Original Article Received: August 27, 2013 Accepted after revision: April 24, 2014 Published online: December 20, 2014
Gynecol Obstet Invest DOI: 10.1159/000363235
In vitro Fertilization Outcome following Embryo Transfer with or without Preinstillation of Human Chorionic Gonadotropin into the Uterine Cavity: A Randomized Controlled Trial Ashraf Aaleyasin Marzieh Aghahosseini Mandana Rashidi Leila Safdarian Fatemeh Sarvi Zahra Najmi Alireza Mobasseri Behzad Amoozgar Department of Infertility, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran
Abstract Background: Intrauterine injection of human chorionic gonadotropin (hCG) at embryo transfer (ET) has been shown to improve the outcome of assisted reproductive techniques. The aim of this study was to confirm previous findings. Methods: In this randomized controlled trial, 483 infertile women who were candidates for in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) for the first time were randomly assigned to receive an intrauterine injection of 500 IU hCG or placebo (tissue culture media) before ET. The main outcome measures were implantation and clinical pregnancy rates. Results: Both the hCG-treated group (n = 240) and control group (n = 243) were similar at baseline in terms of demographic and obstetrical characteristics. There were significant differences between the two groups regarding the implantation rate (23.6 vs. 12.2%, p < 0.001), pregnancy rate (54.6 vs. 35.8%, p < 0.001), clinical pregnancy rate (50 vs. 32.1%, p < 0.001), ongoing pregnancy rate (15.3 vs. 9.2%, p
17 mm were observed. Oocyte retrieval was performed under transvaginal ultrasound guidance 34–36 h after the administration of hCG. Oocytes were fertilized through ICSI and cleavage-stage ET was performed on the 2nd or 3rd day. At this time, by a computergenerated list, patients were randomly assigned to receive intrauterine hCG (study group) or placebo (control group) as described below. A technician, not belonging to the study personnel, prepared and coded the drugs according to the list. All patients and clinical care providers were blinded to the list until the end of the study. In the study group, 500 IU of hCG in a volume of 50 μl was injected into the uterus prior to ET. To prepare 500 IU of hCG for intrauterine injection, 0.5 ml of tissue culture media (Vitrolife, Göteborg, Sweden) was added to one vial containing 5,000 IU of hCG (Choragon, Ferring). We then injected 0.05 ml of this solution into the uterus. In the study group, in the lithotomy position, the cervical mucosa of the vagina was wiped out using sterile gauze. Then 500 IU of hCG was injected into the uterine cavity using a soft ET catheter (K-Soft-5000 Catheter; Cook Medical, Inc., USA). In difficult transfer cases a firmer catheter (malleable stylet catheter) was used. 5–7 min after the hCG injection, two or more high-grade embryos were transferred by another catheter. The process was completed with speculum removal 5 min after ET. The same process was carried out in the control group but with injection of 50 μl tissue culture media instead of hCG at ET. All participants received luteal phase support with 800 mg/day progesterone suppositories (Cyclogest; Actavis, UK) from the day of oocyte retrieval. Outcome Evaluation The β-hCG serum level was analyzed after 14 days of ET. Control transvaginal sonography was performed during the 5th week of gestation. Rising levels of β-hCG in serum was considered as pregnancy, regardless of sonography results. A sonography-confirmed gestational sac with a fetal heart rate was considered as clinical pregnancy. Any fetal loss between 7 and 20 weeks of gestation was considered as an abortion. The fertilization rate was defined as the proportion of all acquired embryos to all injected oocytes. The implantation rate was defined as the number of monographically viewed gestational sacs per transferred embryos. Any pregnancy beyond 20 weeks of gestation was considered as an ongoing pregnancy and calculated per transferred embryos.
Aaleyasin et al.
