2.44±1.71 mIU/ml, P < 0.01), but there was no case of premature LH surge or OHSS in both groups. The consumption of gonadotropin, the number of oocytes retrieved and viable embryos were similar between the two groups. Additionally, no difference was found in the clinical pregnancy rate, miscarriage rate or ectopic pregnancy rate. Conclusion(s): This study shows that LE administration in the PPOS protocol was feasible to improve the implantation rate and alleviate profound pituitary suppression from progestin administration without interfering with its premature LH surge blockade effect but with a non-signicant reduction in gonadotropin consumption in women with PCOS undergoing IVF treatment. No

Introduction Polycystic ovary syndrome (PCOS) is considered to be a highly prevalent endocrine and reproductive disorder and affects 5%-10% of women of reproductive age [1]. It is usually characterized by a heterogeneous syndrome of ovulatory dysfunction, polycystic ovarian morphology and hyperandrogenism [2,3]. In vitro fertilization (IVF) serves as a third-line treatment for infertile PCOS patients who fail to respond to lifestyle modi cation or ovulation induction (OI) therapies [4]. However, women with PCOS treated with IVF often experience a high risk of OHSS because of excessive numbers of oocytes retrieved [5] and poor oocyte quality, leading to repeated transplantation failure [6]. Therefore, safer and more e cacious controlled ovarian hyperstimulation (COH) regimens need to be explored.
Progestin-primed ovarian stimulation (PPOS), a regimen that uses progestin as an alternative to the gonadotropin-releasing hormone (GnRH) analogue in the follicular phase to suppress the premature luteinizing hormone (LH) surge [7], has been approved to be an e cacy and safety in the population of women with PCOS [8] [9]. Nevertheless, the use of progestin in the PPOS regimen tends to inhibit the pituitary in a more profound manner and thus, higher doses of human menopausal gonadotropin (hMG) are required than those in the GnRH agonist short protocol for patients with or without PCOS [7] [8]. Thus, optimizing the PPOS protocol is always the direction we have been trying to explore.
Clomiphene citrate (CC) which was used to increase follicle-stimulating hormone (FSH) and LH secretion from the pituitary by blocking a negative estradiol feedback loop by binding to estradiol receptors in the hypothalamus, was adopted for cotreatment with PPOS in our previous study, and the results showed that CC could increase LH values and diminish hMG consumption but with a harvest of fewer oocytes [10,11] and fewer top-quality embryos among women with PCOS [11]. Currently, letrozole appears to be a good alternative option in women with ovarian stimulation of PCOS, especially for those with CC resistance, due to its higher ovulation rate, pregnancy rate and live birth rate among patients with PCOS [12][13][14]. Different from the mechanism by which CC acts on hypothalamic estrogen receptors, LE decreases estradiolproduction and normal central negative feedback mechanisms remain intact [12].
Therefore, the clinical e cacy of letrozole combined with PPOS is worth exploring.  [15]: 1) oligo-and/or anovulation; 2) biochemical and/or clinical evidence of hyperandrogenism; or 3) polycystic ovarian morphology on ultrasound. The following diseases were excluded: other aetiologies of hyperandrogenism and ovulatory dysfunction, including congenital adrenal hyperplasia, androgensecreting tumours, hyperprolactinemia and thyroid disease. A owchart of the study is shown in Figure 1. . All follicles with a diameter over 10 mm were aspirated [16].

Controlled Ovarian Stimulation
After oocyte retrieval, fertilization was carried out in vitro by either standard IVF or intracytoplasmic sperm injection (ICSI) according to semen quality and previous fertilization conditions [17][18][19]. Embryos were examined for the number or regularity of blastomeres and the degree of fragmentation. Top-quality embryos (including grade-1 and grade-2 6-cell embryos and above) were frozen by means of vitri cation within three days after oocyte retrieval according to the criteria described by Cummins et al. [7,20]. The non-top-quality embryos were placed in extended culture, and blastocysts with good morphology were also frozen on day 5 or 6 [7,16,21,22].

