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The effect of intra-ovarian androgen priming on ovarian reserve parameters in Bologna poor responders
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The Effect of Intra-Ovarian Androgen Priming on Ovarian Reserve Parameters in Bologna Poor Responders Lan N Vuong , Manh T Ho , Anh N Ha , Toan D Pham , Tam TN Le , Claus Yding Andersen , Peter Humaidan PII: DOI: Reference:
S1472-6483(19)30829-6 https://doi.org/10.1016/j.rbmo.2019.11.005 RBMO 2296
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Reproductive BioMedicine Online
Received date: Revised date: Accepted date:
25 July 2019 10 October 2019 20 November 2019
Please cite this article as: Lan N Vuong , Manh T Ho , Anh N Ha , Toan D Pham , Tam TN Le , Claus Yding Andersen , Peter Humaidan , The Effect of Intra-Ovarian Androgen Priming on Ovarian Reserve Parameters in Bologna Poor Responders, Reproductive BioMedicine Online (2019), doi: https://doi.org/10.1016/j.rbmo.2019.11.005
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The Effect of Intra-Ovarian Androgen Priming on Ovarian Reserve Parameters in Bologna Poor Responders Lan N Vuong,a,b,c* Manh T Ho,b,c Anh N Hab,c, Toan D Phamb,c, Tam TN Leb,c, Claus Yding Andersend, Peter Humaidane,f a
University of Medicine and Pharmacy at Ho Chi Minh City, 217 Hong Bang Street, District
5, Ho Chi Minh City, Vietnam ([email protected]; [email protected]) b
IVFMD, My Duc Hospital, 4 Duong Nui Thanh, Phuong 13, Tan Binh, Ho Chí Minh City,
Vietnam ([email protected]; [email protected]; [email protected]; [email protected]; [email protected]) c
HOPE Research Center, 4 Nui Thanh Street, Ward 13, Tan Binh District, Ho Chi Minh City,
Vietnam ([email protected]; [email protected]; [email protected]; [email protected]; [email protected]) d
Laboratory of Reproductive Biology, The Copenhagen University Hospital and Faculty of
Health Science, Copenhagen University, Blegdamsvej 3B, 2200 København, Copenhagen, Denmark ([email protected]) e
The Fertility Clinic, Skive Regional Hospital, Øster Fælled Vej 5, 7800 Skive, Denmark
([email protected]) f
Faculty of Health, Aarhus University, Denmark and Faculty of Health, University of
Southern Denmark, Nordre Ringgade 1, 8000, Aarhus C, Denmark ([email protected])
*Correspondence: Dr LN Vuong, Department of Obstetrics and Gynaecology, University of Medicine and Pharmacy at Ho Chi Minh City, 217 Hong Bang Street, District 5, Ho Chi Minh City, Vietnam; Ph: +84 903008889; Email: [email protected]; [email protected]
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Abstract Research question: What are the effects of long-term androgen priming in Bologna criteria poor ovarian reserve (POR) patients undergoing IVF. Design: This open-label pilot study was conducted at IVFMC, My Duc Hospital, Ho Chi Minh City, Vietnam. It included consecutive patients aged 18–41 years who fulfilled Bologna criteria for POR undergoing intra-ovarian androgen priming and ultra-long down-regulation with a gonadotropin-releasing hormone agonist (GnRHa), followed by stimulation with gonadotropins and GnRH antagonist co-treatment for IVF (n=30). Priming consisted of lowdose recombinant human chorionic gonadotropin (rhCG) 260 IU every second day plus letrozole 2.5 mg/day, both for 8 weeks; priming stopped on the first day of ovarian stimulation. The primary endpoint was serum anti-Müllerian hormone (AMH) concentrations 8 weeks after priming. Secondary endpoints included AFC (2-10 mm), serum hCG, testosterone, and progesterone levels. Results: Circulating testosterone, progesterone, estradiol and hCG levels remained unchanged during androgen priming; the mean AMH level decreased steadily from 0.49 ng/mL (baseline) to 0.33 ng/mL (8 weeks). AFC was 4–5 throughout the study. A mean of 1.10.9 good transferable embryos were obtained; embryo transfer was performed in 15 patients; no ongoing pregnancies were obtained. Conclusions: Long-term intra-ovarian androgen priming in the current set-up had no significant effect on hormone levels, AFC and recruitable follicles after ovarian stimulation in Bologna POR patients undergoing IVF. Further studies are needed to explore other androgen priming protocols and the clinical value of priming regimens in IVF.
Key words: ovarian reserve; poor response; in vitro fertilization; intra-ovarian androgen priming; Bologna criteria
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Key message In poor ovarian reserve patients who underwent ultra-long GnRHa down-regulation an intraovarian androgen priming regimen for 8 weeks before stimulation had no benefit in terms of ovarian reserve parameters and steroid levels.