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Among the molecules involved in implantation, human chorionic gonadotropin (hCG) is a unique glycoprotein hormone with biological functions in the corpus luteum and endometrium [9]. The first embryonic signal initiates invasion and implantation of trophoblastic cells into the uterine myometrium while driving the growth and differentiation of cytotrophoblasts to syncytiotrophoblasts. It also promotes the formation of villous trophoblast tissue and provokes the development of uterine spiral arteries and their protrusion to villous trophoblasts. The corpus luteum continues to secrete progesterone under hCG stimulation [10, 11]. Different cytokines mediate these broad-spectrum functions of hCG [12]. Systemic administration of hCG at embryo transfer (ET) has resulted in an increased pregnancy rate in animal models [13]. Intrauterine injection of hCG has been associated with significant pro-implantation changes of the endometrium in primates [14]. Moreover, in a recent study it has been shown that intrauterine injection of hCG before ET improves implantation and pregnancy rates [15]. Based on these findings, in this study we assessed the effect of intrauterine injection of hCG before ET on the implantation and pregnancy rates in IVF patients.
Table 1. Baseline demographic and clinical characteristics of the hCG-treated group and control group
Age, years Duration of infertility, years BMI Type of infertility Primary Secondary Cause of infertility Male factor Tubal factor Anovulation Unexplained Male and female factors AFC FSH level, IU/l AMH level, ng/ml
hCG-treated group (n = 240)
Control group (n = 243) p value
29.1 ± 5.6 7.1 ± 4 25.4 ± 3.6
28.7 ± 5.4 7.3 ± 4.5 25.8 ± 3.3
195 (81.2) 45 (18.8)
202 (83.1) 41 (16.9)
90 (37.5) 45 (18.8) 38 (15.8) 42 (17.5) 25 (10.4) 12.3 ± 2.5 7.22 ± 2.8 3.5 ± 1.5
80 (33) 37 (15.2) 30 (12.3) 66 (27.2) 30 (12.3) 12.6 ± 2.3 7.44 ± 3.3 3.5 ± 1.6
n.s. n.s. n.s. n.s. n.s.
n.s. n.s. n.s.
Results
In total, 610 out of 764 women referred to the infertility clinic were eligible. Among these patients, 41 did not wish to participate, 17 withdrew due to personal reasons, and IVF/ICSI cycles were cancelled in 69 patients (21 for nonresponsiveness and 48 for ovarian hyperstimulation syndrome). The remaining women (n = 483) were randomly assigned to treatment (hCG-treated, n = 240) and control (placebo, n = 243) groups. Figure 1 represents the flow diagram of participants throughout the trial. Table 1 shows the baseline demographic and clinical characteristics of participants. There were no significant differences in age, duration of infertility, BMI, and type of infertility between the two groups. Similarly, ovarian reserve tests such as the antral follicle count (AFC), FSH level, and AMH level were not statistically different in the two groups. The number of retrieved oocytes, metaphase 2 oocytes, and injected oocytes were not statistically different in the two groups (11.7 ± 3.9 vs. 12.4 ± 4.3, p > 0.05; 8.3 ± 2.8 vs. 8.2 ± 2.7, p > 0.05, and 8.9 ± 2.8 vs. 9.1 ± 2.9, p > 0.05, reIVF Outcome after Embryo Transfer with or without Preinstillation of hCG
Assessed for eligibility (n = 764) Did not meet the criteria (n = 154)
Did not participate (n = 58)
IVF/ICSI cancelled (n = 69) Randomized (n = 483)
hCG-treated group (n = 240)
Control group (n = 243)
Fig.1. Flow diagram of participants throughout the trial.
spectively). Also, the fertilization rate was similar in the two groups (69.2 vs. 67%, p = 0.2). The mean transferred embryos in the hCG-treated group and control group were 2.85 ± 0.52 and 2.93 ± 0.57, respectively (p = 0.08). In hCG-treated women, the rate of pregnancy, clinical pregnancy, and implantation were significantly higher than those in control group (54.6 vs. 35.8%, 50 vs. 32.1%, and 23.6 vs. 12.2%, p < 0.001, respectively). Intrauterine injection of hCG was associated with a higher rate of twin Gynecol Obstet Invest DOI: 10.1159/000363235
3
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Statistics Statistical analysis was performed using SPSS version 19 software (SPSS, Inc., Chicago, Ill., USA). Numerical variables were reported as mean ± SD when normally distributed, otherwise as median plus range. We used an independent sample t test and χ2 test to compare quantitative and qualitative variables, respectively. p ≤ 0.05 was considered to be statistically significant.
