key: cord-0824245-6recw7pb
authors: Hoang, Quoc Huy; Ho, Hung Sy; Do, Huong Thuy; Nguyen, Tien Viet; Nguyen, Hong Phuong; Le, Minh Tam
title: Therapeutic effect of prolonged testosterone pretreatment in women with poor ovarian response: A randomized control trial
date: 2021-03-27
journal: Reprod Med Biol
DOI: 10.1002/rmb2.12383
sha: 6024b836a4509100210be9ac32a99bdf478cad52
doc_id: 824245
cord_uid: 6recw7pb

PURPOSE: This study compared the therapeutic effects of transdermal testosterone gel (TTG) application at 4 and 6 weeks before controlled ovarian hyperstimulation (COH) in women with poor ovarian response (POR). METHODS: In this randomized control trial, infertile women with POR who underwent in vitro fertilization (IVF) were recruited and randomly classified into 4 week (n = 42) and 6 week (n = 38) TTG treatment groups and control group (n = 42). The primary outcome was total number of retrieved mature oocytes. The secondary outcomes were the biochemical pregnancy rate, clinical pregnancy rate, and ongoing pregnancy rate. RESULTS: No significant differences were observed in the number of oocytes retrieved, mature oocytes and embryos between all groups. Human chorionic gonadotropin (hCG) positive, clinical, and ongoing pregnancy rates were significantly higher in the TTG pretreatment groups than in the control group but no differences were observed between the 4‐ and 6 week groups. CONCLUSIONS: Applying TTG in infertile women with POR may ameliorate the outcomes of IVF. The extended application of TTG to 6 weeks did not improve the response to ovarian stimulation regarding the number of retrieved oocytes nor pregnancy outcomes compared to the 4 week pretreatment.

dosages, implementation of a modified natural IVF cycle, and addition of adjuvant agents during ovarian stimulation. 1 One of the promising interventions is the administration of additional testosterone before or during ovarian stimulation because the low response to ovarian stimulation frequently reflects an agerelated decline in testosterone levels. There is a positive association between testosterone levels and the number of oocytes retrieved. 4, 5 Moreover, patients with higher testosterone baseline levels require a lower follicle-stimulating hormone (FSH) dose, a shorter duration of ovarian stimulation, and are more likely to achieve pregnancy than females with lower testosterone levels. 6 Testosterone supplementation in POR treatment could facilitate the transition of follicles from the dormant to the growing pool, during the early and intermediate stages of follicular maturation. 7 Available data suggest that testosterone may increase the number of pre-antral and antral follicles and augment the expression of FSH receptors in granulosa cells, potentially enhancing ovarian sensitivity to FSH. 1 Regarding transdermal testosterone gel (TTG) supplementation, evidence suggests the effect of time-effectiveness ratio is more than the dose-effectiveness ratio. 8 Androgen treatment duration might be critical in stimulating follicular growth. However, limited evidence exists on the optimal treatment duration. While several studies do not support the use of testosterone for less than 21 days due to negative results, [9] [10] [11] only one study evaluated the effects of testosterone pretreatment based on different application durations. 12 Compared with the control group, TTG pretreatment for 3 or 4 weeks increased antral follicle count (AFC) and ovarian stromal blood flow, therefore, augmenting the numbers of oocytes retrieved and matured oocytes. However, significantly better results in terms of clinical pregnancy rate (CPR) and live birth rate (LBR) were only achieved in patients treated with transdermal testosterone extensively (4 weeks). 12 These results seem to agree with the fact that the transition period from the pre-antral to the antral follicular stage lasts approximately 70 days in humans. 13 To evaluate the effect of a longer duration of androgen supplementation, we hypothesized that increasing the testosterone treatment duration to 6 weeks may potentially increase the number of recruited follicles. Therefore, the present study aimed to determine the comparative therapeutic effect of 6 week and 4 week TTG application in women with POR.