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Abbreviations AFC
antral follicle count
AMH
Anti-Müllerian hormone
AR
androgen receptor
ART
assisted reproductive technology
BMI
body mass index
E2
estradiol
ESHRE
European Society of Human Reproduction and Embryology
ET
embryo transfer
FF
follicular fluid
FSH
follicle-stimulating hormone
FSHR
follicle-stimulating hormone receptor
GnRH
gonadotropin-releasing hormone
GnRHa
gonadotropin-releasing hormone agonist
hCG
human chorionic gonadotropin
IVF
in vitro fertilization
LH
luteinizing hormone
OPU
oocyte pick-up
P4
progesterone
POR
poor ovarian response
rhCG
recombinant human chorionic gonadotropin
rLH
recombinant luteinizing hormone
SC
subcutaneous
SD
standard deviation
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Introduction The management of patients with impaired or poor ovarian response (POR) undergoing infertility treatment has challenged reproductive specialists for decades. Different classifications of POR, including the Bologna Criteria (Ferraretti et al. 2011) and more recently the POSEIDON criteria (Humaidan et al. 2016), have helped standardize the definition of the poor responder population. However, this subset of in vitro fertilization (IVF) patients still remains a therapeutic challenge, experiencing disappointing overall outcomes in assisted reproductive technology (ART) due to the retrieval of a very low number of oocytes after exogenous stimulation with gonadotropins (Papathanasiou et al. 2016). In general, patients with POR have a low ovarian reserve characterized by few antral follicles and low circulating anti-Müllerian hormone (AMH) levels. Androgens play a pivotal role in folliculogenesis, facilitating follicle-stimulating hormone (FSH) receptor (FSHR) expression on granulosa cells via the androgen receptor (AR). In turn, increased FSHR expression increases follicular responsiveness to FSH (either endogenous or exogenous) (Weil et al. 1999). In addition, androgens act synergistically with insulin-like growth factor to stimulate growth of the follicle (Vendola et al. 1999). Interestingly, long-term exogenous androgen treatment has been shown to increase the number of pre-antral and antral follicles and, thus, the number of recruitable follicles in both animal models and in humans (Spinder et al. 1989, Vendola et al. 1998). Furthermore, intrafollicular concentrations of androgens and expression of ARs on granulosa cells are highly significantly associated with FSHR expression in humans (Nielsen et al. 2011), suggesting that circulating androgens in humans may facilitate a greater responsiveness of the ovarian follicle when exposed to exogenous FSH stimulation. Short-term intra-ovarian ‘androgen priming’ using a combination of human chorionic gonadotropin (hCG) and aromatase inhibitors during the early follicular phase was shown to
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significantly increase the proportion of cycle day 2 antral follicles reaching 14 mm in diameter, as well as serum estradiol (E2), and follicular fluid (FF) levels of E2 and testosterone; however, short time priming had no effect on the number of oocytes retrieved nor on reproductive outcomes (Lossl et al. 2006, Lossl et al. 2008). In this study we wanted to explore the effect of a novel long-term intra-ovarian androgen priming protocol, based on the “two cell-two gonadotropin model” in which luteinizing hormone (LH) activity regulates theca cell conversion of cholesterol into androgens, and androgens are converted to E2 in granulosa cells due to the combined actions of FSH and aromatase. In this new protocol, patients are subjected to long-term (8 weeks) low-level LH-like activity provided by repeated micro doses of recombinant hCG [rhCG]) to boost local endogenous androgen production by theca cells. To block the conversion of androgens into E2 in the granulosa cell, an aromatase inhibitor is administered on a daily basis, thus, increasing the amount of intra-ovarian and circulating endogenous androgens. We call this concept “intra ovarian priming”. The present pilot study investigated the effects of the proposed long-term androgen priming regimen on ovarian reserve parameters in patients considered to have the lowest ovarian reserve in IVF, the so-called European Society of Human Reproduction and Embryology (ESHRE) Bologna criteria POR patient.
Materials and methods Study design and population This single center pilot study was conducted at IVFMD, My Duc Hospital, Ho Chi Minh City, Vietnam between February 2018 and March 2019 (NCT03447184). Consecutive patients who fulfilled the ESHRE Bologna criteria for POR (Ferraretti et al. 2011), determined within the previous 2 months, were eligible to participate. Other inclusion criteria were: age 18–41
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years; body mass index (BMI) <30 kg/m2; and co-treatment with a gonadotropin-releasing hormone (GnRH) antagonist during ovarian stimulation. All participants provided informed consent. Women were excluded if they had any chronic medical conditions (e.g. diabetes, Crohn’s disease, thyroid disease, hepatitis B or a sexually transmitted disease) or were participating in another interventional clinical trial.
Ethical approval The study was performed in accordance with the ICH Harmonised Tripartite Guideline for Good Clinical Practice and the ethical principles of the Declaration of Helsinki. Ethics approval was obtained from the Ethical Board of My Duc Hospital (approval number 01/18/ĐĐ-BVMĐ, dated 22 January 2018). The trial was registered in clinicaltrials.gov (NCT03447184), Unique Protocol ID: CS/BVMD/18/01
Endpoints The primary endpoint was serum AMH levels after 8 weeks of priming. Secondary endpoints included antral follicle count (AFC) (2-10 mm) after 8 weeks of priming, at ovulation trigger and at oocyte retrieval; serum hCG, testosterone, and progesterone (P4) levels after 8 weeks of priming, on the day of ovulation trigger, and oocyte retrieval.
Study protocol Androgen priming Androgen priming started 8 weeks prior to stimulation for IVF, on day two of menses. From this day onwards patients received low-dose rhCG (Ovitrelle) 260 IU subcutaneously (SC) every second day in addition to letrozole 2.5 mg daily for 8 weeks, stopping on the first stay of stimulation for IVF with exogenous gonadotropins. Alongside androgen priming, pituitary
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down-regulation with a GnRH agonist (GnRHa) started (depot injection of triptorelin 3.75 mg); a second depot dose of triptorelin was given 28 days later. Thus, the total priming period lasted for 8 weeks (Figure 1).
Stimulation, monitoring and oocyte pick-up Stimulation with exogenous gonadotropins started after 8 weeks of androgen priming, irrespective of menses. A fixed dose of recombinant FSH (rFSH; Gonal F, Merck) was used for the first 5 days, either 300 IU rFSH alone in patients aged <35 years or 300 IU rFSH and 150 IU rLH (follitropin alfa/lutropin alfa, Pergoveris, Merck) in patients aged 35 years (Figure 1). Monitoring was undertaken according to the standard clinical procedure. Cotreatment with a GnRH antagonist started on the fifth day of stimulation. A bolus of 6,500 IU rhCG (Ovitrelle, Merck) was given for triggering of final oocyte maturation (Figure 1). Oocyte pick-up (OPU) and embryo transfer were performed based on local policies and procedures. Luteal phase support was provided by vaginal micronized progesterone, 180 mg daily (Crinone, Merck).
Blood sampling A total of six blood samples (2 ml each) were collected during the study period: at baseline, during priming after 4 and 8 weeks, on the day of starting stimulation, at ovulation trigger and at OPU. The first sample at baseline was used to determine FSH, LH, E2, testosterone, and AMH levels. The other five samples were used to determine FSH, LH, E2, testosterone, P4, hCG and AMH levels.
Ultrasound monitoring
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Ultrasound examination to count all antral follicles of 2-10 mm in diameter in each ovary was performed at each of the same time points.
Statistical analysis No previous data exist on long-term endogenous androgen priming in IVF patients, and due to the explorative character of this pilot study a sample size calculation was not feasible; Instead we decided to recruit a total of 30 ESHRE Bologna criteria POR patients. Data were analyzed using SPSS version 20 software. Continuous variables are presented as mean standard deviation (SD) and were compared using Student’s t-test. Categorical data are expressed as numbers and were compared using the Chi-square test. All tests were two tailed, and a p-value of <0.05 was considered statistically significant.
Results Patient population A total of 30 patients were enrolled in this pilot study. Patients were lean, the majority had primary infertility, and the mean number of previous unsuccessful IVF attempts was two (Table 1). A summary of stimulation details and outcomes is provided in Table 2.