Hormone Measurement
Serum FSH, LH, E 2 and P levels were measured on MC3, MC8, MC10-12, the trigger day and the day after trigger. Hormone concentrations were measured by means of chemiluminescence (Abbott Biologicals B.V., the Netherlands). The lower limits of sensitivity were as follows: FSH, 0.06 mIU/ml; LH, 0.09 mIU/ml; E 2 , 10 pg/ml; and P 0.1 ng/ml. The upper limit for the E 2 measurement was 5000 pg/ml. If the E 2 level on the trigger day or the day after trigger was greater than the upper limit, it was recorded as 5000 pg/ml without repeating the assay after sample dilution.

Endometrial Preparation and Frozen Embryo Transfer
Endometrial preparation and FET were arranged for the second cycle after oocyte retrieval as previously described [7,21]. Mild stimulation with letrozole was recommended for all patients, while hormone replacement therapy (HRT) was conducted for those who failed to become pregnant with mild stimulation cycles or with a history of a thin endometrium (endometrial thickness ≤6 mm). In the mild stimulation cycle, women were administered letrozole 2.5/5 mg for 5 days beginning at MC3. When the dominant follicle was ≥17 mm in diameter with an endometrial lining >8 mm, an E 2 level >150 pg/ml and a P level <1 ng/ml, a bolus of urinary hCG (5000 IU) was injected for ovulation triggering. Progesterone commenced 2 and 3 days later, followed by day 3 embryo transfer 4 and 5 days later or blastocyst transfer 6 and 7 days later via abdominal ultrasound guidance.

Outcome Measures
The primary outcome measure of this study was the implantation rate in FET cycles. The secondary measures included the Gn dose and duration, the number of oocytes retrieved, the number of viable embryos, profound LH suppression (a serum LH level less than 1 mIU/ml on the trigger day during ovarian stimulation [23]) and incidence of premature LH surge (an increased serum LH level of more than twice the baseline level or a serum LH >15 mIU/ml and increased serum progesterone level >2.5 ng/ml on the trigger day during ovarian stimulation [24]). As well as the clinical pregnancy rate, the miscarriage rate and ectopic pregnancy rate were recorded. The viable embryo rate per oocyte was de ned as the number of viable embryos divided by the number of oocytes retrieved. The cycle cancellation rate was estimated based on the number of patients who had no viable embryos after complete oocyte retrieval. The implantation rate was calculated according to the number of gestational sacs divided by the number of embryos transferred. Clinical pregnancy was de ned as the presence of at least one gestational sac with or without foetal heart activity by ultrasound examination at 4 weeks after FET divided by FET cycles.
The miscarriage rate was based on the proportion of eventuated pregnancies in spontaneous or therapeutic abortion.

Statistical Analysis
A propensity score matching (PSM) model was established to balance differences in baseline characteristics between the two groups. To estimate the propensity score, we selected 10 covariates, namely, age, antral follicle count (AFC), basal levels of FSH, LH, E 2 and P, type of infertility (primary or secondary), infertility duration, previous IVF attempts (0, 1-2 or ≥3), and body mass index (BMI).
Patients using MPA+hMG+LE were matched with the MPA+hMG group using the nearest-neighbour random matching algorithm at a ratio of 1:

