- Email: [email protected]
ICSI-ET with Poor Ovarian Response to Gonadotropin
Journal of Reproduction & Contraception 2014 Mar.; 25(1):32-40
doi: 10.7669/j.issn.1001-7844.2014.01.0032 E-mail: [email protected]
Effects of Growth Hormone Supplementation in Patients Undergoing IVF/ICSI-ET with Poor Ovarian Response to Gonadotropin Zhi-ping HU, Ying WANG, Xiao-guo DU, Rong LI, Xin-na CHEN, Hai-yan WANG, Ping LIU, Cai-hong MA, Jie QIAO Reproductive Medical Center, Peking University Third Hospital, Beijing 100191, China
Objective To analyze the effects of growth hormone (GH) supplementation during IVF/ ICSI-ET in Chinese patients who had prior IVF cycle with poor response to gonadotropin (Gn). Methods Ovulation was stimulated in 389 consecutive patients who all had poor ovarian response, among them, 102 patients (GH cycle) received 4 IU GH and the other 287 patients (non-GH cycle) underwent IVF without GH. Fisher’s exact test, Chi square test and Student’s t-test were used to analyze IVF/ICSI-ET outcomes. Results After GH treatment, 102 patients had significantly more large- and mediumsized follicles, oocytes retrieved, 2 pronucleus oocytes, metaphase II stage (MII) oocytes, and high-quality embryos than in previous cycles without GH. However, the number of embryos transferred, clinical pregnancy rate, transfer rate and biochemical pregnancy rate were not significantly different. Furthermore, the 102 patients given GH had significantly lower luteinizing hormone levels and biochemical pregnancy rates; thicker endometrium and more Gn administration days; and more large- and medium-sized follicles and MII oocytes than 287 other patients undergoing IVF/ICSI-ET without GH. However, these groups did not differ significantly in clinical pregnancies, high-quality embryos, MII oocytes, and embryo implantation rates. Conclusion GH may improve some IVF/ICSI-ET outcomes for women with poor ovarian response. Key words: assisted reproductive technology (ART); growth factor; in vitro fertilization (IVF); superovulation
Corresponding author: Ying WANG; Tel: +86-13911688652; E-mail: [email protected] 32
During in vitro fertilization and embryo transfer (IVF-ET), 9%-24% patients have poor ovarian response (POR)[1-3]. POR is defined according to the Bologna consensus criteria by two of three factors: 1) maternal age ≥ 40 years or another risk factor for poor ovarian response, 2) previous POR, and 3) an abnormal ovarian reserve test[4]. In this pathological condition, the ovary responds poorly to the standard protocol of stimulating ovulation with gonadotropin-releasing hormone (GnRH). This poor response leads to few developing follicles, low peak 17-β estradiol (E2) levels, increased requirement for gonadotropin (Gn), a high cycle cancellation rate, few oocytes harvested, and low pregnancy and live birth rates[5]. Poor ovarian function remains an issue in IVF-ET, which often leads to couples giving up the treatment or using donated eggs. As assisted reproductive techniques (ART) have become more advanced, POR has presented a greater challenge to infertility treatment. Despite several interventions to improve POR, the pregnancy rate after IVF-ET in these patients is still quite low[3,6]. A common approach is to increase the dose of Gn; however, this treatment remains controversial because of its negative effects on implantation and fetal development [7]. In some IVF-ET protocols, a treatment considered beneficial is growth hormone (GH)[8]. Administering GH during ovarian stimulation increased the number of oocytes and fertilization rate in patients with poor response to GnRH agonists (GnRH-a) or human menopausal gonadotropin (hMG), but not the clinical pregnancy rate[9]. However, a Meta-analysis of 22 randomized clinical trials on improving pregnancy in poor responders found evidence suggesting that GH treatment combined with embryo transfer on day 2 instead of day 3 increased the probability of pregnancy[6]. In that Meta-analysis, only 5 randomized clinical trials assessed the effect of GH on pregnancy rate. Additionally, these 5 trials were relatively small scale, limiting their statistical power. Thus, the ability of GH to increase pregnancy rates during stimulation of ovulation in IVF-ET requires further research. In this study, we investigated the effects of adding GH as an adjuvant treatment on IVF/ ICSI-ET outcomes in 102 patients with POR.
Materials & Methods Patient selection For this retrospective study, we analyzed the outcomes of 389 consecutive, infertile female Chinese patients with POR who visited the Reproductive Medical Center of Peking University Third Hospital from January 1, 2008 to December 31, 2011 for IVF-ET or intracytoplasmic sperm injection (ICSI) treatment. The main reasons for infertility were tubal problems, male infertility, endometriosis, and other factors (including ovulation failure and unexplained infertility); all patients had normal uterine and cervical morphology. All 389 patients had POR in the prior IVF cycle, as defined by the Bologna consensus criteria[4]. 33
Stimulation of ovulation In fact, no protocol is effective to patients with POR, including long, ultralong, antagonists, short and mild stimulation approach. Each protocol with GH was used in our center according to doctor experience. Long GnRH protocol is used mostly and the clinical data are the most complete. Our study was a retrospective study, so we select the patients who underwent long GnRH protocol, with 0.1 mg GnRH-a (diphereline; Ipsen, Paris, France) per day or 1.8 mg one time if injected during the midterm luteal phase of the previous menstrual period. Ovulation was stimulated 14 d later by recombinant FSH (Gonal-f; Merck Serono, Geneva, Switzerland and Purigon, Organon, P.O. Box 20 OssNL5340BH, Netherlands) or hMG (Livzon Pharmaceutical Group, Zhuhai, China). The 389 patients were divided into two groups: those who received GH (n=102, GH cycle) and those who did not (n=287, non-GH cycle). Patients in GH group received 4 IU/d of GH (Saizen; Merck Serono, Geneva, Switzerland)[10,11], beginning on the initial day of Gn until the day of human chorionic gonadotropin (hCG) injection. Except for GH administration, each group received the same protocol to induce ovulation. Follicle development was monitored by ultrasound. On the fifth day after Gn administration, the serum was assessed for concentrations of E2, progesterone (P) and luteinizing hormone (LH). The blood serum concentrations of these hormones were measured again when 3 follicles were detected with diameters>17 mm. At 8 pm on the same day, patients received 250 µg hCG (Ovidrio; Merck Serono, Geneva, Switzerland) via intramuscular injection, and the ova were harvested 36 h later. Oocyte harvest and fertilization Oocytes were harvested under anesthesia by venous administration of propofol (Fresenius Kabi, Hombury, Germany) and ultrasound guidance. After harvest, granular cells and corona radiata of the cumulus oophorus were removed, the maturity of the ova were evaluated, and ova were naturally fertilized or ICSI treated, depending on the semen condition. After fertilization, the zygote was incubated for 18 h in IVF nutrient solution at 37 ℃ with a 5% CO2 atmosphere. Fertilization status was observed at 24 h and the nutrient solution was renewed. Three days after natural fertilization or ICSI, embryos were transplanted. The number of available embryos was defined as the sum of the number of embryos transferred and the number of embryos frozen. Clinical pregnancy was defined as a positive pregnancy test at 14 d post transfer, followed by a vaginal ultrasound 2 weeks later demonstrating an embryonic cardiac pulse. Statistical analysis The results were expressed as mean ± standard deviation (x- ± s) or percentage (%). SPSS 13.0 (SPSS Inc., Chicago, IL, USA) was used to analyze numerical data. Student’s t test was used to compare paired data and χ2 test was used for proportional data. P<0.05 34
was considered to be significantly different.