Effects of priming Mean AMH levels decreased steadily over time (Table 3) and in most patients the AMH value after 8 weeks of androgen priming was lower than that at baseline (Figure 2). Longterm androgen priming had no significant effects on the AFC (Table 3). FSH levels decreased, from baseline to 4 weeks, increased slightly at 8 weeks, and then increased markedly at ovulation trigger, following exogenous stimulation (Table 3). Levels of circulating testosterone and P4 remained unchanged during the androgen priming period, to increase at
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ovulation trigger and OPU (Table 3). No changes were seen in E2 and hCG levels during androgen priming; as expected E2 levels increased during stimulation, reaching peak levels at ovulation trigger and decreasing at OPU (Table 3). LH levels decreased from baseline after 4 and 8 weeks during androgen priming, to increase slightly at ovulation trigger and, as expected, decrease at OPU (Table 3).
Embryo transfer and reproductive outcomes The mean number of good-quality embryos per patient was 1.050.90. Zero, one, two and three good quality embryos were available in 7 (23/3%), 8 (26.7%), 6 (20.0%) and 1 (3.3%) patients, respectively; seven patients (23.3%) had no good-quality embryos available. Embryo transfer (ET) was performed on day 3 in 15 patients (fresh ET in 5 and frozen ET in 10 patients). The reasons for no ET in the remaining 15 patients included cycle cancellation due to inadequate response (n=6), no oocytes retrieved (n=2), patient request to delay ET (n=1) or not to transfer a single poor-quality embryo (n=2), ET not performed during the study period (n=3), and no embryo (n=1). The characteristics of the seven patients who did not undergo ET were similar to those in the overall study population. A mean of 1.60.5 embryos were transferred per patient. One clinical pregnancy was achieved, but this ended as a miscarriage before 12 weeks’ gestation.
Discussion In this study we explored a novel concept of long-term “intra-ovarian” androgen priming in Bologna criteria POR IVF patients. The idea of the protocol was to possibly boost the residual follicular pool in the POR patient during long-term androgen stimulation, which might physiologically increase the number of recruitable follicles as well as the follicular response to exogenous FSH stimulation. We clearly showed that the regimen used in this
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study had no effect on steroid levels and follicles prior to and after priming. Thus, the response to stimulation in this poor ovarian reserve population was similar to what has previously been reported (Humaidan et al. 2017). The stimulation of androgen production by theca cells was performed by micro-dose administration of hCG, while aromatization into estrogens was prevented by coadministration of the aromatase inhibitor letrozole. In addition, women underwent pituitary down-regulation using a GnRHa depot preparation for the entire duration of the 8-week priming period. There were two main reasons for using the ultra-long protocol. Firstly, because letrozole might induce follicular recruitment and development during the priming period, and secondly because we wanted to have a synchronous cohort of follicles before starting stimulation with exogenous FSH. Although previous studies suggested that the sensitivity of developing follicles to FSH stimulation might be enhanced by androgens via stimulation of the AR (Lossl et al. 2006, Lossl et al. 2008), the results of the present study clearly showed that the approach used was insufficient to cause an endogenous elevation of circulating testosterone, and that the number of antral follicles available for ovarian stimulation did not increase. Nevertheless, the circulating androgen level sufficient to act at the ovarian level on pre-antral and small antral follicles is unknown and cannot be determined from our data. Clinical outcomes in the small group of women in our study were clearly not satisfactory. However, this is consistent with earlier studies, showing no significant improvement in implantation or ongoing pregnancy rates after use of short-term androgen priming regimens (Lossl et al. 2006, Lossl et al. 2008). There are several potential explanations for our findings. Firstly, the pituitary GnRHa downregulation effectively reduced endogenous FSH and LH levels. Therefore, the rhCG given only replaced the endogenously produced LH and did not increase the LH drive on theca cell androgen production, resulting in stable low AMH levels. It would, therefore, appear that the
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rhCG dosage of 260 IU administered every second day was too low to cause an augmented drive for theca cell androgen synthesis despite the presence of the aromatase inhibitor blocking conversion into estrogens. Equally important, the drive from endogenous FSH to augment theca cell androgen synthesis was absent because FSH was effectively downregulated, and from a physiological point of view FSH secures synthesis of granulosa cell paracrine hormones that act synergistically with LH to increase theca cell androgen output including inhibin-A, inhibin-B and insulin-like growth factor-2 (Hillier et al. 1991, Nahum et al. 1995). This synergistic effect is pronounced and the presence of both IGF1, inhibin-A and LH results in a more than 20-times higher secretion of androstenedione from cultured human theca cells than LH alone (Hillier et al. 1991, Nahum et al. 1995). Thus, in order to improve the present protocol, it could be suggested that pituitary GnRHa down regulation should be avoided during the priming period to maintain pituitary function, allowing physiological levels of circulating FSH and LH. Moreover, the hCG dosage might be increased to provide stronger theca cell stimulation. A rhCG dosage of 260 IU every second day is approximately equivalent to a daily dosage of 130 IU. A previous randomized controlled trial (RCT) of GnRHa down-regulated women undergoing ovarian stimulation with recombinant FSH titrated the hCG dosage by providing 0, 50, 100 or 150 IU hCG daily (Thuesen et al. 2013). Steady-state hCG concentrations were achieved after 6 days, being approximately 3, 6 and 11 IU/L after the 50, 100 and 150 IU dosages, respectively. In that study, increasing hCG levels in the context of a constant FSH stimulation was associated with a dose-dependent increase in androgen, P4, and E2 levels. Therefore, in the current study, according to the abovementioned previous findings, it was expected that a daily rhCG dosage of 130 IU would result in a steady-state concentration of around 8-9 IU/L which theoretically should have been sufficient to increase the androgen production. In contrast to the RCT described above
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(Thuesen et al. 2013), the steady-state concentration of hCG in the current study was about 5 IU/L. A clear explanation for this discrepancy cannot readily be given, but it might relate to ethnic differences between the Asian and European population of the two studies; however, these findings indicate that the hCG administration might benefit form an increase. Despite the negative findings of the present study, we see it as a preliminary study and, thus, still consider intra-ovarian priming to be an interesting future model for the poor ovarian reserve patient, which after modifications of the present protocol should induce an increase in local intraovarian testosterone production and granulosa cell FSHR expression. We believe that such modifications would be superior to exogenous testosterone supplementation (Massin et al. 2006, Fabregues et al. 2009, Bosdou et al. 2016), in which the effect of exogenous testosterone is primarily systemic, with only a fraction of the testosterone supplemented reaching the theca-granulosa cell compartment and with potentially unwanted systemic side effects. This proof-of-concept study provides the first data on the effects of long-term intra- ovarian androgen priming in patients with POR. However, the results need to be interpreted in light of several important limitations. Firstly, because this was a pilot study, the number of patients enrolled was small. Secondly, the study design did not include a control group, making it difficult to determine whether the results seen could be attributed to the androgen priming regimen or were caused by other factors. Finally, the single ethnicity population included in this study could limit the external validity of our findings. In conclusion, long-term intra-ovarian androgen priming regimen was not associated with any increase in AMH or AFC in Bologna criteria POR patients undergoing IVF. No ongoing pregnancies or live births were achieved in the 15 patients who underwent ET. It is suggested that androgen priming should be performed without GnRHa down-regulation. Thus, the present approach to androgen priming should not currently be used in patients with POR, but
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rather await the results of protocol modifications. Importantly, additional research is needed in this area before clear recommendations about the value of intra-ovarian androgen priming in a clinical setting can be made.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Conflict of interest All authors state that they have no conflicts of interest to disclose.