Results
Patient Characteristics Figure 1 shows a pro le summary of the study. Brie y, a total of 2453 IVF/ICSI cycles were screened from our database, and 401 cycles were excluded as described in the Materials and Methods section. Of the remaining 2052 patients, 224 patients undergoing the MPA+ hMG +LE protocol were matched with 224 patients who used the MPA+ hMG protocol. All of these patients completed oocyte retrieval cycles and succeeded in producing oocytes (range, 1-62), while 34 patients had no viable embryos. Among the remaining patients, 371 women completed FET cycles. No signi cant between-group differences were found in the post-matching analysis with regard to any baseline characteristics (all P >0.05) ( Table 1). The basic characteristics before matching are presented in Supplementary Table 1.   Hormone Pro les During Treatment The endocrine dynamics of FSH, LH, E 2 and P during ovarian stimulation are presented in Figure 2. The FSH levels increased dramatically after hMG administration for 5 days and then remained stable until the trigger day. After the dual trigger, the FSH levels increased dramatically to nearly 20 mIU/ml. No signi cant differences were observed in FSH levels at any time point between the two groups.
During the whole process of COH, LH in the two groups always maintained a low level. However, there were differences in the change trend of LH between the two groups. LH in the control group showed a downward trend. However, in the study group, LH remained basically unchanged in the rst ve days, followed by a downward trend until the trigger day.. The LH values were signi cantly higher in the study group than those in the control group at each observation point during the COH (P < 0.01). In addition, there were signi cantly fewer patients experiencing profound LH suppression in the study group (7.14%, 16/224) than in the control group (14.29%, 32/224, P <0.05). None of the patients in either group experienced a premature LH surge.
Serum E 2 levels increased consistently with the development of multiple follicles and were signi cantly higher in the control group than those in the study group at each observation point (P < 0.01).
In addition, the P level increased gradually during ovarian stimulation, especially on the day after trigger in the two groups. On the trigger day and the day after trigger, the P values in the study group were signi cantly higher than those in the control group (P < 0.01).

Pregnancy Outcomes After FET Cycles
In this study, 382 women completed a total of 574 FET cycles. A total of 189 patients completed 280 FET cycles in the MPA+hMG+ LE group, and 193 patients completed 294 FET cycles in the MPA + hMG group.
Before the end of the study, 252 women completed one FET cycle, 83 women completed two FET cycles, 35 women completed three FET cycles, 9 women completed four FET cycles, 3 women completed ve FET cycles, and the remaining 32 women did not complete FET cycles due to personal reasons. A total of 970 embryos were thawed, and the rate of viable frozen-thawed embryos was 100%. The implantation rate in the study group (42.14%, 201/477) was signi cantly higher than that in the control group (34.69%, 171/493) (P < 0.05). The clinical pregnancy rate, miscarriage rate and ectopic pregnancy rate were comparable between the two groups (P > 0.05). The live birth rate and neonatal status require further statistical analysis.