Results Patient characteristics Patients in the two groups did not differ significantly in age, body mass index (BMI), duration of infertility, baseline hormone levels, and baseline antral follicles (Table 1). The 102 patients who underwent several cycles of IVF/ICSI-ET with and without GH did not differ significantly in any factor related to controlled ovarian hyperstimulation status (Table 2). Comparison of IVF/ICSI-ET outcomes in patients before and after treatment with GH The 102 patients who received GH during IVF/ICSI-ET had significantly more largeand medium-sized follicles on the day of hCG injection (P<0.001), oocytes retrieved (P<0.001), 2 pronucleus (2PN) oocytes (P=0.003), metaphase II stage (MII) oocytes (P=0.015), and high-quality embryos (P=0.016) than those during IVF/ICSI-ET cycles without GH (Table 3). The clinical pregnancy rate of patients who received GH was 2.3 times higher than during IVF/ICSI-ET cycles without GH, but this difference was only borderline significant (P=0.054). Other outcomes were not significantly different (Table 3). Comparison of IVF/ICSI-ET outcomes in patients who received and did not receive GH The 102 patients who received GH during IVF/ICSI-ET had significantly lower levels of LH on the day of hCG injection (P=0.014) and lower biochemical pregnancy rate (P=0.034)
Table 1 Characteristics of patients who received and did not receive growth hormone during IVF/ICSI-ET (x- ± s) Characteristic n Age (year)
GH cycle 102
Non-GH cycle 287
Z(t) or χ2
P
37.7 ± 4.2
37.9 ± 4.8
-0.309
0.758
22.91 ± 3.16
22.52 ± 2.81
-0.446
0.054
5.9 ± 4.2
6.4 ± 4.7
-0.622
0.534
FSH (mIU/L)
10.01 ± 4.10
9.94 ± 4.19
-0.400
0.689
LH (mIU/L)
3.83 ± 2.03
3.97 ± 3.34
-0.044
0.965
172.75 ± 107.09
162.52 ± 85.09
-0.727
0.467
13.36 ± 9.05
26.21 ± 74.61
-1.740
0.082
Testosterone (nmol/L)
2.38 ± 7.15
3.52 ± 11.31
-0.346
0.729
Androstenidione (nmol/L)
9.85 ± 26.16
6.45 ± 2.84
-0.060
0.952
BMI (kg/m2) Duration of infertility (year) Baseline hormone levels
Estradiol (pmol/L) Prolactin (ng/ml)
Baseline antral follicles Left (n)
2.6 ± 1.3
2.8 ± 1.5
-0.593
0.553
Right (n)
2.7 ± 1.4
2.9 ± 1.5
-0.709
0.478
Total (n)
4.6 ± 2.5
4.5 ± 3.0
-0.521
0.602 35
Table 2 Controlled ovarian hyperstimulation status of patients before and after receiving GH during IVF/ICSI-ET (n=102) (x- ± s) GH cycle
Z(t) or χ2
P
-0.609
0.544
1.364
0.176
1.302
0.196
1.45 ± 0.92
1.311
0.194
4.0 ± 2.4
-0.254
0.800
11.3 ± 3.1
-0.971
0.334
0.229
0.820
-1.596
0.114
IVF/ICSI-ET parameter FSH on Gn initiation day (mIU/L)
7.55 ± 5.36
Non-GH cycle 7.07 ± 4.43
LH on Gn initiation day (mIU/L)
2.26 ± 2.09
3.09 ± 5.92
E2 on Gn initiation day (pmol/L)
124.45 ± 49.43
134.22 ± 58.68
P on the initiation day (nmol/L)
1.31 ± 0.63 4.1 ± 2.0
Total follicles on Gn initiation day (n) Gn days (d)
11.8 ± 3.0 4 544.12 ± 1 696.62 4 596.54 ± 1 625.65 5 240.45 ± 3 955.27 4 643.69 ± 2 591.66
Total Gn dose (IU) E2 on hCG injection day (pmol/L) LH on hCG injection day (mIU/L)
2.27 ± 2.98
2.49 ± 2.72
0.607
0.545
P on hCG injection day (nmol/L)
2.46 ± 1.33
2.47 ± 1.24
0.059
0.953
Endometrial thickness on hCG injection day (mm)
10.5 ± 1.5
10.3 ± 1.6
-1.411
0.161
Table 3 IVF/ICSI-ET outcomes for patients before and after receiving growth hormone (n=102) (x- ± s) Outcome Large- and medium-sized follicles
Non-GH cycle
Z(t) or χ2
3.0 ± 1.3
-5.224
<0.001
4.6 ± 3.0
3.0 ± 1.5
-4.979
<0.001
2.3 ± 1.9
1.7 ± 1.3
-3.002
0.003
GH cycle 4.2 ± 2.2
P
on day of hCG injection (n) Number of oocytes retrieved (n) Number of 2PN oocytes (n) 2PN oocyte rate*
0.80 ± 0.31
0.81 ± 0.36
0.312
0.756
MII oocytes (n)
3.59 ± 2.74
2.05 ± 0.95
-2.664
0.015
Number of high-quality embryos (n)
1.5 ± 1.6
1.0 ± 1.1
-2.442
0.016
Number of embryos transferred (n)
2.1 ± 0.8
1.9 ± 0.7
-1.879
0.065
0.713
0.399
0.337
0.561
Cycle outcome (transfer rate) (%)
80.39 (82/102)
75.49 (77/102)
Biochemical pregnancy rate (%)
20.73 (17/82)
17.11 (13/76)
Clinical pregnancy rate (%)
3.700 0.054 7.89 (6/76) 18.29 (15/82) *: 2PN oocyte rate is the value, which is calculated from 2PN oocyte (oocyte fertilized normally) number divided by oocyte fertilized number in each subject
than those in the 287 patients who did not receive GH during IVF/ICSI-ET. Furthermore, the patients who received GH had significantly higher endometrial thickness (P=0.006), days of Gn administration (P=0.043), the number of large- and medium-sized follicles on the day of hCG injection (P=0.028), and the number of MII oocytes (P=0.016) than those in the 287 patients who did not receive GH during IVF/ICSI-ET. These two groups did not differ significantly in clinical pregnancy rate, high-quality embryo rate, MII oocyte rate, and embryo implantation rate (Tables 4 and 5).