Data Availability All data generated or analyzed during this study are included in this published article.
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Tables Table 1. Characteristics of participants at baseline Characteristic Age, years Body mass index, kg/m2 Duration of infertility, months
Androgen priming (n=30) 34.74.1 21.57 (2.09) 56.641.1
Type of infertility, n (%) Primary
18 (60.0)
Secondary
12 (40.0)
IVF indication, n (%) Low ovarian reserve
20 (66.7)
Male factor
5 (16.7)
Tubal factor
4 (13.3)
Advanced female age
1 (3.3)
Number of previous IVF attempts, n (%) 1
2 (6.7)
2
15 (50.0)
3
8 (26.7)
4
3 (10.0)
5
2 (6.7)
Values are mean standard deviation, or number of patients (%). IVF, in vitro fertilization.
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Table 2. Stimulation details and outcomes Patients (n=30) Duration of stimulation, days Total dose of FSH, IU
14.42.4 5290.0971.3
Type of rFSH, n (%) Gonal F
13 (43.3)
Pergoveris
17 (56.7)
Trigger, n (%)
24 (80.0)
Oocytes retrieved, n
3.42.2
MII oocytes, n
3.01.4
Fertilized oocytes, n
2.11.2
Cleavage embryos, n
1.60.7
Good embryos, n
1.10.9
Adverse event, n (%) Vasomotor
9 (30)
Cycle cancelled due to inadequate response
6 (20.0)
No oocyte retrieval
2 (6.7)
Values are mean standard deviation, or number of patients (%). FSH, follicle-stimulating hormone; MII, metaphase II; rFSH, recombination folliclestimulating hormone.
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Table 3. Follicular fluid hormone levels Androgen priming
At ovulation trigger
At oocyte retrieval
Baseline (n=30) At 4 weeks (n=30)
At 8 weeks (n=30)
(n=24)
(n=24)
0.49 (0.20, 0.92)
0.36 (0.19, 0.57)
0.33 (0.13, 0.57)
0.15 (0.08, 0.28)
0.13 (0.08, 0.24)
FSH, IU/L
12.48 (8.16, 16.37)
2.23 (1.52, 3.12)
4.22 (3.08, 5.00)
33.05 (24.37, 36.38)
14.96 (12.63, 19.02)
LH, IU/L
5.07 (3.58, 6.89)
1.01 (0.79, 1.16)
0.44 (0.31, 0.64)
1.74 (0.50, 2.67)
0.65 (0.48, 0.91)
28.30 (11.18, 42.52)
5.00 (5.00, 5.00)
5.00 (5.00, 5.00)
322.35 (159.33, 788.00)
176.15 (71.80, 257.40)
Progesterone, ng/mL
NA
0.06 (0.05, 0.11)
0.05 (0.05, 0.09)
0.77 (0.38, 1.13)
6.89 (3.67, 11.27)
Testosterone, ng/mL
0.30 (0.12, 0.51)
0.14 (0.09, 0.56)
0.24 (0.09, 0.38)
0.47 (0.34, 0.89)
1.00 (0.66, 1.24)
NA
4.93 (4.31, 6.15)
3.54 (2.00, 4.09)
0.10 (0.10, 0.10)
100.26 (78.17, 133.85)
4.00 (2.00, 6.00)
4.00 (3.00, 6.00)
5.00 (3.00, 6.00)
4.00 (2.00, 5.00)
4.00 (2.00, 5.00)
AMH, ng/mL
Estradiol, pg/mL
hCG, mIU/mL AFC, n
Values are median (interquartile range). AFC, antral follicle count; AMH, anti-Müllerian hormone; FSH, follicle-stimulating hormone; hCG, human chorionic gonadotropin; LH, luteinizing hormone; NA, not applicable.
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Figure Legends
Figure 1. Intra-ovarian androgen priming and stimulation protocol (8 weeks’ androgen priming, followed by stimulation (gonadotropin-releasing hormone antagonist protocol) and trigger. GnRH, gonadotropin-releasing hormone; GnRHa, GnRH agonist; rFSH, recombinant follicle-stimulating hormone; rhCG, recombinant human chorionic gonadotropin; rLH, recombinant luteinizing hormone.
Figure 2. Bland-Altman plot showing relationship between anti-Müllerian hormone (AMH) levels at baseline and end of treatment. Each dot represents one patient. The x-axis is the mean of two AMH values (at baseline and after 8 weeks of androgen priming) and the y-axis is the AMH value at 8 weeks minus the AMH value at baseline.
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Thuesen, L.L., Smitz, J., Loft, A., Nyboe Andersen, A., 2013. Endocrine effects of hCG supplementation to recombinant FSH throughout controlled ovarian stimulation for IVF: a dose-response study. Clin. Endocrinol. (Oxf). 79, 708-715. Vendola, K.A., Zhou, J., Adesanya, O.O., Weil, S.J., Bondy, C.A., 1998. Androgens stimulate early stages of follicular growth in the primate ovary. J. Clin. Invest. 101, 2622-2629. Vendola, K., Zhou, J., Wang, J., Famuyiwa, O.A., Bievre, M., Bondy, C.A., 1999. Androgens promote oocyte insulin-like growth factor I expression and initiation of follicle development in the primate ovary. Biol. Reprod. 61, 353-357. Weil, S., Vendola, K., Zhou, J., Bondy, C.A., 1999. Androgen and follicle-stimulating hormone interactions in primate ovarian follicle development. J. Clin. Endocrinol. Metab. 84, 2951-2956.
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Vitae Dr Vuong is Head of OB/GYN Department, University of Medicine and Pharmacy at HCM City, Vietnam. She was one of the IVF pioneers in Vietnam, starting in 1997. She has performed about 20,000 ART cycles. Her areas of research activity include ovulation induction, poor responder, IVM, and frozen embryo transfer.