This randomized control trial (RCT) was conducted between January 2018 and September 2019 at the National Center for Reproductive Medicine, Hanoi, Vietnam. In total, 165 women with POR undergoing IVF treatment in the study was assessed. For eligibility, inclusion criteria based on Bologna criteria included patients who met at least two of the following criteria: (a) advanced maternal age (40 years) or any other POR risk factor; (b) A previous incident of POR (cycles canceled or 3 oocytes with a conventional ovarian stimulation protocol); (c) A low ovarian reserve test (AFC ≤ 5-7 or AMH ≤ 0.5-1.1 ng/mL). In contrast, exclusion criteria included severe primary ovarian insufficiency (POI) (which was defined as basal FSH level over 40 mIU/mL, basal estradiol level under 5 pg/mL, and female age < 40 years), donated gamete IVF cycles, male factor infertility, severe endometriosis or unexplained infertility, thyroid disease, liver and kidney dysfunction, and abnormal genitalia. After screening, eligible patients were randomly allocated into two TTG intervention groups (4 week and 6 week groups) and one control group through a manual lottery. Allocation concealment was by sequentially numbered, opaque, sealed envelopes. All participants were blinded to group assignment; however, researchers were not blinded.

The two TTG groups were administered 12.5 mg transdermal testosterone 1% (AndroGel 50 mg, Besin Healthcare, Belgium) onto clean, dry, healthy skin over both upper arms, once daily in the evening. All women did not take any form of oral contraceptive pills before or during the pretreatment period. Patients were instructed to avoid washing the skin on which the gel had been applied for 5 hours after application. The duration of TTG pretreatment was 4 or 6 weeks, depending on the group. In the 4 week group, TTG was applied from the first day of the menstrual cycle until the first day of the next menstrual cycle. In the 6 week group, TTG application was started 2 weeks earlier than in the 4 week group and ended on the first day of the menstrual cycle. Ovarian stimulation was started the day after the last testosterone gel application. In the control group, con- Healthcare, France) was administered (800 mg/day) from the day of oocyte retrieval until 12 weeks of gestational age. 

Ultrasonic scans were performed using a GE Voluson p6 ultrasound machine (2018, Korea) equipped with a 5-7 MHz endovaginal probe.

The AFC ultrasound procedure was performed on day 2 of the menstrual cycle. All follicles measuring 2-10 mm in diameter in both ovaries were counted and recorded with the total number of AFCs. In IVF cycles, patients were scanned by ultrasound at least three times on day 2 of the cycle, day 6 of FSH, and day 8 of FSH. The last ultrasound was performed on the day of the trigger. The follicular diameter was calculated as the average length of the longest follicular axis and that of the axis perpendicular to it in the same scanning plane.

The sample size was calculated for the difference in 2 independent means as follows:

Notably, μ 1 ± σ 1 = 5.4 ± 1.9 was considered the number of oocytes retrieved in the testosterone group, and μ 2 ± σ 2 = 3.8 ± 1.4 was the number of oocytes retrieved in the control group according to Kim's study. 2 Z 1−α/2 = 1.96 corresponds to 95% confidence interval; Z 1−β = 1.64 corresponds to 95% strength, and the minimum sample size was 28 cycles per group. In total, 80 cycles were recruited for the study population during the recruitment period.

The primary outcome of this study was the total number of retrieved mature oocytes. The secondary outcomes were the ongoing pregnancy rate per cycle, pregnancy rate, biochemical pregnancy rate, and clinical pregnancy rate per cycle. Similarly, we also evaluated and analyzed the total dose and days of rFSH administration. Pregnancy was defined as positive when the serum beta-hCG level was ≥50 IU/L 14 days after embryo transfer. Clinical pregnancy was defined as the presence of a gestational sac identified by transvaginal ultrasound 4 weeks after embryo transfer. Biochemical pregnancy was defined as the absence of an identifiable pregnancy on ultrasound examination despite a positive serum hCG pregnancy test, and ongoing pregnancy was defined as a viable intrauterine pregnancy after 12 weeks of gestation. Multivariable logistic regressions were used to quantify the effects of the treatment group, adjusted for all potential confounders including maternal age, body mass index (BMI), infertility duration, primary or secondary infertility, and history of IVF treatment. All statistical tests were two-sided, and P values <0.05 were considered statistically significant.