Discussion The primary indicators of ovarian responsiveness include the number of retrieved 36
Table 4 Controlled ovarian hyperstimulation status of patients who received and did not receive GH during IVF/ICSI-ET (x- ± s) Status n
GH cycle 102
Non-GH cycle 287
Z(t) or χ2
P
FSH on Gn initiation day (mIU/L)
7.48 ± 5.28
7.93 ± 4.80
-1.145
0.252
LH on Gn initiation day (mIU/L)
2.35 ± 2.12
2.66 ± 1.98
-1.740
0.082
E2 on Gn initiation day (pmol/L)
126.26 ± 54.63
142.95 ± 72.34
-1.809
0.070
P on Gn initiation day (nmol/L)
1.31 ± 0.63
1.22 ± 0.56
-0.928
0.353
Gn days (d)
11.8 ± 3.0
11.1 ± 2.7
-2.022
0.043
4 194.32 ± 1 784.97 1.677 4 939.83 ± 4 085.99 -1.069 3.09 ± 4.28 -2.464
0.094
Total GnRH dose (IU)
4 537.88 ± 1 710.25
E2 on hCG injection day (pmol/L)
5 264.87 ± 3 940.70
LH on hCG injection day (mIU/L)
2.30 ± 3.00
P on hCG injection day (nmol/L)
2.47 ± 1.34
Endometrial thickness on hCG injection day (mm) 10.5 ± 1.5
0.285 0.014
2.55 ± 1.50
-0.254
0.800
10.1 ± 1.5
-2.773
0.006
Table 5 IVF/ICSI-ET outcomes for patients who received and did not receive GH (x- ± s) Outcome n Large- and medium-sized
GH cycle 102 4.2 ± 2.2
Non-GH cycle 287
Z(t) or χ2
P
3.7 ± 2.4
-2.196
0.028
follicles on hCG injection day (n) Number of oocytes retrieved (n)
4.6 ± 3.0
4.4 ± 3.3
-1.080
0.280
Number of 2PN oocytes (n)
2.3 ± 1.9
2.5 ± 2.4
-0.158
0.875
0.79 ± 0.31
0.78 ± 0.33
-0.114
0.909
3.8 ± 2.7
2.6 ± 1.9
-2.412
0.016
Number of high-quality embryos (n) 1.5 ± 1.6 Number of embryos transferred (n) 2.2 ± 0.8
1.5 ± 1.8
-0.004
0.997
2.1 ± 0.8
2PN oocyte rate* MII oocytes (n)
-0.692
0.489
Cycle outcome (transfer rate) (%)
80.00 (80/100)
77.16 (223/289)
0.347
0.556
Biochemical pregnancy rate (%)
20.00 (16/80)
32.58 (72/221)
4.493
0.034
Clinical pregnancy rate (%)
17.50 (14/80)
0.134 2.241 25.79 (57/221) 0.298 0.51 ± 0.23 -1.040 0.61 ± 0.28 *: 2PN oocyte rate is the value, which is calculated from 2PN oocyte (oocyte fertilized normally) number divided Implantation rate**
by oocyte fertilized number in each subject **: Implantation rate is the value, which is calculated from surviving embryo number divided by transferred embryo number in each subject Note: Because our study is retrospective study, some clinical data is not very complete, there is some lacunary in data, which were disposaled in statistics process. There are some tiny variances in GH cycle results between Table 4 and Table 2, between Table 5 and Table 3, according to different comparison
oocytes and E2 level on the day of hCG injection. The results of this study showed that patients who had poor response to Gn during IVF/ICSI-ET without GH treatment perhaps had better outcomes in later cycles when they were treated with GH. These patients had more largeand medium-sized follicles on the day of hCG injection, more oocytes retrieved and MII oocytes which facilitate better IVF/ICSI-ET outcomes in GH cycle than in non-GH cycle. These GH37
treated patients also had significantly more large- and medium-sized follicles and MII oocytes than other patients without GH treated. Our results echo another study showing that GH significantly increased the number of mature oocytes, suggesting that GH reduces follicular atresia and stimulates antral follicular recruitment and growth, enhances the responsiveness of the antral follicles to Gn[9,10]. Similarly, adjuvant GH during ovarian stimulation in patients with polycystic ovary syndrome (PCOS) increased the number of dominant follicles[11]. This possible explanation is supported by a report that GH selectively increased the FSH sensitivity of the dominant follicle and promoted oocyte maturation[12]. In addition to the proposed ability of GH to improve ovarian responsiveness, GH increases oocyte and embryo quality in IVF-ET cycles[10]. We found that patients treated with GH had significantly more 2PN oocytes and high-quality embryos than in IVF-ET cycles without GH, but no significant change in the number of embryos transferred. Furthermore, GH-treated patients had a clinical pregnancy rate that was 2.3 times higher than during IVFET cycles without GH, but this difference was only borderline significant (P=0.054). The rate of clinical pregnancy probably improves significantly by optimizing the dose or timing of GH according to the situation in the body or by changing some other part of the IVF-ET treatment. Furthermore, our result on clinical pregnancy is consistent with previous reports that GH did not improve the clinical pregnancy rate[9,13], but it may improve this rate if embryos are transferred on day 2 instead of day 3[6]. Our results indicated that GH controlled follicle growth at an appropriate rate, and maintained a relatively low LH level during controlled ovarian hyperstimulation. These results were consistent with a report that GH-treated patients had significantly higher levels of IGF-1 in follicular fluid than in the control, suggesting that GH can increase estrogen synthesis by increasing IGF-1 levels in follicular fluid, which promoted the synthesis of granular cell steroidogenic acute regulatory protein and effects of FSH[10]. A study to evaluate the effects of equine growth hormone (eGH) on nuclear and cytoplasmic maturation of equine oocytes in vitro, steroid production by cumulus cells, revealed that cumulus cells incubated with eGH had more oocytes that reached metaphase II; greater concentrations of oestradiol in the culture medium, concluded that addition of eGH to maturation medium increased rates of cytoplasmic maturation and had an important role in equine oocyte maturation[14]. These data indicated that GH directly impacts the ovum, which in turn affects embryo quality[15]. In a previous study, GH treatment led to a lower recurrent pregnancy loss rate, and higher parturition and live birth rates than a placebo group[16]. These effects were not observed in our parallel comparison with other patients undergoing IVF-ET during the same time period, which may be related to influencing factors such as variance in the general characteristics of the two patient groups. These results also suggest that follicle growth and embryo development are influenced by a variety of factors, so that simply adding GH or IGF-I is not sufficient to improve the outcomes of IVF-ET. 38