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The Effect of Intra-Ovarian Androgen Priming on Ovarian Reserve Parameters in Bologna Poor Responders Lan N Vuong , Manh T Ho , Anh N Ha , Toan D Pham , Tam TN Le , Claus Yding Andersen , Peter Humaidan PII: DOI: Reference:
S1472-6483(19)30829-6 https://doi.org/10.1016/j.rbmo.2019.11.005 RBMO 2296
To appear in:
Reproductive BioMedicine Online
Received date: Revised date: Accepted date:
25 July 2019 10 October 2019 20 November 2019
Please cite this article as: Lan N Vuong , Manh T Ho , Anh N Ha , Toan D Pham , Tam TN Le , Claus Yding Andersen , Peter Humaidan , The Effect of Intra-Ovarian Androgen Priming on Ovarian Reserve Parameters in Bologna Poor Responders, Reproductive BioMedicine Online (2019), doi: https://doi.org/10.1016/j.rbmo.2019.11.005
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The Effect of Intra-Ovarian Androgen Priming on Ovarian Reserve Parameters in Bologna Poor Responders Lan N Vuong,a,b,c* Manh T Ho,b,c Anh N Hab,c, Toan D Phamb,c, Tam TN Leb,c, Claus Yding Andersend, Peter Humaidane,f a
University of Medicine and Pharmacy at Ho Chi Minh City, 217 Hong Bang Street, District
5, Ho Chi Minh City, Vietnam ([email protected]; [email protected]) b
IVFMD, My Duc Hospital, 4 Duong Nui Thanh, Phuong 13, Tan Binh, Ho Chí Minh City,
Vietnam ([email protected]; [email protected]; [email protected]; [email protected]; [email protected]) c
HOPE Research Center, 4 Nui Thanh Street, Ward 13, Tan Binh District, Ho Chi Minh City,
Vietnam ([email protected]; [email protected]; [email protected]; [email protected]; [email protected]) d
Laboratory of Reproductive Biology, The Copenhagen University Hospital and Faculty of
Health Science, Copenhagen University, Blegdamsvej 3B, 2200 København, Copenhagen, Denmark ([email protected]) e
The Fertility Clinic, Skive Regional Hospital, Øster Fælled Vej 5, 7800 Skive, Denmark
([email protected]) f
Faculty of Health, Aarhus University, Denmark and Faculty of Health, University of
Southern Denmark, Nordre Ringgade 1, 8000, Aarhus C, Denmark ([email protected])
*Correspondence: Dr LN Vuong, Department of Obstetrics and Gynaecology, University of Medicine and Pharmacy at Ho Chi Minh City, 217 Hong Bang Street, District 5, Ho Chi Minh City, Vietnam; Ph: +84 903008889; Email: [email protected]; [email protected]
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Abstract Research question: What are the effects of long-term androgen priming in Bologna criteria poor ovarian reserve (POR) patients undergoing IVF. Design: This open-label pilot study was conducted at IVFMC, My Duc Hospital, Ho Chi Minh City, Vietnam. It included consecutive patients aged 18–41 years who fulfilled Bologna criteria for POR undergoing intra-ovarian androgen priming and ultra-long down-regulation with a gonadotropin-releasing hormone agonist (GnRHa), followed by stimulation with gonadotropins and GnRH antagonist co-treatment for IVF (n=30). Priming consisted of lowdose recombinant human chorionic gonadotropin (rhCG) 260 IU every second day plus letrozole 2.5 mg/day, both for 8 weeks; priming stopped on the first day of ovarian stimulation. The primary endpoint was serum anti-Müllerian hormone (AMH) concentrations 8 weeks after priming. Secondary endpoints included AFC (2-10 mm), serum hCG, testosterone, and progesterone levels. Results: Circulating testosterone, progesterone, estradiol and hCG levels remained unchanged during androgen priming; the mean AMH level decreased steadily from 0.49 ng/mL (baseline) to 0.33 ng/mL (8 weeks). AFC was 4–5 throughout the study. A mean of 1.10.9 good transferable embryos were obtained; embryo transfer was performed in 15 patients; no ongoing pregnancies were obtained. Conclusions: Long-term intra-ovarian androgen priming in the current set-up had no significant effect on hormone levels, AFC and recruitable follicles after ovarian stimulation in Bologna POR patients undergoing IVF. Further studies are needed to explore other androgen priming protocols and the clinical value of priming regimens in IVF.
Key words: ovarian reserve; poor response; in vitro fertilization; intra-ovarian androgen priming; Bologna criteria
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Key message In poor ovarian reserve patients who underwent ultra-long GnRHa down-regulation an intraovarian androgen priming regimen for 8 weeks before stimulation had no benefit in terms of ovarian reserve parameters and steroid levels.