Color version available online
Values represent mean ± SD or n (%).
Table 2. Fertilization, implantation, and pregnancy outcomes of the hCG-treated group and control group
Retrieved oocytes Oocytes in metaphase II Injected oocytes Fertilization rate Transferred fetus Implantation rate Biochemical pregnancies Clinical pregnancies Pregnancy sac Single Multiple Abortion Ongoing pregnancies per clinical pregnancies Ongoing pregnancies per transferred fetus Live delivery per clinical pregnancies Live delivery per transferred fetus
hCG-treated group (n = 240)
Control group (n = 243)
p value
11.7 ± 3.9 8.3 ± 2.8 8.9 ± 2.8 1,413/2,043 (69.2) 2.85 ± 0.52 162/685 (23.6) 131 (54.6) 120 (50)
12.4 ± 4.3 8.2 ± 2.7 9.1 ± 2.9 1,481/2,211 (67) 2.93 ± 0.57 87/715 (12.2) 87 (35.8) 78 (32.1)
n.s. n.s. n.s. n.s. n.s. 0.0001 0.0001 0.0001 0.007
87 (36.3) 33 (13.7) 15/120 (12.5) 105/120 (87.5) 105/685 (15.3) 98/120 (81.6) 98/685 (14.3)
69 (28.4) 9 (3.7) 12/78 (15.4) 66/78 (84.6) 66/715 (9.2) 60/78 (77) 60/715 (8.4)
n.s. n.s. 0.0005 n.s. 0.0005
Values represent mean ± SD or n (%).
Discussion
In the present study we found a significant improvement in the implantation and pregnancy rates of women treated with intrauterine injection of 500 IU hCG at ET. Additionally, hCG injection was associated with a higher rate of multiple gestations as compared with the control group. No other adverse effect of intrauterine injection of hCG was found. A normal implantation process encompasses continuous highly regulated steps. Biosynthesis of multiple prostaglandins, adhesion molecules, proteinases, and growth factors is a necessary step for a successful implantation. To stabilize an implanted embryo, adequate progesterone production, immunomodulation at implantation site, 4
Gynecol Obstet Invest DOI: 10.1159/000363235
placental growth and differentiation are also needed [1]. hCG is probably a critical signal in both implantation process and pregnancy stabilization: it increases expression of the cyclooxygenase-2 gene in endometrial glands, resulting in increased production of prostaglandins [18]. hCG provokes secretion of matrix metalloproteinases while suppressing their inhibitors, to induce endometrial tissue remodeling [19]. It reduces endometrial IGFBP-1 biosynthesis, resulting in an increased IGF-II level and angiogenesis. Neoangiogenesis and placental formation are also stimulated through increased intrauterine VEGF levels by hCG [19]. hCG inhibits apoptosis of cytotrophoblast cells while promoting their growth and invasion into the myometrium by blocking TGF-β receptor [20]. As a paracrine agent, it replaces LH function in promoting the production of progesterone during the first 3–4 weeks of gestation [20]. hCG presents its immunomodulatory effect by inhibiting M-CSF and macrophage phagocytosis activity, while stimulating leukocyte inhibitory factor [19]. The concept of our current study was based on previous animal investigations evaluating implantation and pregnancy rates after hCG injection. Wallace et al. [13] demonstrated improved pregnancy rates after an intramuscular injection of hCG at ET in cows. In a study by Banerjee and Fazleabas [14], intrauterine injection of hCG in baboons induced a favorable uterine environment for embryo implantation. Although hCG has been commerAaleyasin et al.