Our patients treated with GH during IVF/ICSI-ET had significantly greater endometrial thickness than those patients who did not receive GH, suggesting that GH improved the endometrial receptivity, which had potential promotion on endometrial adhesion, blastocystendometrium communication, and embryo implantation. This possibility is supported by a report that adding GH during IVF/ICSI-ET of women with underdeveloped endometrium (<6 mm thickness) significantly improved the morphology and thickness of the endometrium, and led to a significantly higher clinical pregnancy rate. In our study, however, endometrial thickness did not increase significantly in the same 102 women after receiving GH compared with IVF/ICSI-ET cycles without GH. These different results on the endometrium thickness between with and without GH group patients could not be explained, perhaps due to not being prospect study and not the parallel data from the patients. Overall, the results of this study suggest that administering GH probably improves some IVF/ICSI-ET outcomes of women with POR. Because of sample size restriction, we did not study the effects of different etiological factors and different ovarian stimulation protocols on the results. However, the effects of GH during IVF/ICSI-ET need to be confirmed in prospective, randomized controlled studies with a larger sample. Such studies are required to determine the optimal dose, time and duration of GH administration, etiological factor grouping and to investigate the safety of GH on the patients and their offspring. Additionally, no data are available on the long-term effects of GH during ovarian stimulation on the offspring born as a result of ART.
Conclusion Adding GH to ovarian stimulation protocols probably can lead to an increased number of ovum and the number of high-quality embryos in patients with poor ovarian response undergoing IVF/ICSI-ET. Although GH doubled the clinical pregnancy rate in this study, this effect was only borderline significant.
Acknowledgments We are grateful to the staff of the Reproductive Medical Center, Peking University Third Hospital for their support and comments during preparation of this manuscript. We thank Yan GAO and Tao SHI for their assistance.
References 1. Ben-Rafael Z, Bider D, Dan U, et al. Combined gonadotropin releasing hormone agonist/human menopausal gonadotropin therapy (GnRH-a/ hMG) in normal, high, and poor responders to hMG. J In Vitro Fert Embryo Transf, 1991, 8(1):33-6. 39
2. Hellberg D, Waldenstrom U, Nilsson S. Defining a poor responder in in vitro fertilization. Fertil Steril, 2004, 82(2):488-90. 3.
Aghahosseini M, Aleyassin A, Khodaverdi S, et al. Estradiol supplementation during the luteal phase in poor responder patients undergoing in vitro fertilization: a randomized clinical trial. J Assist Reprod Genet, 2011, 28(9):785-90.
4. Ferraretti AP, La Marca A, Fauser BC, et al. ESHRE consensus on the definition of ‘poor response’to ovarian stimulation for in vitro fertilization: the Bologna criteria. Hum Reprod, 2011, 26(7):1616-24. 5.
Keay SD. Poor ovarian response to gonadotrophin stimulation-The role of adjuvant treatments. Hum Fertil, 2002, 5(1):46-52.
6. Kyrou D, Kolibianakis EM, Venetis CA, et al. How to improve the probability of pregnancy in poor responders undergoing in vitro fertilization: a systematic review and meta-analysis. Fertil Steril, 2009, 91(3):749-66. 7.
Horsthemke B, Ludwig M. Assisted reproduction: the epigenetic perspective. Hum Reprod Update, 2005, 11(5pp):473-82.
8. Jacobs HS, Shoham Z, Schachter M, et al. Cotreatment with growth hormone and gonadotropin for ovulation induction in hypogonadotropic patients: a prospective, randomized, placebo-controlled, dose-response study. Fertil Steril, 1995, 64(5):917-23. 9. Kucuk T, Kozinoglu H, Kaba A. Growth hormone co-treatment within a GnRH agonist long protocol in patients with poor ovarian response: a prospective, randomized, clinical trial. J Assist Reprod Genet, 2008, 25(4): 123-7. 10. Xu HL, Li J, Ou JP, et al. Growth hormone cotreatment in poor responders undergoing IVF-ET. Chin J Clinicians (Electronic Edition), 2009, 3(11):1823-30. 11. Wang Y, Li MZ, Yang CS, et al. Effect of growth hormone on the outcome of ovulation induction in patients with polycystic ovary syndrome. Chin J Obstet Gynecol, 2001, 36(1):36-9. 12. Kiapekou E, Loutradis D, Drakakis P, et al. Effects of GH and IGF-I on the in vitro maturation of mouse oocytes. Endocrinology, 2005, 4(3):155-60. 13. Eftekhar M, Aflatoonian A, Mohammadian F, et al. Adjuvant growth hormone therapy in antagonist protocol in poor responders undergoing assisted reproductive technology. Arch Gynecol Obstet, 2013, 287(5): 1017-21. 14. Pereira GR, Lorenzo PL, Carneiro GF, et al. The involvement of growth hormone in equine oocyte maturation, receptor localization and steroid production by cumulus-oocyte complexes in vitro. Res Vet Sci, 2013, 95(2): 667-74. 15. Yovich JL, Stanger JD. Growth hormone supplementation improves implantation and pregnancy productivity rates for poor-prognosis patients undertaking IVF. Reprod BioMed Online, 2010, 21(1):37-49. 16. Tesarik J, Hazout A, Mendoza C. Improvement of delivery and live birth rates after ICSI in women aged > 40 years by ovarian co-stimulation with growth hormone. Hum Reprod, 2005, 20(9):2536-41.