The protocol for this study was approved by the local Scientific and Ethical Committee in Biomedical Research, Hanoi Medical University (26NCS17/HMU IRB dated February 8, 2018) and the registration number at Clinical trials was NCT04602143. Written consent was obtained from all participants before data collection, following the Declaration of Helsinki.

We invited 165 potentially eligible women for screening, among which 6 were excluded before random assignment because they did not meet the study criteria. Among the 159 women who were eligible and agreed to participate, 37 were lost to follow-up because they received a donor egg or quit treatment before ovarian stimulation ( Figure 1 ).

No differences were observed between the TTG pretreatment and control groups in general characteristics, including age, BMI,

infertility duration, infertility classification, history of IVF treatment, AFC, and basal endocrine profile (Table 1) . No difference in the characteristics of the study sample was observed among the groups.

Two cycles (4.7%) of the control group, two cycles (4.7%) of the 4 week group, and one cycle (2.6%) of the 6 week TTG group were canceled because of the lack of follicular development after stimulation or unproductive embryo obtained after culture (Table 2 ). No significant difference in the overall cycle cancellation rate was observed between the TTG pretreatment and control groups.

Compared to the control group, the treatment groups (either 4 or 6 weeks) have a significantly high endometrial thickness on the day of hCG injection (P = .00), short time of recombinant folliclestimulating hormone (rFSH) administration (P = .00), and a low total rFSH dose (P = .00) ( Table 2) . Similarity, all these differences are observed when comparing the 6 week group with the 4 week group on the day of hCG injection (Table 2) .

Regarding the primary outcomes, no significant differences in the number of oocytes retrieved were observed between groups (5.5 ± 2.4 vs 5.4 ± 2.8 and 5.6 ± 3.4 in control and 4 week or 6 week group with P = .9 and P = .88, respectively). Similar findings were also obtained in the secondary outcomes, including mature oocytes Table 3) .

The hCG positive and ongoing pregnancy rates were significantly higher in the TTG pretreatment groups (32.5% and 30% in 4 week;

29.7% and 21.6% in 6 week group, respectively) than in the control group (only 10% and 7.5%, respectively) as shown in Table 3 .

Additionally, the clinical pregnancy rate was higher in the 4 week pretreatment groups (32.5% vs 10%) than in the control group (P = .01). No differences in hCG positive, clinical, and ongoing pregnancy rates were observed between the 4 and 6 week TTG pretreatment groups. Only one case presented with light itching for a short time, which disappeared after a few days.

The effects of TTG treatment were further examined in multivariable logistic regression models, adjusted for all potential confounders ( Table 4 ). The statistically significant differences were observed in the TTG treatment groups, unlike the BMI and female age groups.

The 4 week pretreatment group has a better pregnancy outcome in hCG positive rate (odds ratio (OR): 4.14, 95% CI (confidence inter- 