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and triplet pregnancies (p < 0.05). Triplet pregnancy occurred in 3 of the cases, but none of the control group. In hCG-treated women, ongoing pregnancy and live delivery rate were significantly higher than those in the control group when calculated per ET (15.3 vs. 9.2% and 14.3 vs. 8.4%, p < 0.001, respectively). However, they did not reach statistical significance when calculated per clinical pregnancy. Table 2 demonstrates the outcomes of fertilization, implantation, and pregnancy in the two groups. Seven intrauterine deaths in the hCG-treated group and 6 deaths in the control group were observed.
cially available for a long time, a few human trials have addressed the value of hCG injection in ET. In a study by Ben-Meir et al. [21], standard endometrial preparation with progesterone was compared with an additional 250 μg of subcutaneous recombinant hCG on the day of progesterone initiation, the day of ET, and 6 days later. Implantation and pregnancy rates did not differ between the two methods (14.9 vs. 12.7% and 32.2 vs. 28.2%, respectively, p > 0.05). In a study by Järvelä et al. [22], intramuscular injection of hCG 3–5 min after the intrauterine insemination procedure resulted in an improved pregnancy rate compared with 24–36 h injection before intrauterine insemination. Mansour et al. [15] evaluated the intrauterine injection of hCG prior to ET in three experimental doses. While 100 and 200 IU of hCG did not change clinical pregnancy and implantation rates, 500 IU of hCG improved both outcomes significantly. Our results were in accordance with the findings of these last two trials. Despite an increased rate of multiple gestations with hCG injection, it was not associated with lower ongoing
pregnancy and abortion rates. It could be concluded that hCG injection has no adverse effect on pregnancy outcomes even if it is associated with multiple gestations. Triplet pregnancy, which is rather a complication than a positive result, occurred in 3 of cases but none of the control group and could be due to the higher implantation rate in the HCG-treated group. Using hCG may be more suitable in single ET methods, where the risk of multiple gestations induced by hCG can be minimized and the implantation and pregnancy rates could improve. The main limitation of the present trial was its singlecenter design. We also did not compare different doses of hCG on pregnancy outcomes. However, our results showed the effectiveness of 500 IU of hCG without serious complications. Further studies are needed to figure out its cost effectiveness at the time of ET. In conclusion, intrauterine injection of 500 IU hCG at ET increases implantation and pregnancy rates. These findings suggest that intrauterine injection of hCG could be considered an adjuvant to traditional ET protocols.
References
IVF Outcome after Embryo Transfer with or without Preinstillation of hCG
10 Banerjee P, Fazleabas AT: Extragonadal actions of chorionic gonadotropin. Rev Endocr Metab Disord 2011;12:323–332. 11 Cole LA: hCG, the wonder of today’s science. Reprod Biol Endocrinol 2012;10:24. 12 Licht P, Russu V, Wildt L: On the role of human chorionic gonadotropin (hCG) in the embryo-endometrial microenvironment: implications for differentiation and implantation. Semin Reprod Med 2001;19:37–47. 13 Wallace LD, Breiner CA, Breiner RA, Spell AR, Carter JA, Lamb GC, et al: Administration of human chorionic gonadotropin at embryo transfer induced ovulation of a first wave dominant follicle, and increased progesterone and transfer pregnancy rates. Theriogenology 2011;75:1506–1515. 14 Banerjee P, Fazleabas AT: Endometrial responses to embryonic signals in the primate. Int J Dev Biol 2010;54:295–302. 15 Mansour R, Tawab N, Kamal O, El-Faissal Y, Serour A, Aboulghar M, et al: Intrauterine injection of human chorionic gonadotropin before embryo transfer significantly improves the implantation and pregnancy rates in in vitro fertilization/intracytoplasmic sperm injection: a prospective randomized study. Fertil Steril 2011;96:1370–1374. 16 Weissman A, Ravhon A, Steinfeld Z, Nahum H, Golan A, Levran D: Controlled ovarian stimulation using a long gonadotropin-releasing hormone antagonist protocol: a proof of concept and feasibility study. Gynecol Obstet Invest 2013;76:113–118.