(Received on December 14, 2013)
40
doi: 10.7669/j.issn.1001-7844.2014.01.0032 E-mail: [email protected]
Effects of Growth Hormone Supplementation in Patients Undergoing IVF/ICSI-ET with Poor Ovarian Response to Gonadotropin Zhi-ping HU, Ying WANG, Xiao-guo DU, Rong LI, Xin-na CHEN, Hai-yan WANG, Ping LIU, Cai-hong MA, Jie QIAO Reproductive Medical Center, Peking University Third Hospital, Beijing 100191, China
Objective To analyze the effects of growth hormone (GH) supplementation during IVF/ ICSI-ET in Chinese patients who had prior IVF cycle with poor response to gonadotropin (Gn). Methods Ovulation was stimulated in 389 consecutive patients who all had poor ovarian response, among them, 102 patients (GH cycle) received 4 IU GH and the other 287 patients (non-GH cycle) underwent IVF without GH. Fisher’s exact test, Chi square test and Student’s t-test were used to analyze IVF/ICSI-ET outcomes. Results After GH treatment, 102 patients had significantly more large- and mediumsized follicles, oocytes retrieved, 2 pronucleus oocytes, metaphase II stage (MII) oocytes, and high-quality embryos than in previous cycles without GH. However, the number of embryos transferred, clinical pregnancy rate, transfer rate and biochemical pregnancy rate were not significantly different. Furthermore, the 102 patients given GH had significantly lower luteinizing hormone levels and biochemical pregnancy rates; thicker endometrium and more Gn administration days; and more large- and medium-sized follicles and MII oocytes than 287 other patients undergoing IVF/ICSI-ET without GH. However, these groups did not differ significantly in clinical pregnancies, high-quality embryos, MII oocytes, and embryo implantation rates. Conclusion GH may improve some IVF/ICSI-ET outcomes for women with poor ovarian response. Key words: assisted reproductive technology (ART); growth factor; in vitro fertilization (IVF); superovulation
Corresponding author: Ying WANG; Tel: +86-13911688652; E-mail: [email protected] 32
During in vitro fertilization and embryo transfer (IVF-ET), 9%-24% patients have poor ovarian response (POR)[1-3]. POR is defined according to the Bologna consensus criteria by two of three factors: 1) maternal age ≥ 40 years or another risk factor for poor ovarian response, 2) previous POR, and 3) an abnormal ovarian reserve test[4]. In this pathological condition, the ovary responds poorly to the standard protocol of stimulating ovulation with gonadotropin-releasing hormone (GnRH). This poor response leads to few developing follicles, low peak 17-β estradiol (E2) levels, increased requirement for gonadotropin (Gn), a high cycle cancellation rate, few oocytes harvested, and low pregnancy and live birth rates[5]. Poor ovarian function remains an issue in IVF-ET, which often leads to couples giving up the treatment or using donated eggs. As assisted reproductive techniques (ART) have become more advanced, POR has presented a greater challenge to infertility treatment. Despite several interventions to improve POR, the pregnancy rate after IVF-ET in these patients is still quite low[3,6]. A common approach is to increase the dose of Gn; however, this treatment remains controversial because of its negative effects on implantation and fetal development [7]. In some IVF-ET protocols, a treatment considered beneficial is growth hormone (GH)[8]. Administering GH during ovarian stimulation increased the number of oocytes and fertilization rate in patients with poor response to GnRH agonists (GnRH-a) or human menopausal gonadotropin (hMG), but not the clinical pregnancy rate[9]. However, a Meta-analysis of 22 randomized clinical trials on improving pregnancy in poor responders found evidence suggesting that GH treatment combined with embryo transfer on day 2 instead of day 3 increased the probability of pregnancy[6]. In that Meta-analysis, only 5 randomized clinical trials assessed the effect of GH on pregnancy rate. Additionally, these 5 trials were relatively small scale, limiting their statistical power. Thus, the ability of GH to increase pregnancy rates during stimulation of ovulation in IVF-ET requires further research. In this study, we investigated the effects of adding GH as an adjuvant treatment on IVF/ ICSI-ET outcomes in 102 patients with POR.
Materials & Methods Patient selection For this retrospective study, we analyzed the outcomes of 389 consecutive, infertile female Chinese patients with POR who visited the Reproductive Medical Center of Peking University Third Hospital from January 1, 2008 to December 31, 2011 for IVF-ET or intracytoplasmic sperm injection (ICSI) treatment. The main reasons for infertility were tubal problems, male infertility, endometriosis, and other factors (including ovulation failure and unexplained infertility); all patients had normal uterine and cervical morphology. All 389 patients had POR in the prior IVF cycle, as defined by the Bologna consensus criteria[4]. 33
Stimulation of ovulation In fact, no protocol is effective to patients with POR, including long, ultralong, antagonists, short and mild stimulation approach. Each protocol with GH was used in our center according to doctor experience. Long GnRH protocol is used mostly and the clinical data are the most complete. Our study was a retrospective study, so we select the patients who underwent long GnRH protocol, with 0.1 mg GnRH-a (diphereline; Ipsen, Paris, France) per day or 1.8 mg one time if injected during the midterm luteal phase of the previous menstrual period. Ovulation was stimulated 14 d later by recombinant FSH (Gonal-f; Merck Serono, Geneva, Switzerland and Purigon, Organon, P.O. Box 20 OssNL5340BH, Netherlands) or hMG (Livzon Pharmaceutical Group, Zhuhai, China). The 389 patients were divided into two groups: those who received GH (n=102, GH cycle) and those who did not (n=287, non-GH cycle). Patients in GH group received 4 IU/d of GH (Saizen; Merck Serono, Geneva, Switzerland)[10,11], beginning on the initial day of Gn until the day of human chorionic gonadotropin (hCG) injection. Except for GH administration, each group received the same protocol to induce ovulation. Follicle development was monitored by ultrasound. On the fifth day after Gn administration, the serum was assessed for concentrations of E2, progesterone (P) and luteinizing hormone (LH). The blood serum concentrations of these hormones were measured again when 3 follicles were detected with diameters>17 mm. At 8 pm on the same day, patients received 250 µg hCG (Ovidrio; Merck Serono, Geneva, Switzerland) via intramuscular injection, and the ova were harvested 36 h later. Oocyte harvest and fertilization Oocytes were harvested under anesthesia by venous administration of propofol (Fresenius Kabi, Hombury, Germany) and ultrasound guidance. After harvest, granular cells and corona radiata of the cumulus oophorus were removed, the maturity of the ova were evaluated, and ova were naturally fertilized or ICSI treated, depending on the semen condition. After fertilization, the zygote was incubated for 18 h in IVF nutrient solution at 37 ℃ with a 5% CO2 atmosphere. Fertilization status was observed at 24 h and the nutrient solution was renewed. Three days after natural fertilization or ICSI, embryos were transplanted. The number of available embryos was defined as the sum of the number of embryos transferred and the number of embryos frozen. Clinical pregnancy was defined as a positive pregnancy test at 14 d post transfer, followed by a vaginal ultrasound 2 weeks later demonstrating an embryonic cardiac pulse. Statistical analysis The results were expressed as mean ± standard deviation (x- ± s) or percentage (%). SPSS 13.0 (SPSS Inc., Chicago, IL, USA) was used to analyze numerical data. Student’s t test was used to compare paired data and χ2 test was used for proportional data. P<0.05 34
was considered to be significantly different.