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Abbreviations AFC
antral follicle count
AMH
Anti-Müllerian hormone
AR
androgen receptor
ART
assisted reproductive technology
BMI
body mass index
E2
estradiol
ESHRE
European Society of Human Reproduction and Embryology
ET
embryo transfer
FF
follicular fluid
FSH
follicle-stimulating hormone
FSHR
follicle-stimulating hormone receptor
GnRH
gonadotropin-releasing hormone
GnRHa
gonadotropin-releasing hormone agonist
hCG
human chorionic gonadotropin
IVF
in vitro fertilization
LH
luteinizing hormone
OPU
oocyte pick-up
P4
progesterone
POR
poor ovarian response
rhCG
recombinant human chorionic gonadotropin
rLH
recombinant luteinizing hormone
SC
subcutaneous
SD
standard deviation
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Introduction The management of patients with impaired or poor ovarian response (POR) undergoing infertility treatment has challenged reproductive specialists for decades. Different classifications of POR, including the Bologna Criteria (Ferraretti et al. 2011) and more recently the POSEIDON criteria (Humaidan et al. 2016), have helped standardize the definition of the poor responder population. However, this subset of in vitro fertilization (IVF) patients still remains a therapeutic challenge, experiencing disappointing overall outcomes in assisted reproductive technology (ART) due to the retrieval of a very low number of oocytes after exogenous stimulation with gonadotropins (Papathanasiou et al. 2016). In general, patients with POR have a low ovarian reserve characterized by few antral follicles and low circulating anti-Müllerian hormone (AMH) levels. Androgens play a pivotal role in folliculogenesis, facilitating follicle-stimulating hormone (FSH) receptor (FSHR) expression on granulosa cells via the androgen receptor (AR). In turn, increased FSHR expression increases follicular responsiveness to FSH (either endogenous or exogenous) (Weil et al. 1999). In addition, androgens act synergistically with insulin-like growth factor to stimulate growth of the follicle (Vendola et al. 1999). Interestingly, long-term exogenous androgen treatment has been shown to increase the number of pre-antral and antral follicles and, thus, the number of recruitable follicles in both animal models and in humans (Spinder et al. 1989, Vendola et al. 1998). Furthermore, intrafollicular concentrations of androgens and expression of ARs on granulosa cells are highly significantly associated with FSHR expression in humans (Nielsen et al. 2011), suggesting that circulating androgens in humans may facilitate a greater responsiveness of the ovarian follicle when exposed to exogenous FSH stimulation. Short-term intra-ovarian ‘androgen priming’ using a combination of human chorionic gonadotropin (hCG) and aromatase inhibitors during the early follicular phase was shown to
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significantly increase the proportion of cycle day 2 antral follicles reaching 14 mm in diameter, as well as serum estradiol (E2), and follicular fluid (FF) levels of E2 and testosterone; however, short time priming had no effect on the number of oocytes retrieved nor on reproductive outcomes (Lossl et al. 2006, Lossl et al. 2008). In this study we wanted to explore the effect of a novel long-term intra-ovarian androgen priming protocol, based on the “two cell-two gonadotropin model” in which luteinizing hormone (LH) activity regulates theca cell conversion of cholesterol into androgens, and androgens are converted to E2 in granulosa cells due to the combined actions of FSH and aromatase. In this new protocol, patients are subjected to long-term (8 weeks) low-level LH-like activity provided by repeated micro doses of recombinant hCG [rhCG]) to boost local endogenous androgen production by theca cells. To block the conversion of androgens into E2 in the granulosa cell, an aromatase inhibitor is administered on a daily basis, thus, increasing the amount of intra-ovarian and circulating endogenous androgens. We call this concept “intra ovarian priming”. The present pilot study investigated the effects of the proposed long-term androgen priming regimen on ovarian reserve parameters in patients considered to have the lowest ovarian reserve in IVF, the so-called European Society of Human Reproduction and Embryology (ESHRE) Bologna criteria POR patient.
Materials and methods Study design and population This single center pilot study was conducted at IVFMD, My Duc Hospital, Ho Chi Minh City, Vietnam between February 2018 and March 2019 (NCT03447184). Consecutive patients who fulfilled the ESHRE Bologna criteria for POR (Ferraretti et al. 2011), determined within the previous 2 months, were eligible to participate. Other inclusion criteria were: age 18–41
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years; body mass index (BMI) <30 kg/m2; and co-treatment with a gonadotropin-releasing hormone (GnRH) antagonist during ovarian stimulation. All participants provided informed consent. Women were excluded if they had any chronic medical conditions (e.g. diabetes, Crohn’s disease, thyroid disease, hepatitis B or a sexually transmitted disease) or were participating in another interventional clinical trial.
Ethical approval The study was performed in accordance with the ICH Harmonised Tripartite Guideline for Good Clinical Practice and the ethical principles of the Declaration of Helsinki. Ethics approval was obtained from the Ethical Board of My Duc Hospital (approval number 01/18/ĐĐ-BVMĐ, dated 22 January 2018). The trial was registered in clinicaltrials.gov (NCT03447184), Unique Protocol ID: CS/BVMD/18/01
Endpoints The primary endpoint was serum AMH levels after 8 weeks of priming. Secondary endpoints included antral follicle count (AFC) (2-10 mm) after 8 weeks of priming, at ovulation trigger and at oocyte retrieval; serum hCG, testosterone, and progesterone (P4) levels after 8 weeks of priming, on the day of ovulation trigger, and oocyte retrieval.
Study protocol Androgen priming Androgen priming started 8 weeks prior to stimulation for IVF, on day two of menses. From this day onwards patients received low-dose rhCG (Ovitrelle) 260 IU subcutaneously (SC) every second day in addition to letrozole 2.5 mg daily for 8 weeks, stopping on the first stay of stimulation for IVF with exogenous gonadotropins. Alongside androgen priming, pituitary
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down-regulation with a GnRH agonist (GnRHa) started (depot injection of triptorelin 3.75 mg); a second depot dose of triptorelin was given 28 days later. Thus, the total priming period lasted for 8 weeks (Figure 1).
Stimulation, monitoring and oocyte pick-up Stimulation with exogenous gonadotropins started after 8 weeks of androgen priming, irrespective of menses. A fixed dose of recombinant FSH (rFSH; Gonal F, Merck) was used for the first 5 days, either 300 IU rFSH alone in patients aged <35 years or 300 IU rFSH and 150 IU rLH (follitropin alfa/lutropin alfa, Pergoveris, Merck) in patients aged 35 years (Figure 1). Monitoring was undertaken according to the standard clinical procedure. Cotreatment with a GnRH antagonist started on the fifth day of stimulation. A bolus of 6,500 IU rhCG (Ovitrelle, Merck) was given for triggering of final oocyte maturation (Figure 1). Oocyte pick-up (OPU) and embryo transfer were performed based on local policies and procedures. Luteal phase support was provided by vaginal micronized progesterone, 180 mg daily (Crinone, Merck).
Blood sampling A total of six blood samples (2 ml each) were collected during the study period: at baseline, during priming after 4 and 8 weeks, on the day of starting stimulation, at ovulation trigger and at OPU. The first sample at baseline was used to determine FSH, LH, E2, testosterone, and AMH levels. The other five samples were used to determine FSH, LH, E2, testosterone, P4, hCG and AMH levels.
Ultrasound monitoring
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Ultrasound examination to count all antral follicles of 2-10 mm in diameter in each ovary was performed at each of the same time points.
Statistical analysis No previous data exist on long-term endogenous androgen priming in IVF patients, and due to the explorative character of this pilot study a sample size calculation was not feasible; Instead we decided to recruit a total of 30 ESHRE Bologna criteria POR patients. Data were analyzed using SPSS version 20 software. Continuous variables are presented as mean standard deviation (SD) and were compared using Student’s t-test. Categorical data are expressed as numbers and were compared using the Chi-square test. All tests were two tailed, and a p-value of <0.05 was considered statistically significant.
Results Patient population A total of 30 patients were enrolled in this pilot study. Patients were lean, the majority had primary infertility, and the mean number of previous unsuccessful IVF attempts was two (Table 1). A summary of stimulation details and outcomes is provided in Table 2.