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17 Bozdag G, Esinler I, Yaralı H: Pretreatment with oral contraceptive pills does not influence the pregnancy rate in the long leuprolide acetate protocol. Gynecol Obstet Invest 2012; 73:53–57. 18 Zhou XL, Lei ZM, Rao CV: Treatment of human endometrial gland epithelial cells with chorionic gonadotropin/luteinizing hormone increases the expression of the cyclooxygenase-2 gene. J Clin Endocrinol Metab 1999;84: 3364–3377. 19 Licht P, Fluhr H, Neuwinger J, Wallwiener D, Wildt L: Is human chorionic gonadotropin directly involved in the regulation of human implantation? Mol Cell Endocrinol 2007;269: 85–92. 20 Cole LA: Biological functions of hCG and hCG-related molecules. Reprod Biol Endocrinol 2010;8:102. 21 Ben-Meir A, Aboo-Dia M, Revel A, Eizenman E, Laufer N, Simon A: The benefit of human chorionic gonadotropin supplementation throughout the secretory phase of frozenthawed embryo transfer cycles. Fertil Steril 2010;93:351–354. 22 Järvelä IY, Tapanainen JS, Martikainen H: Improved pregnancy rate with administration of hCG after intrauterine insemination: a pilot study. Reprod Biol Endocrinol 2010; 8: 18.
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1 Norwitz ER, Schust DJ, Fisher SJ: Implantation and the survival of early pregnancy. N Engl J Med 2001;345:1400–1408. 2 Bourgain C, Devroey P: Histologic and functional aspects of the endometrium in the implantatory phase. Gynecol Obstet Invest 2007; 64:131–133. 3 Simon A, Laufer N: Repeated implantation failure: clinical approach. Fertil Steril 2012;97: 1039–1043. 4 Di Micco P, D’Uva M, Lodigiani C, Rota LL: Thrombophilia and repeated in vitro fertilisation and embryo transfer failure: an open issue. Thromb Haemost 2010;103:472–473. 5 Simur A, Ozdemir S, Acar H, Colakoğlu MC, Görkemli H, Balci O, Nergis S: Repeated in vitro fertilization failure and its relation with thrombophilia. Gynecol Obstet Invest 2009; 67:109–112. 6 Margalioth EJ, Ben-Chetrit A, Gal M, EldarGeva T: Investigation and treatment of repeated implantation failure following IVFET. Hum Reprod 2006;21:3036–3043. 7 Penzias AS: Recurrent IVF failure: other factors. Fertil Steril 2012;97:1033–1038. 8 Lédée N, Dubanchet S, Oger P, Meynant C, Lombroso R, Ville Y, Chaouat G: Uterine receptivity and cytokines: new concepts and new applications. Gynecol Obstet Invest 2007;64:138–143. 9 Makrigiannakis A, Minas V, Kalantaridou SN, Nikas G, Chrousos GP: Hormonal and cytokine regulation of early implantation. Trends Endocrinol Metab 2006;17:178–185.
Gynecol Obstet Invest DOI: 10.1159/000363235
In vitro Fertilization Outcome following Embryo Transfer with or without Preinstillation of Human Chorionic Gonadotropin into the Uterine Cavity: A Randomized Controlled Trial Ashraf Aaleyasin Marzieh Aghahosseini Mandana Rashidi Leila Safdarian Fatemeh Sarvi Zahra Najmi Alireza Mobasseri Behzad Amoozgar Department of Infertility, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran
Abstract Background: Intrauterine injection of human chorionic gonadotropin (hCG) at embryo transfer (ET) has been shown to improve the outcome of assisted reproductive techniques. The aim of this study was to confirm previous findings. Methods: In this randomized controlled trial, 483 infertile women who were candidates for in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) for the first time were randomly assigned to receive an intrauterine injection of 500 IU hCG or placebo (tissue culture media) before ET. The main outcome measures were implantation and clinical pregnancy rates. Results: Both the hCG-treated group (n = 240) and control group (n = 243) were similar at baseline in terms of demographic and obstetrical characteristics. There were significant differences between the two groups regarding the implantation rate (23.6 vs. 12.2%, p < 0.001), pregnancy rate (54.6 vs. 35.8%, p < 0.001), clinical pregnancy rate (50 vs. 32.1%, p < 0.001), ongoing pregnancy rate (15.3 vs. 9.2%, p