Results Patient characteristics Patients in the two groups did not differ significantly in age, body mass index (BMI), duration of infertility, baseline hormone levels, and baseline antral follicles (Table 1). The 102 patients who underwent several cycles of IVF/ICSI-ET with and without GH did not differ significantly in any factor related to controlled ovarian hyperstimulation status (Table 2). Comparison of IVF/ICSI-ET outcomes in patients before and after treatment with GH The 102 patients who received GH during IVF/ICSI-ET had significantly more largeand medium-sized follicles on the day of hCG injection (P<0.001), oocytes retrieved (P<0.001), 2 pronucleus (2PN) oocytes (P=0.003), metaphase II stage (MII) oocytes (P=0.015), and high-quality embryos (P=0.016) than those during IVF/ICSI-ET cycles without GH (Table 3). The clinical pregnancy rate of patients who received GH was 2.3 times higher than during IVF/ICSI-ET cycles without GH, but this difference was only borderline significant (P=0.054). Other outcomes were not significantly different (Table 3). Comparison of IVF/ICSI-ET outcomes in patients who received and did not receive GH The 102 patients who received GH during IVF/ICSI-ET had significantly lower levels of LH on the day of hCG injection (P=0.014) and lower biochemical pregnancy rate (P=0.034)
Table 1 Characteristics of patients who received and did not receive growth hormone during IVF/ICSI-ET (x- ± s) Characteristic n Age (year)
GH cycle 102
Non-GH cycle 287
Z(t) or χ2
P
37.7 ± 4.2
37.9 ± 4.8
-0.309
0.758
22.91 ± 3.16
22.52 ± 2.81
-0.446
0.054
5.9 ± 4.2
6.4 ± 4.7
-0.622
0.534
FSH (mIU/L)
10.01 ± 4.10
9.94 ± 4.19
-0.400
0.689
LH (mIU/L)
3.83 ± 2.03
3.97 ± 3.34
-0.044
0.965
172.75 ± 107.09
162.52 ± 85.09
-0.727
0.467
13.36 ± 9.05
26.21 ± 74.61
-1.740
0.082
Testosterone (nmol/L)
2.38 ± 7.15
3.52 ± 11.31
-0.346
0.729
Androstenidione (nmol/L)
9.85 ± 26.16
6.45 ± 2.84
-0.060
0.952
BMI (kg/m2) Duration of infertility (year) Baseline hormone levels
Estradiol (pmol/L) Prolactin (ng/ml)
Baseline antral follicles Left (n)
2.6 ± 1.3
2.8 ± 1.5
-0.593
0.553
Right (n)
2.7 ± 1.4
2.9 ± 1.5
-0.709
0.478
Total (n)
4.6 ± 2.5
4.5 ± 3.0
-0.521
0.602 35
Table 2 Controlled ovarian hyperstimulation status of patients before and after receiving GH during IVF/ICSI-ET (n=102) (x- ± s) GH cycle
Z(t) or χ2
P
-0.609
0.544
1.364
0.176
1.302
0.196
1.45 ± 0.92
1.311
0.194
4.0 ± 2.4
-0.254
0.800
11.3 ± 3.1
-0.971
0.334
0.229
0.820
-1.596
0.114
IVF/ICSI-ET parameter FSH on Gn initiation day (mIU/L)
7.55 ± 5.36
Non-GH cycle 7.07 ± 4.43
LH on Gn initiation day (mIU/L)
2.26 ± 2.09
3.09 ± 5.92
E2 on Gn initiation day (pmol/L)
124.45 ± 49.43
134.22 ± 58.68
P on the initiation day (nmol/L)
1.31 ± 0.63 4.1 ± 2.0
Total follicles on Gn initiation day (n) Gn days (d)
11.8 ± 3.0 4 544.12 ± 1 696.62 4 596.54 ± 1 625.65 5 240.45 ± 3 955.27 4 643.69 ± 2 591.66
Total Gn dose (IU) E2 on hCG injection day (pmol/L) LH on hCG injection day (mIU/L)
2.27 ± 2.98
2.49 ± 2.72
0.607
0.545
P on hCG injection day (nmol/L)
2.46 ± 1.33
2.47 ± 1.24
0.059
0.953
Endometrial thickness on hCG injection day (mm)
10.5 ± 1.5
10.3 ± 1.6
-1.411
0.161
Table 3 IVF/ICSI-ET outcomes for patients before and after receiving growth hormone (n=102) (x- ± s) Outcome Large- and medium-sized follicles
Non-GH cycle
Z(t) or χ2
3.0 ± 1.3
-5.224
<0.001
4.6 ± 3.0
3.0 ± 1.5
-4.979
<0.001
2.3 ± 1.9
1.7 ± 1.3
-3.002
0.003
GH cycle 4.2 ± 2.2
P
on day of hCG injection (n) Number of oocytes retrieved (n) Number of 2PN oocytes (n) 2PN oocyte rate*
0.80 ± 0.31
0.81 ± 0.36
0.312
0.756
MII oocytes (n)
3.59 ± 2.74
2.05 ± 0.95
-2.664
0.015
Number of high-quality embryos (n)
1.5 ± 1.6
1.0 ± 1.1
-2.442
0.016
Number of embryos transferred (n)
2.1 ± 0.8
1.9 ± 0.7
-1.879
0.065
0.713
0.399
0.337
0.561
Cycle outcome (transfer rate) (%)
80.39 (82/102)
75.49 (77/102)
Biochemical pregnancy rate (%)
20.73 (17/82)
17.11 (13/76)
Clinical pregnancy rate (%)
3.700 0.054 7.89 (6/76) 18.29 (15/82) *: 2PN oocyte rate is the value, which is calculated from 2PN oocyte (oocyte fertilized normally) number divided by oocyte fertilized number in each subject
than those in the 287 patients who did not receive GH during IVF/ICSI-ET. Furthermore, the patients who received GH had significantly higher endometrial thickness (P=0.006), days of Gn administration (P=0.043), the number of large- and medium-sized follicles on the day of hCG injection (P=0.028), and the number of MII oocytes (P=0.016) than those in the 287 patients who did not receive GH during IVF/ICSI-ET. These two groups did not differ significantly in clinical pregnancy rate, high-quality embryo rate, MII oocyte rate, and embryo implantation rate (Tables 4 and 5).