This study was designed to evaluate the optimal duration of androgen supplementation before controlled ovarian stimulation using a GnRH F I G U R E 1 Study population enrollment flow chart From 165 women with poor ovarian response, 159 cases satisfied the inclusion and exclusion criteria. After screening, eligible patients were randomly allocated into two transdermal testosterone gel intervention groups (53 cases in 4 wk group and 53 cases in 6 wk group) and one control group (53 cases) through a manual lottery. During the time of study, loss of follow-up and discontinue of treatment results in the final data with 42, 38, and 42 women in 4 wk group, 6 wk group, and the control group, respectively antagonist in poor responders undergoing IVF. Our data confirms that androgen supplementation improves response to ovarian stimulation, regarding the number of retrieved oocytes. However, the 4 week protocol appeared to be as good as 6 week administration in term of ovarian response and pregnancy outcomes. Therefore, it is unnecessary to extend the duration of androgen supplementation to 6 weeks. substrate in the steroid making process of the ovaries based on the two-cell, two-gonadotropin theory. 14 Androgens supposedly play an important role in early follicular development and granulosa cell proliferation. [15] [16] [17] Additionally, they have been shown to increase the expression of FSH receptors in granulosa cells and increase the number of pre-antral and antral follicles. 16 In studies of human granulosa cells and follicular fluid of primary follicles, a significant correlation was observed between androgen receptors and FSH receptors in mRNA expression. Furthermore, follicle fluid testosterone concentration was observed to be highly correlated with FSH receptor mRNA expression. 18 Although there have been many studies on the use of testosterone before ovarian stimulation, the results are limited and controversial. Similarly, studies have evaluated the efficacy of the bioactive androgen, testosterone, and its use in patients with POR to improve ovarian response and IVF outcomes. [9] [10] [11] 19 Our study found that androgen supplementation did not improve the retrieved oocytes number, however, better pregnancy outcomes were recognized in testosterone treatment groups. Although the number of embryos transferred was similar, clinical and ongoing pregnancy rates were lower in the control group than in the treatment groups. Because the endometrium thickness was higher in the 4 week group than in the control group, the "quality" of the oocytes and, therefore, quality of the embryos might be improved with testosterone supplement.

The duration and total dose of testosterone supplementation were suggested as key factors before controlled ovarian stimulation. 12 The ovarian bioavailability of systemic androgen is recommended to be studied further. 20 Similarly, the appropriate timing of androgen supplementation should be carefully considered.

Considering the effect of androgen in the early stages of follicular development, supplementation can be taken for 1-3 weeks before ovarian stimulation. 12, 17, 21, 22 To evaluate the effect of a longer duration of androgen supplementation, the present RCT compared 4 and 6 week interventions to a control group to evaluate the optimum duration of testosterone supplementation in patients with POR. Our data showed that the duration of FSH administration and the total FSH dose in the 4 week group were significantly lower than those in the 6 week group. Positive effects of TTG pretreatment on ovarian stimulation were observed in the pregnancy and ongoing pregnancy rates in the treatment groups compared to the control group.

The main findings of this study were the nonsignificant differences observed in pregnancy rate, CPR per cycle recruited, and ongoing pregnancy rates per cycle recruited between the 4 and 6 week TTG Therefore, a short course of androgens has no significant effect on ovarian response and IVF outcome.

A possible limitation of this study is that it was not doubleblinded as the clinicians were aware of the groups the patients were classified. This study was conducted in a tertiary hospital, a national center for assisted reproduction; therefore, clinical activities were monitored closely by the principal investigator, and the study design was strictly followed. The study sample was homogeneous (by random selection using sealed envelopes), based on general characteristics between groups. Although the sample size of 122 patients can be considered a strength of this study, we did not include severe POI cases into the inclusion criteria because they all received donated oocytes after consultation. Furthermore, several previous studies reported that there was a positive association between testosterone levels and the number of oocytes retrieved 4,5 ; however, we did not collect data on serum testosterone. Therefore, this relation was not revealed by our data. In this study, some cases were lost to follow-up because they withdrew from treatment to have their oocytes donated and some other cycles were paused due to the need to adhere to the social distancing guideline because of the Covid-19 pandemic.

In conclusion, this study confirmed that TTG pretreatment could improve IVF outcomes in poor responders undergoing IVF/ICSI.

Particularly, a 4 week dose of 12.5 mg androgen supplementation appeared to be a better approach than an extended 6 week treatment as it guarantees a similar number of retrieved mature oocytes and pregnancy outcomes with shorter application time and lower cost.

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Therapeutic effect of prolonged testosterone pretreatment in women with poor ovarian response: A randomized control trial

The authors are grateful to physicians, administrative staff at the National Assisted Reproductive Technology Centre, NationalHospital of Obstetrics and Gynecology, Vietnam, for allowing and supporting us to undertake this research. Minh Tam Le https://orcid.org/0000-0001-6225-3108