17 mm were observed. Oocyte retrieval was performed under transvaginal ultrasound guidance 34–36 h after the administration of hCG. Oocytes were fertilized through ICSI and cleavage-stage ET was performed on the 2nd or 3rd day. At this time, by a computergenerated list, patients were randomly assigned to receive intrauterine hCG (study group) or placebo (control group) as described below. A technician, not belonging to the study personnel, prepared and coded the drugs according to the list. All patients and clinical care providers were blinded to the list until the end of the study. In the study group, 500 IU of hCG in a volume of 50 μl was injected into the uterus prior to ET. To prepare 500 IU of hCG for intrauterine injection, 0.5 ml of tissue culture media (Vitrolife, Göteborg, Sweden) was added to one vial containing 5,000 IU of hCG (Choragon, Ferring). We then injected 0.05 ml of this solution into the uterus. In the study group, in the lithotomy position, the cervical mucosa of the vagina was wiped out using sterile gauze. Then 500 IU of hCG was injected into the uterine cavity using a soft ET catheter (K-Soft-5000 Catheter; Cook Medical, Inc., USA). In difficult transfer cases a firmer catheter (malleable stylet catheter) was used. 5–7 min after the hCG injection, two or more high-grade embryos were transferred by another catheter. The process was completed with speculum removal 5 min after ET. The same process was carried out in the control group but with injection of 50 μl tissue culture media instead of hCG at ET. All participants received luteal phase support with 800 mg/day progesterone suppositories (Cyclogest; Actavis, UK) from the day of oocyte retrieval. Outcome Evaluation The β-hCG serum level was analyzed after 14 days of ET. Control transvaginal sonography was performed during the 5th week of gestation. Rising levels of β-hCG in serum was considered as pregnancy, regardless of sonography results. A sonography-confirmed gestational sac with a fetal heart rate was considered as clinical pregnancy. Any fetal loss between 7 and 20 weeks of gestation was considered as an abortion. The fertilization rate was defined as the proportion of all acquired embryos to all injected oocytes. The implantation rate was defined as the number of monographically viewed gestational sacs per transferred embryos. Any pregnancy beyond 20 weeks of gestation was considered as an ongoing pregnancy and calculated per transferred embryos.
Aaleyasin et al.
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Among the molecules involved in implantation, human chorionic gonadotropin (hCG) is a unique glycoprotein hormone with biological functions in the corpus luteum and endometrium [9]. The first embryonic signal initiates invasion and implantation of trophoblastic cells into the uterine myometrium while driving the growth and differentiation of cytotrophoblasts to syncytiotrophoblasts. It also promotes the formation of villous trophoblast tissue and provokes the development of uterine spiral arteries and their protrusion to villous trophoblasts. The corpus luteum continues to secrete progesterone under hCG stimulation [10, 11]. Different cytokines mediate these broad-spectrum functions of hCG [12]. Systemic administration of hCG at embryo transfer (ET) has resulted in an increased pregnancy rate in animal models [13]. Intrauterine injection of hCG has been associated with significant pro-implantation changes of the endometrium in primates [14]. Moreover, in a recent study it has been shown that intrauterine injection of hCG before ET improves implantation and pregnancy rates [15]. Based on these findings, in this study we assessed the effect of intrauterine injection of hCG before ET on the implantation and pregnancy rates in IVF patients.
Table 1. Baseline demographic and clinical characteristics of the hCG-treated group and control group
Age, years Duration of infertility, years BMI Type of infertility Primary Secondary Cause of infertility Male factor Tubal factor Anovulation Unexplained Male and female factors AFC FSH level, IU/l AMH level, ng/ml
hCG-treated group (n = 240)
Control group (n = 243) p value
29.1 ± 5.6 7.1 ± 4 25.4 ± 3.6
28.7 ± 5.4 7.3 ± 4.5 25.8 ± 3.3
195 (81.2) 45 (18.8)
202 (83.1) 41 (16.9)
90 (37.5) 45 (18.8) 38 (15.8) 42 (17.5) 25 (10.4) 12.3 ± 2.5 7.22 ± 2.8 3.5 ± 1.5
80 (33) 37 (15.2) 30 (12.3) 66 (27.2) 30 (12.3) 12.6 ± 2.3 7.44 ± 3.3 3.5 ± 1.6
n.s. n.s. n.s. n.s. n.s.
n.s. n.s. n.s.