Discussion The primary indicators of ovarian responsiveness include the number of retrieved 36
Table 4 Controlled ovarian hyperstimulation status of patients who received and did not receive GH during IVF/ICSI-ET (x- ± s) Status n
GH cycle 102
Non-GH cycle 287
Z(t) or χ2
P
FSH on Gn initiation day (mIU/L)
7.48 ± 5.28
7.93 ± 4.80
-1.145
0.252
LH on Gn initiation day (mIU/L)
2.35 ± 2.12
2.66 ± 1.98
-1.740
0.082
E2 on Gn initiation day (pmol/L)
126.26 ± 54.63
142.95 ± 72.34
-1.809
0.070
P on Gn initiation day (nmol/L)
1.31 ± 0.63
1.22 ± 0.56
-0.928
0.353
Gn days (d)
11.8 ± 3.0
11.1 ± 2.7
-2.022
0.043
4 194.32 ± 1 784.97 1.677 4 939.83 ± 4 085.99 -1.069 3.09 ± 4.28 -2.464
0.094
Total GnRH dose (IU)
4 537.88 ± 1 710.25
E2 on hCG injection day (pmol/L)
5 264.87 ± 3 940.70
LH on hCG injection day (mIU/L)
2.30 ± 3.00
P on hCG injection day (nmol/L)
2.47 ± 1.34
Endometrial thickness on hCG injection day (mm) 10.5 ± 1.5
0.285 0.014
2.55 ± 1.50
-0.254
0.800
10.1 ± 1.5
-2.773
0.006
Table 5 IVF/ICSI-ET outcomes for patients who received and did not receive GH (x- ± s) Outcome n Large- and medium-sized
GH cycle 102 4.2 ± 2.2
Non-GH cycle 287
Z(t) or χ2
P
3.7 ± 2.4
-2.196
0.028
follicles on hCG injection day (n) Number of oocytes retrieved (n)
4.6 ± 3.0
4.4 ± 3.3
-1.080
0.280
Number of 2PN oocytes (n)
2.3 ± 1.9
2.5 ± 2.4
-0.158
0.875
0.79 ± 0.31
0.78 ± 0.33
-0.114
0.909
3.8 ± 2.7
2.6 ± 1.9
-2.412
0.016
Number of high-quality embryos (n) 1.5 ± 1.6 Number of embryos transferred (n) 2.2 ± 0.8
1.5 ± 1.8
-0.004
0.997
2.1 ± 0.8
2PN oocyte rate* MII oocytes (n)
-0.692
0.489
Cycle outcome (transfer rate) (%)
80.00 (80/100)
77.16 (223/289)
0.347
0.556
Biochemical pregnancy rate (%)
20.00 (16/80)
32.58 (72/221)
4.493
0.034
Clinical pregnancy rate (%)
17.50 (14/80)
0.134 2.241 25.79 (57/221) 0.298 0.51 ± 0.23 -1.040 0.61 ± 0.28 *: 2PN oocyte rate is the value, which is calculated from 2PN oocyte (oocyte fertilized normally) number divided Implantation rate**
by oocyte fertilized number in each subject **: Implantation rate is the value, which is calculated from surviving embryo number divided by transferred embryo number in each subject Note: Because our study is retrospective study, some clinical data is not very complete, there is some lacunary in data, which were disposaled in statistics process. There are some tiny variances in GH cycle results between Table 4 and Table 2, between Table 5 and Table 3, according to different comparison
oocytes and E2 level on the day of hCG injection. The results of this study showed that patients who had poor response to Gn during IVF/ICSI-ET without GH treatment perhaps had better outcomes in later cycles when they were treated with GH. These patients had more largeand medium-sized follicles on the day of hCG injection, more oocytes retrieved and MII oocytes which facilitate better IVF/ICSI-ET outcomes in GH cycle than in non-GH cycle. These GH37
treated patients also had significantly more large- and medium-sized follicles and MII oocytes than other patients without GH treated. Our results echo another study showing that GH significantly increased the number of mature oocytes, suggesting that GH reduces follicular atresia and stimulates antral follicular recruitment and growth, enhances the responsiveness of the antral follicles to Gn[9,10]. Similarly, adjuvant GH during ovarian stimulation in patients with polycystic ovary syndrome (PCOS) increased the number of dominant follicles[11]. This possible explanation is supported by a report that GH selectively increased the FSH sensitivity of the dominant follicle and promoted oocyte maturation[12]. In addition to the proposed ability of GH to improve ovarian responsiveness, GH increases oocyte and embryo quality in IVF-ET cycles[10]. We found that patients treated with GH had significantly more 2PN oocytes and high-quality embryos than in IVF-ET cycles without GH, but no significant change in the number of embryos transferred. Furthermore, GH-treated patients had a clinical pregnancy rate that was 2.3 times higher than during IVFET cycles without GH, but this difference was only borderline significant (P=0.054). The rate of clinical pregnancy probably improves significantly by optimizing the dose or timing of GH according to the situation in the body or by changing some other part of the IVF-ET treatment. Furthermore, our result on clinical pregnancy is consistent with previous reports that GH did not improve the clinical pregnancy rate[9,13], but it may improve this rate if embryos are transferred on day 2 instead of day 3[6]. Our results indicated that GH controlled follicle growth at an appropriate rate, and maintained a relatively low LH level during controlled ovarian hyperstimulation. These results were consistent with a report that GH-treated patients had significantly higher levels of IGF-1 in follicular fluid than in the control, suggesting that GH can increase estrogen synthesis by increasing IGF-1 levels in follicular fluid, which promoted the synthesis of granular cell steroidogenic acute regulatory protein and effects of FSH[10]. A study to evaluate the effects of equine growth hormone (eGH) on nuclear and cytoplasmic maturation of equine oocytes in vitro, steroid production by cumulus cells, revealed that cumulus cells incubated with eGH had more oocytes that reached metaphase II; greater concentrations of oestradiol in the culture medium, concluded that addition of eGH to maturation medium increased rates of cytoplasmic maturation and had an important role in equine oocyte maturation[14]. These data indicated that GH directly impacts the ovum, which in turn affects embryo quality[15]. In a previous study, GH treatment led to a lower recurrent pregnancy loss rate, and higher parturition and live birth rates than a placebo group[16]. These effects were not observed in our parallel comparison with other patients undergoing IVF-ET during the same time period, which may be related to influencing factors such as variance in the general characteristics of the two patient groups. These results also suggest that follicle growth and embryo development are influenced by a variety of factors, so that simply adding GH or IGF-I is not sufficient to improve the outcomes of IVF-ET. 38