Effects of priming Mean AMH levels decreased steadily over time (Table 3) and in most patients the AMH value after 8 weeks of androgen priming was lower than that at baseline (Figure 2). Longterm androgen priming had no significant effects on the AFC (Table 3). FSH levels decreased, from baseline to 4 weeks, increased slightly at 8 weeks, and then increased markedly at ovulation trigger, following exogenous stimulation (Table 3). Levels of circulating testosterone and P4 remained unchanged during the androgen priming period, to increase at
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ovulation trigger and OPU (Table 3). No changes were seen in E2 and hCG levels during androgen priming; as expected E2 levels increased during stimulation, reaching peak levels at ovulation trigger and decreasing at OPU (Table 3). LH levels decreased from baseline after 4 and 8 weeks during androgen priming, to increase slightly at ovulation trigger and, as expected, decrease at OPU (Table 3).
Embryo transfer and reproductive outcomes The mean number of good-quality embryos per patient was 1.050.90. Zero, one, two and three good quality embryos were available in 7 (23/3%), 8 (26.7%), 6 (20.0%) and 1 (3.3%) patients, respectively; seven patients (23.3%) had no good-quality embryos available. Embryo transfer (ET) was performed on day 3 in 15 patients (fresh ET in 5 and frozen ET in 10 patients). The reasons for no ET in the remaining 15 patients included cycle cancellation due to inadequate response (n=6), no oocytes retrieved (n=2), patient request to delay ET (n=1) or not to transfer a single poor-quality embryo (n=2), ET not performed during the study period (n=3), and no embryo (n=1). The characteristics of the seven patients who did not undergo ET were similar to those in the overall study population. A mean of 1.60.5 embryos were transferred per patient. One clinical pregnancy was achieved, but this ended as a miscarriage before 12 weeks’ gestation.
Discussion In this study we explored a novel concept of long-term “intra-ovarian” androgen priming in Bologna criteria POR IVF patients. The idea of the protocol was to possibly boost the residual follicular pool in the POR patient during long-term androgen stimulation, which might physiologically increase the number of recruitable follicles as well as the follicular response to exogenous FSH stimulation. We clearly showed that the regimen used in this
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study had no effect on steroid levels and follicles prior to and after priming. Thus, the response to stimulation in this poor ovarian reserve population was similar to what has previously been reported (Humaidan et al. 2017). The stimulation of androgen production by theca cells was performed by micro-dose administration of hCG, while aromatization into estrogens was prevented by coadministration of the aromatase inhibitor letrozole. In addition, women underwent pituitary down-regulation using a GnRHa depot preparation for the entire duration of the 8-week priming period. There were two main reasons for using the ultra-long protocol. Firstly, because letrozole might induce follicular recruitment and development during the priming period, and secondly because we wanted to have a synchronous cohort of follicles before starting stimulation with exogenous FSH. Although previous studies suggested that the sensitivity of developing follicles to FSH stimulation might be enhanced by androgens via stimulation of the AR (Lossl et al. 2006, Lossl et al. 2008), the results of the present study clearly showed that the approach used was insufficient to cause an endogenous elevation of circulating testosterone, and that the number of antral follicles available for ovarian stimulation did not increase. Nevertheless, the circulating androgen level sufficient to act at the ovarian level on pre-antral and small antral follicles is unknown and cannot be determined from our data. Clinical outcomes in the small group of women in our study were clearly not satisfactory. However, this is consistent with earlier studies, showing no significant improvement in implantation or ongoing pregnancy rates after use of short-term androgen priming regimens (Lossl et al. 2006, Lossl et al. 2008). There are several potential explanations for our findings. Firstly, the pituitary GnRHa downregulation effectively reduced endogenous FSH and LH levels. Therefore, the rhCG given only replaced the endogenously produced LH and did not increase the LH drive on theca cell androgen production, resulting in stable low AMH levels. It would, therefore, appear that the
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rhCG dosage of 260 IU administered every second day was too low to cause an augmented drive for theca cell androgen synthesis despite the presence of the aromatase inhibitor blocking conversion into estrogens. Equally important, the drive from endogenous FSH to augment theca cell androgen synthesis was absent because FSH was effectively downregulated, and from a physiological point of view FSH secures synthesis of granulosa cell paracrine hormones that act synergistically with LH to increase theca cell androgen output including inhibin-A, inhibin-B and insulin-like growth factor-2 (Hillier et al. 1991, Nahum et al. 1995). This synergistic effect is pronounced and the presence of both IGF1, inhibin-A and LH results in a more than 20-times higher secretion of androstenedione from cultured human theca cells than LH alone (Hillier et al. 1991, Nahum et al. 1995). Thus, in order to improve the present protocol, it could be suggested that pituitary GnRHa down regulation should be avoided during the priming period to maintain pituitary function, allowing physiological levels of circulating FSH and LH. Moreover, the hCG dosage might be increased to provide stronger theca cell stimulation. A rhCG dosage of 260 IU every second day is approximately equivalent to a daily dosage of 130 IU. A previous randomized controlled trial (RCT) of GnRHa down-regulated women undergoing ovarian stimulation with recombinant FSH titrated the hCG dosage by providing 0, 50, 100 or 150 IU hCG daily (Thuesen et al. 2013). Steady-state hCG concentrations were achieved after 6 days, being approximately 3, 6 and 11 IU/L after the 50, 100 and 150 IU dosages, respectively. In that study, increasing hCG levels in the context of a constant FSH stimulation was associated with a dose-dependent increase in androgen, P4, and E2 levels. Therefore, in the current study, according to the abovementioned previous findings, it was expected that a daily rhCG dosage of 130 IU would result in a steady-state concentration of around 8-9 IU/L which theoretically should have been sufficient to increase the androgen production. In contrast to the RCT described above
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(Thuesen et al. 2013), the steady-state concentration of hCG in the current study was about 5 IU/L. A clear explanation for this discrepancy cannot readily be given, but it might relate to ethnic differences between the Asian and European population of the two studies; however, these findings indicate that the hCG administration might benefit form an increase. Despite the negative findings of the present study, we see it as a preliminary study and, thus, still consider intra-ovarian priming to be an interesting future model for the poor ovarian reserve patient, which after modifications of the present protocol should induce an increase in local intraovarian testosterone production and granulosa cell FSHR expression. We believe that such modifications would be superior to exogenous testosterone supplementation (Massin et al. 2006, Fabregues et al. 2009, Bosdou et al. 2016), in which the effect of exogenous testosterone is primarily systemic, with only a fraction of the testosterone supplemented reaching the theca-granulosa cell compartment and with potentially unwanted systemic side effects. This proof-of-concept study provides the first data on the effects of long-term intra- ovarian androgen priming in patients with POR. However, the results need to be interpreted in light of several important limitations. Firstly, because this was a pilot study, the number of patients enrolled was small. Secondly, the study design did not include a control group, making it difficult to determine whether the results seen could be attributed to the androgen priming regimen or were caused by other factors. Finally, the single ethnicity population included in this study could limit the external validity of our findings. In conclusion, long-term intra-ovarian androgen priming regimen was not associated with any increase in AMH or AFC in Bologna criteria POR patients undergoing IVF. No ongoing pregnancies or live births were achieved in the 15 patients who underwent ET. It is suggested that androgen priming should be performed without GnRHa down-regulation. Thus, the present approach to androgen priming should not currently be used in patients with POR, but
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rather await the results of protocol modifications. Importantly, additional research is needed in this area before clear recommendations about the value of intra-ovarian androgen priming in a clinical setting can be made.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Conflict of interest All authors state that they have no conflicts of interest to disclose.