Results
In total, 610 out of 764 women referred to the infertility clinic were eligible. Among these patients, 41 did not wish to participate, 17 withdrew due to personal reasons, and IVF/ICSI cycles were cancelled in 69 patients (21 for nonresponsiveness and 48 for ovarian hyperstimulation syndrome). The remaining women (n = 483) were randomly assigned to treatment (hCG-treated, n = 240) and control (placebo, n = 243) groups. Figure 1 represents the flow diagram of participants throughout the trial. Table 1 shows the baseline demographic and clinical characteristics of participants. There were no significant differences in age, duration of infertility, BMI, and type of infertility between the two groups. Similarly, ovarian reserve tests such as the antral follicle count (AFC), FSH level, and AMH level were not statistically different in the two groups. The number of retrieved oocytes, metaphase 2 oocytes, and injected oocytes were not statistically different in the two groups (11.7 ± 3.9 vs. 12.4 ± 4.3, p > 0.05; 8.3 ± 2.8 vs. 8.2 ± 2.7, p > 0.05, and 8.9 ± 2.8 vs. 9.1 ± 2.9, p > 0.05, reIVF Outcome after Embryo Transfer with or without Preinstillation of hCG
Assessed for eligibility (n = 764) Did not meet the criteria (n = 154)
Did not participate (n = 58)
IVF/ICSI cancelled (n = 69) Randomized (n = 483)
hCG-treated group (n = 240)
Control group (n = 243)
Fig.1. Flow diagram of participants throughout the trial.
spectively). Also, the fertilization rate was similar in the two groups (69.2 vs. 67%, p = 0.2). The mean transferred embryos in the hCG-treated group and control group were 2.85 ± 0.52 and 2.93 ± 0.57, respectively (p = 0.08). In hCG-treated women, the rate of pregnancy, clinical pregnancy, and implantation were significantly higher than those in control group (54.6 vs. 35.8%, 50 vs. 32.1%, and 23.6 vs. 12.2%, p < 0.001, respectively). Intrauterine injection of hCG was associated with a higher rate of twin Gynecol Obstet Invest DOI: 10.1159/000363235
3
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Statistics Statistical analysis was performed using SPSS version 19 software (SPSS, Inc., Chicago, Ill., USA). Numerical variables were reported as mean ± SD when normally distributed, otherwise as median plus range. We used an independent sample t test and χ2 test to compare quantitative and qualitative variables, respectively. p ≤ 0.05 was considered to be statistically significant.
Color version available online
Values represent mean ± SD or n (%).
Table 2. Fertilization, implantation, and pregnancy outcomes of the hCG-treated group and control group
Retrieved oocytes Oocytes in metaphase II Injected oocytes Fertilization rate Transferred fetus Implantation rate Biochemical pregnancies Clinical pregnancies Pregnancy sac Single Multiple Abortion Ongoing pregnancies per clinical pregnancies Ongoing pregnancies per transferred fetus Live delivery per clinical pregnancies Live delivery per transferred fetus
hCG-treated group (n = 240)
Control group (n = 243)
p value
11.7 ± 3.9 8.3 ± 2.8 8.9 ± 2.8 1,413/2,043 (69.2) 2.85 ± 0.52 162/685 (23.6) 131 (54.6) 120 (50)
12.4 ± 4.3 8.2 ± 2.7 9.1 ± 2.9 1,481/2,211 (67) 2.93 ± 0.57 87/715 (12.2) 87 (35.8) 78 (32.1)
n.s. n.s. n.s. n.s. n.s. 0.0001 0.0001 0.0001 0.007
87 (36.3) 33 (13.7) 15/120 (12.5) 105/120 (87.5) 105/685 (15.3) 98/120 (81.6) 98/685 (14.3)
69 (28.4) 9 (3.7) 12/78 (15.4) 66/78 (84.6) 66/715 (9.2) 60/78 (77) 60/715 (8.4)
n.s. n.s. 0.0005 n.s. 0.0005
Values represent mean ± SD or n (%).