Our patients treated with GH during IVF/ICSI-ET had significantly greater endometrial thickness than those patients who did not receive GH, suggesting that GH improved the endometrial receptivity, which had potential promotion on endometrial adhesion, blastocystendometrium communication, and embryo implantation. This possibility is supported by a report that adding GH during IVF/ICSI-ET of women with underdeveloped endometrium (<6 mm thickness) significantly improved the morphology and thickness of the endometrium, and led to a significantly higher clinical pregnancy rate. In our study, however, endometrial thickness did not increase significantly in the same 102 women after receiving GH compared with IVF/ICSI-ET cycles without GH. These different results on the endometrium thickness between with and without GH group patients could not be explained, perhaps due to not being prospect study and not the parallel data from the patients. Overall, the results of this study suggest that administering GH probably improves some IVF/ICSI-ET outcomes of women with POR. Because of sample size restriction, we did not study the effects of different etiological factors and different ovarian stimulation protocols on the results. However, the effects of GH during IVF/ICSI-ET need to be confirmed in prospective, randomized controlled studies with a larger sample. Such studies are required to determine the optimal dose, time and duration of GH administration, etiological factor grouping and to investigate the safety of GH on the patients and their offspring. Additionally, no data are available on the long-term effects of GH during ovarian stimulation on the offspring born as a result of ART.
Conclusion Adding GH to ovarian stimulation protocols probably can lead to an increased number of ovum and the number of high-quality embryos in patients with poor ovarian response undergoing IVF/ICSI-ET. Although GH doubled the clinical pregnancy rate in this study, this effect was only borderline significant.
Acknowledgments We are grateful to the staff of the Reproductive Medical Center, Peking University Third Hospital for their support and comments during preparation of this manuscript. We thank Yan GAO and Tao SHI for their assistance.
References 1. Ben-Rafael Z, Bider D, Dan U, et al. Combined gonadotropin releasing hormone agonist/human menopausal gonadotropin therapy (GnRH-a/ hMG) in normal, high, and poor responders to hMG. J In Vitro Fert Embryo Transf, 1991, 8(1):33-6. 39
2. Hellberg D, Waldenstrom U, Nilsson S. Defining a poor responder in in vitro fertilization. Fertil Steril, 2004, 82(2):488-90. 3.
Aghahosseini M, Aleyassin A, Khodaverdi S, et al. Estradiol supplementation during the luteal phase in poor responder patients undergoing in vitro fertilization: a randomized clinical trial. J Assist Reprod Genet, 2011, 28(9):785-90.
4. Ferraretti AP, La Marca A, Fauser BC, et al. ESHRE consensus on the definition of ‘poor response’to ovarian stimulation for in vitro fertilization: the Bologna criteria. Hum Reprod, 2011, 26(7):1616-24. 5.
Keay SD. Poor ovarian response to gonadotrophin stimulation-The role of adjuvant treatments. Hum Fertil, 2002, 5(1):46-52.
6. Kyrou D, Kolibianakis EM, Venetis CA, et al. How to improve the probability of pregnancy in poor responders undergoing in vitro fertilization: a systematic review and meta-analysis. Fertil Steril, 2009, 91(3):749-66. 7.
Horsthemke B, Ludwig M. Assisted reproduction: the epigenetic perspective. Hum Reprod Update, 2005, 11(5pp):473-82.
8. Jacobs HS, Shoham Z, Schachter M, et al. Cotreatment with growth hormone and gonadotropin for ovulation induction in hypogonadotropic patients: a prospective, randomized, placebo-controlled, dose-response study. Fertil Steril, 1995, 64(5):917-23. 9. Kucuk T, Kozinoglu H, Kaba A. Growth hormone co-treatment within a GnRH agonist long protocol in patients with poor ovarian response: a prospective, randomized, clinical trial. J Assist Reprod Genet, 2008, 25(4): 123-7. 10. Xu HL, Li J, Ou JP, et al. Growth hormone cotreatment in poor responders undergoing IVF-ET. Chin J Clinicians (Electronic Edition), 2009, 3(11):1823-30. 11. Wang Y, Li MZ, Yang CS, et al. Effect of growth hormone on the outcome of ovulation induction in patients with polycystic ovary syndrome. Chin J Obstet Gynecol, 2001, 36(1):36-9. 12. Kiapekou E, Loutradis D, Drakakis P, et al. Effects of GH and IGF-I on the in vitro maturation of mouse oocytes. Endocrinology, 2005, 4(3):155-60. 13. Eftekhar M, Aflatoonian A, Mohammadian F, et al. Adjuvant growth hormone therapy in antagonist protocol in poor responders undergoing assisted reproductive technology. Arch Gynecol Obstet, 2013, 287(5): 1017-21. 14. Pereira GR, Lorenzo PL, Carneiro GF, et al. The involvement of growth hormone in equine oocyte maturation, receptor localization and steroid production by cumulus-oocyte complexes in vitro. Res Vet Sci, 2013, 95(2): 667-74. 15. Yovich JL, Stanger JD. Growth hormone supplementation improves implantation and pregnancy productivity rates for poor-prognosis patients undertaking IVF. Reprod BioMed Online, 2010, 21(1):37-49. 16. Tesarik J, Hazout A, Mendoza C. Improvement of delivery and live birth rates after ICSI in women aged > 40 years by ovarian co-stimulation with growth hormone. Hum Reprod, 2005, 20(9):2536-41.
(Received on December 14, 2013)
40