Discussion
This retrospective cohort study rst evaluated the e cacy of PPOS protocols with or without letrozole in infertile women with PCOS. Our study showed that compared with PPOS protocol, letrozole supplementation could effectively improve the implantation rate and alleviate the profound pituitary suppression from progestin administration, but with a non-signi cant reduction in Gn consumption in women with PCOS undergoing IVF treatment.
The continuous supply of progestin in PPOS protocol can lead to strong pituitary suppression, presented as a sustained low level of LH during COH [7], which was also demonstrated by the present study. After combined letrozole treatment, the LH level kept steady in the rst ve days of treatment and then slowly declined until the trigger day. This may be related to the biological mechanism of letrozole, which can block oestrogen biosynthesis, thereby reducing negative oestrogenic feedback to the hypothalamic/pituitary axis and increasing the LH level [25]. Thus, the mean LH levels in the MPA+hMG+LE group were higher than those in the MPA + hMG group during the whole process of COH, and it follows that letrozole supplementation can release the pituitary from the deep inhibition of progestin. In addition, the large changes in LH levels (either an increase or decrease from basal levels) have been demonstrated to be associated with a decreased chance of clinical pregnancy [26]. Consistent with this, our present study also showed that relatively high levels and smaller changes in LH in the letrozole cotreatment with PPOS group were accompanied by an advanced implantation rate. While opposite conclusions also drawn that high follicular-phase LH levels appear to have a deleterious effect on the pregnancy outcome in women with PCOS [27]. Therefore, the optimal LH level required for the PPOS protocol in PCOS patients is still worth exploring.
Although letrozole alleviated the deep inhibitory effect of progestin on pituitary and improved LH levels during the COH, it does not interfere with the suppression of progestin on the premature LH surge. Indeed, there was no premature LH surge in either group of the present research. In rodent, hypothalamic GnRH/LH secretion is divided into GnRH/LH surge secretion, which is controlled by anteroventral periventricular (AVPV), and GnRH/LH pulse secretion, which is controlled by arcuate (ARC) nuclei, respectively [28]. Progestin's inhibitory effect on the GnRH/LH surge is mediated by AVPV [29], while letrozole plays a role to enhance LH pulse secretion through ARC in rodent [30]. The different action sites might explain why letrozole does not interfere with the blocking effect of progestin on LH surge. Whether this theory is applicable to humans still needs further exploration, as LH pulse and surge was regulated via different neuroendocrinology mechanisms in primate [28].
However, it should be mentioned that in our study, although letrozole coadministration with the PPOS protocol could signi cantly improve LH values in women with PCOS, the FSH levels were similar between the two groups during the COH. This may be related to the impaired steroid hormone feedback in females with PCOS [31,32]. Hyperactive GnRH neuron in females with PCOS [33] result in partial pituitary desensitization, mainly manifested by a relative decline of FSH responsiveness [31,32]. Because of this, the increase of FSH induced by LE through blocking oestrogen production [12] cannot be re ected in this study, and thus exogenous Gn consumption in both groups was comparable during the whole process of COH.
During oestrogen biosynthesis pathway, progesterone converts into androgen and then subsequently transformed into oestrogens by aromatase in brief [34]. As an aromatase inhibitor, letrozole coadministration results in decreased oestrogens and accumulation of precursors such as progesterone, testosterone as well as 17α-progesterone [12,34,35].Thus, the lower serum E 2 concentration and higher P levels were observed after cotreatment with LE in PPOS protocol. In addition, the accumulation of androgens promotes recruitment of small antral follicles [36,37]. Under the stimulation of exogenous Gn, the number of follicles with diameter >10 mm or >14 mm was signi cantly higher after LE coadministration, and more growing follicles may also result in higher progesterone levels in the late follicular phase during COH. However, the number of follicles that could reach the preovulation state may be similar although there were more follicles with diameter >10 mm, so comparable number of oocytes received was observed in both groups.
What we cannot ignore is that the rates of mature and fertilized oocytes were signi cantly lower in the study group, which may be related with the different follicles development mode between the two groups. After adding LE, higher LH level during the whole follicular phase in the study group probably advanced the dominant follicles selection [38], resulting in the polarization of the subsequent development of follicles. Thus, follicle sizes on the trigger day in the study group exhibit larger variation indicated by the B ultrasonic observation. Previous studies showed that patients with a greater proportion of their follicles within appropriate diameter range had more mature oocytes retrieved [39,40]. Hence, a more disparate distribution of follicular size in the study group means that fewer follicular could reach this appropriate diameter range, which may lead to lower mature oocyte and fertilization rates. Nevertheless, in this follicle polarization development model, the dominant follicles may be more fully developed and could obtain high qualityoocytes [39,40]. Moreover, higher progesterone level may also have a positive effect on oocyte development, which has been demonstrated in the previous study [36]. Therefore, the more developed of oocytes obtained, the better result of the implantation rate.
In our study, there was a waiting period between oocyte retrieval and embryo transfer as we used the "freeze-all" strategy, and 1462 extra embryos were not transferred, so we cannot show a complete data of pregnancy rates. Moreover, some women were less than three months pregnant, and some have not yet given birth, so we cannot provide data of ongoing pregnancy rates and live birth rate. Besides of that, this retrospective study may have potential unknown or unmeasured covariates, which may lead to incomplete or inexact matching and thus confound the robustness of the study ndings. Prospective randomized controlled study with larger sample sizes and basic research should be performed in the future. Flow chart of the study.