Discussion
In the present study we found a significant improvement in the implantation and pregnancy rates of women treated with intrauterine injection of 500 IU hCG at ET. Additionally, hCG injection was associated with a higher rate of multiple gestations as compared with the control group. No other adverse effect of intrauterine injection of hCG was found. A normal implantation process encompasses continuous highly regulated steps. Biosynthesis of multiple prostaglandins, adhesion molecules, proteinases, and growth factors is a necessary step for a successful implantation. To stabilize an implanted embryo, adequate progesterone production, immunomodulation at implantation site, 4
Gynecol Obstet Invest DOI: 10.1159/000363235
placental growth and differentiation are also needed [1]. hCG is probably a critical signal in both implantation process and pregnancy stabilization: it increases expression of the cyclooxygenase-2 gene in endometrial glands, resulting in increased production of prostaglandins [18]. hCG provokes secretion of matrix metalloproteinases while suppressing their inhibitors, to induce endometrial tissue remodeling [19]. It reduces endometrial IGFBP-1 biosynthesis, resulting in an increased IGF-II level and angiogenesis. Neoangiogenesis and placental formation are also stimulated through increased intrauterine VEGF levels by hCG [19]. hCG inhibits apoptosis of cytotrophoblast cells while promoting their growth and invasion into the myometrium by blocking TGF-β receptor [20]. As a paracrine agent, it replaces LH function in promoting the production of progesterone during the first 3–4 weeks of gestation [20]. hCG presents its immunomodulatory effect by inhibiting M-CSF and macrophage phagocytosis activity, while stimulating leukocyte inhibitory factor [19]. The concept of our current study was based on previous animal investigations evaluating implantation and pregnancy rates after hCG injection. Wallace et al. [13] demonstrated improved pregnancy rates after an intramuscular injection of hCG at ET in cows. In a study by Banerjee and Fazleabas [14], intrauterine injection of hCG in baboons induced a favorable uterine environment for embryo implantation. Although hCG has been commerAaleyasin et al.
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and triplet pregnancies (p < 0.05). Triplet pregnancy occurred in 3 of the cases, but none of the control group. In hCG-treated women, ongoing pregnancy and live delivery rate were significantly higher than those in the control group when calculated per ET (15.3 vs. 9.2% and 14.3 vs. 8.4%, p < 0.001, respectively). However, they did not reach statistical significance when calculated per clinical pregnancy. Table 2 demonstrates the outcomes of fertilization, implantation, and pregnancy in the two groups. Seven intrauterine deaths in the hCG-treated group and 6 deaths in the control group were observed.
cially available for a long time, a few human trials have addressed the value of hCG injection in ET. In a study by Ben-Meir et al. [21], standard endometrial preparation with progesterone was compared with an additional 250 μg of subcutaneous recombinant hCG on the day of progesterone initiation, the day of ET, and 6 days later. Implantation and pregnancy rates did not differ between the two methods (14.9 vs. 12.7% and 32.2 vs. 28.2%, respectively, p > 0.05). In a study by Järvelä et al. [22], intramuscular injection of hCG 3–5 min after the intrauterine insemination procedure resulted in an improved pregnancy rate compared with 24–36 h injection before intrauterine insemination. Mansour et al. [15] evaluated the intrauterine injection of hCG prior to ET in three experimental doses. While 100 and 200 IU of hCG did not change clinical pregnancy and implantation rates, 500 IU of hCG improved both outcomes significantly. Our results were in accordance with the findings of these last two trials. Despite an increased rate of multiple gestations with hCG injection, it was not associated with lower ongoing
pregnancy and abortion rates. It could be concluded that hCG injection has no adverse effect on pregnancy outcomes even if it is associated with multiple gestations. Triplet pregnancy, which is rather a complication than a positive result, occurred in 3 of cases but none of the control group and could be due to the higher implantation rate in the HCG-treated group. Using hCG may be more suitable in single ET methods, where the risk of multiple gestations induced by hCG can be minimized and the implantation and pregnancy rates could improve. The main limitation of the present trial was its singlecenter design. We also did not compare different doses of hCG on pregnancy outcomes. However, our results showed the effectiveness of 500 IU of hCG without serious complications. Further studies are needed to figure out its cost effectiveness at the time of ET. In conclusion, intrauterine injection of 500 IU hCG at ET increases implantation and pregnancy rates. These findings suggest that intrauterine injection of hCG could be considered an adjuvant to traditional ET protocols.
References
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