Data Availability All data generated or analyzed during this study are included in this published article.
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Tables Table 1. Characteristics of participants at baseline Characteristic Age, years Body mass index, kg/m2 Duration of infertility, months
Androgen priming (n=30) 34.74.1 21.57 (2.09) 56.641.1
Type of infertility, n (%) Primary
18 (60.0)
Secondary
12 (40.0)
IVF indication, n (%) Low ovarian reserve
20 (66.7)
Male factor
5 (16.7)
Tubal factor
4 (13.3)
Advanced female age
1 (3.3)
Number of previous IVF attempts, n (%) 1
2 (6.7)
2
15 (50.0)
3
8 (26.7)
4
3 (10.0)
5
2 (6.7)
Values are mean standard deviation, or number of patients (%). IVF, in vitro fertilization.
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Table 2. Stimulation details and outcomes Patients (n=30) Duration of stimulation, days Total dose of FSH, IU
14.42.4 5290.0971.3
Type of rFSH, n (%) Gonal F
13 (43.3)
Pergoveris
17 (56.7)
Trigger, n (%)
24 (80.0)
Oocytes retrieved, n
3.42.2
MII oocytes, n
3.01.4
Fertilized oocytes, n
2.11.2
Cleavage embryos, n
1.60.7
Good embryos, n
1.10.9
Adverse event, n (%) Vasomotor
9 (30)
Cycle cancelled due to inadequate response
6 (20.0)
No oocyte retrieval
2 (6.7)
Values are mean standard deviation, or number of patients (%). FSH, follicle-stimulating hormone; MII, metaphase II; rFSH, recombination folliclestimulating hormone.
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Table 3. Follicular fluid hormone levels Androgen priming
At ovulation trigger
At oocyte retrieval
Baseline (n=30) At 4 weeks (n=30)
At 8 weeks (n=30)
(n=24)
(n=24)
0.49 (0.20, 0.92)
0.36 (0.19, 0.57)
0.33 (0.13, 0.57)
0.15 (0.08, 0.28)
0.13 (0.08, 0.24)
FSH, IU/L
12.48 (8.16, 16.37)
2.23 (1.52, 3.12)
4.22 (3.08, 5.00)
33.05 (24.37, 36.38)
14.96 (12.63, 19.02)
LH, IU/L
5.07 (3.58, 6.89)
1.01 (0.79, 1.16)
0.44 (0.31, 0.64)
1.74 (0.50, 2.67)
0.65 (0.48, 0.91)
28.30 (11.18, 42.52)
5.00 (5.00, 5.00)
5.00 (5.00, 5.00)
322.35 (159.33, 788.00)
176.15 (71.80, 257.40)
Progesterone, ng/mL
NA
0.06 (0.05, 0.11)
0.05 (0.05, 0.09)
0.77 (0.38, 1.13)
6.89 (3.67, 11.27)
Testosterone, ng/mL
0.30 (0.12, 0.51)
0.14 (0.09, 0.56)
0.24 (0.09, 0.38)
0.47 (0.34, 0.89)
1.00 (0.66, 1.24)
NA
4.93 (4.31, 6.15)
3.54 (2.00, 4.09)
0.10 (0.10, 0.10)
100.26 (78.17, 133.85)
4.00 (2.00, 6.00)
4.00 (3.00, 6.00)
5.00 (3.00, 6.00)
4.00 (2.00, 5.00)
4.00 (2.00, 5.00)
AMH, ng/mL
Estradiol, pg/mL
hCG, mIU/mL AFC, n
Values are median (interquartile range). AFC, antral follicle count; AMH, anti-Müllerian hormone; FSH, follicle-stimulating hormone; hCG, human chorionic gonadotropin; LH, luteinizing hormone; NA, not applicable.
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Figure Legends
Figure 1. Intra-ovarian androgen priming and stimulation protocol (8 weeks’ androgen priming, followed by stimulation (gonadotropin-releasing hormone antagonist protocol) and trigger. GnRH, gonadotropin-releasing hormone; GnRHa, GnRH agonist; rFSH, recombinant follicle-stimulating hormone; rhCG, recombinant human chorionic gonadotropin; rLH, recombinant luteinizing hormone.
Figure 2. Bland-Altman plot showing relationship between anti-Müllerian hormone (AMH) levels at baseline and end of treatment. Each dot represents one patient. The x-axis is the mean of two AMH values (at baseline and after 8 weeks of androgen priming) and the y-axis is the AMH value at 8 weeks minus the AMH value at baseline.
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Thuesen, L.L., Smitz, J., Loft, A., Nyboe Andersen, A., 2013. Endocrine effects of hCG supplementation to recombinant FSH throughout controlled ovarian stimulation for IVF: a dose-response study. Clin. Endocrinol. (Oxf). 79, 708-715. Vendola, K.A., Zhou, J., Adesanya, O.O., Weil, S.J., Bondy, C.A., 1998. Androgens stimulate early stages of follicular growth in the primate ovary. J. Clin. Invest. 101, 2622-2629. Vendola, K., Zhou, J., Wang, J., Famuyiwa, O.A., Bievre, M., Bondy, C.A., 1999. Androgens promote oocyte insulin-like growth factor I expression and initiation of follicle development in the primate ovary. Biol. Reprod. 61, 353-357. Weil, S., Vendola, K., Zhou, J., Bondy, C.A., 1999. Androgen and follicle-stimulating hormone interactions in primate ovarian follicle development. J. Clin. Endocrinol. Metab. 84, 2951-2956.
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Vitae Dr Vuong is Head of OB/GYN Department, University of Medicine and Pharmacy at HCM City, Vietnam. She was one of the IVF pioneers in Vietnam, starting in 1997. She has performed about 20,000 ART cycles. Her areas of research activity include ovulation induction, poor responder, IVM, and frozen embryo transfer.
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