Assisted Reproductive Technology Outcomes in Women with Normal Ovarian Response Receiving Recombinant Luteinizing Hormone/Human Menopausal Gonadotropin: An Observational Study.

Infertility affects approximately 15% of couples worldwide, 1 who can achieve pregnancy via assisted reproduction techniques. In patients with a normal ovarian response, the prolonged gonadotropin-releasing hormone agonist (GnRH-a) protocol is the most commonly used protocol for in vitro fertilization-embryo transfer (IVF-ET) procedure. 2 , 3 However, GnRH-a may lead to over-suppression of luteinizing hormone (LH), negatively impacting IVF-ET outcome. 4 Optimal LH activity is critical for the quality and function of oocytes, 5 and cell proliferation and cytoplasmic maturation of oocytes. 6 During ovarian stimulation, LH administration varies in dose and timing. 7 Nevertheless, adding LH to follicle-stimulating hormone (FSH) can improve ovarian function in patients with a poor ovarian response, 8 by increasing the mature egg rate, embryo quality, 9 and implantation rate. 10 Additive recombinant (r)-LH during controlled ovarian stimulation can obtain >5 oocytes. 11 In women aged >35 years, LH supplementation can improve clinical pregnancy and ongoing pregnancy rates. 8 Before r-LH was available, human menopausal gonadotropin (HMG) was once the only option for stimulating LH activity, 12 which significantly improved clinical pregnancy rates than FSH alone in women with normal gonadotropic responses. 13 When using the GnRH-antagonist protocol, r-LH seemed to result in a higher cumulative live birth rate than HMG. 14 However, when using the early-follicular phase prolonged GnRH-a protocol, the optimal choice between r-LH, HMG, or combined is unclear for women with normal ovarian response. Herein, we explored the pregnancy outcomes and medical costs in women with normal ovarian response who received various early-follicular phase prolonged GnRH-a protocols.

Of the total 456 women with normal ovarian response, 115 treated by FSH, 244 received the FSH+HMG/r-LH treatment, and 97 received FSH+HMG+r-LH treatment. Table 1 shows the characteristics of women with normal ovarian response who received the IVF-ET. The age, BMI, progesterone, preliminary GnRH-a dose, maximum daily GnRH-a dose, duration of GnRH-a, endometrial thickness, and blastocyst formation rate were statistical differences among the three groups ( P 0.05). Table 1 Characteristics of Women with Normal Ovarian Response Who Received the IVF-ET Variables Group I (n=115) Group II (n=244) Group III (n=97) P Age (years), mean±SD 30.90±3.75 31.43±3.86 32.38±4.24 ab * 0.023 BMI (kg/m 2 ), mean±SD 22.04±3.11 21.78±3.14 23.61±3.34 ab ** <0.001 Type of infertility, n (%)  Primary 46 (40.0) 113 (49.1) 19 (38.8)  Secondary 69 (60.0) 117 (50.9) 30 (61.2) Baseline FSH (U/L), mean±SD 6.58±1.56 6.82±1.53 6.69±1.53 0.386 Baseline LH (U/L), mean±SD 4.51±2.18 4.75±2.41 4.98±2.46 0.362 Baseline FSH/LH ratio, mean±SD 1.67±0.60 1.66±0.60 1.58±0.62 0.512 Baseline prolactin (ng/mL), mean±SD 13.94±8.34 13.77±9.03 12.20±7.20 0.282 Baseline progesterone (ng/mL), mean±SD 0.54±0.33 0.69±1.52 0.95±2.10 0.214 Baseline estradiol (pg/mL), mean±SD 52.96±56.03 52.02±38.14 45.75±16.87 0.383 Baseline testosterone (ng/mL) 0.27±0.15 0.30±0.18 0.63±3.29 0.170 AMH (ng/mL), mean±SD 4.11±2.42 4.04±2.52 3.96±2.63 0.921 AFC (n), mean±SD 15.72±4.55 14.99±5.30 15.25±4.94 0.667 GnRH-a on day 1 LH (U/L), mean±SD 0.78±0.79 0.91±5.23 0.32±0.35 0.499 Estradiol (pg/mL), mean±SD 27.80±17.93 32.52±89.46 23.47±9.27 0.555 Progesterone (ng/mL), mean±SD 0.41±0.18 0.54±0.47 a * 0.46±0.28 0.025 Follicles (n), mean±SD 16.00±4.47 16.00±5.53 16.62±5.49 0.610 During ovarian stimulation Preliminary GnRH-a dose (IU), mean±SD 168.48±43.44 192.98±52.75 a ** 200.64±67.37 a ** <0.001 Maximum daily GnRH-a dose (IU), mean±SD 188.26±45.17 248.21±59.93 a ** 278.87±65.16 ab ** <0.001 Duration of GnRH-a (days), mean±SD 10.12±2.00 11.43±2.55 a ** 12.53±2.14 ab ** <0.001 Total GnRH-a dose (IU), mean±SD 1789±569 2447±1068 a ** 2834±978 ab ** <0.001 Mature follicles ≥ 14 mm (n), mean±SD 10.88±3.91 11.07±4.44 10.38±4.23 0.431 Estradiol (pg/mL), mean±SD 2821±1256 2764±1314 2568±1426 0.389 LH (U/L), mean±SD 1.69±2.43 1.91±12.62 0.63±0.43 0.554 Progesterone (ng/mL), mean±SD 0.86±0.40 6.01±72.91 0.65±0.42 0.629 Endometrial thickness (mm), mean±SD 10.31±2.81 11.30±2.48 a * 11.14±2.67 a * 0.002 Retrieved oocytes, mean±SD 15.10±6.19 14.61±5.43 14.55±4.67 0.700 Mature oocytes, mean±SD 12.90±5.43 12.72±4.95 12.67±5.66 0.941 Fertilized oocytes, mean±SD 10.57±4.61 10.47±4.41 10.45±4.99 0.975 Fertilization rate (%), mean±SD 83.01±13.07 82.53±13.47 82.17±11.68 0.919 Total embryos, mean±SD 10.35±4.61 10.23±4.27 10.16±4.91 0.955 Total high-quality embryos, mean±SD 8.39±4.16 7.86±3.82 7.91±3.91 0.222 High-quality embryo rate (%), mean±SD 81.21±17.80 76.70±19.26 78.23±21.00 0.116 Blastocysts, mean±SD 5.55±2.98 4.84±2.85 4.73±3.24 0.069 Blastocyst formation rate (%) 54.22±21.02 48.13±22.26 a * 49.77±21.42 0.049 Fresh embryo transfer cycles, n 83 197 66 Pregnancy outcomes Biochemical pregnancy, n (%) 68/83 (81.9) 149/197 (75.6) 53/66 (80.3) 0.426 Clinical pregnancy, n (%) 55/83 (66.3) 123/197 (62.4) 46/66 (69.7) 0.749 Live birth, n (%) 46/83 (55.4) 110/197 (55.8) 40/66 (60.6) 0.819 Cumulative live birth, n (%) 78/115 (67.8) 161/244 (66.0) 56/90 (62.2) 0.978 Notes : * P <0.05, ** P <0.001. a vs. Group I. b vs. Group II. Group I: FSH; group II: FSH+HMG/r-LH; group II: FSH+HMG+r-LH. Abbreviations : SD, standard deviation; BMI, body mass index; FSH, follicle-stimulating hormone; HMG, human menopausal gonadotropin; r-LH, recombinant luteinizing hormone; AMH, anti-Mullerian hormone; AFC, antral follicle count; LH, luteinizing hormone. Characteristics of Women with Normal Ovarian Response Who Received the IVF-ET Notes : * P <0.05, ** P <0.001. a vs. Group I. b vs. Group II. Group I: FSH; group II: FSH+HMG/r-LH; group II: FSH+HMG+r-LH. Abbreviations : SD, standard deviation; BMI, body mass index; FSH, follicle-stimulating hormone; HMG, human menopausal gonadotropin; r-LH, recombinant luteinizing hormone; AMH, anti-Mullerian hormone; AFC, antral follicle count; LH, luteinizing hormone. The characteristics of women aged ≥32 years or overweight were shown in Table 2 . The average age in Group III was 35, versus 34 in Groups 1 and 2 (P<0.05 vs Groups I). BMI was also much bigger in the Group III (P<0.05, vs both groups). Endometrial thickness was still greater in the Groups II and III (both P <0.05 vs Group I). Blastocyst formation rate and biochemical pregnancy rate were highest in Group I; however, the clinical pregnancy rate was highest in Group III. Live birth rate and cumulative live birth rate were higher in Groups II and III, albeit without statistical significance. Table 2 Characteristics of Women Aged ≥32 Years and Overweight Variables Age ≥32 Years Overweight Group I (n=115) Group II (n=244) Group III (n=97) P Group I (n=115) Group II (n=244) Group III (n=97) P Age (years), mean±SD 34.25±1.93 34.61±2.12 35.28±2.57 a * 0.045 31.34±3.32 31.85±4.06 31.67±4.41 0.803 BMI (kg/m 2 ), mean±SD 22.09±2.94 21.76±3.12 23.39±3.40 ab * 0.006 25.67±2.15 25.43±2.03 25.93±1.85 0.293 Endometrial thickness (mm), mean±SD 9.70±2.93 11.10±2.55 a * 11.05±2.16 a * 0.009 10.82±3.29 11.36±2.39 11.79±3.00 0.190 Blastocysts, mean±SD 5.55±2.83 4.73±2.81 4.91±3.24 0.245 5.32±2.78 5.09±2.70 4.55±3.14 0.772 Blastocyst formation rate (%), mean±SD 55.68±20.62 47.99±22.81 45.17±20.61 0.058 55.39±21.18 52.25±23.96 50.16±24.70 0.659 Fresh embryo transfer cycles (n) 37 101 39 28 60 36 Biochemical pregnancy, n (%) 29/37 (78.4) 74/101 (73.3) 30/39 (76.9) 0.793 20/28 (71.4) 46/60 (76.7) 30/36 (83.3) 0.518 Clinical pregnancy, n (%) 22/37 (59.5) 61/101 (60.4) 26/39 (66.7) 0.757 15/28 (53.6) 31/60 (51.7) 26/36 (72.2) 0.122 Live birth, n (%) 17/37 (45.9) 53/101 (52.5) 21/39 (53.8) 0.748 11/28 (39.3) 28/60 (46.7) 24/36 (66.7) 0.063 Cumulative live birth, n (%) 29/51 (56.9) 72/123 (58.5) 31/54 (57.4) 0.976 24/38 (63.2) 54/79 (68.4) 34/51 (66.7) 0.976 Notes : * P <0.05, ** P <0.001. a vs. Group I. b vs. Group II. Group I: FSH; group II: FSH+HMG/r-LH; group II: FSH+HMG+r-LH. Abbreviations : SD, standard deviation; BMI, body mass index. Characteristics of Women Aged ≥32 Years and Overweight Notes : * P <0.05, ** P <0.001. a vs. Group I. b vs. Group II. Group I: FSH; group II: FSH+HMG/r-LH; group II: FSH+HMG+r-LH. Abbreviations : SD, standard deviation; BMI, body mass index. There was no difference in the age or BMI among the 3 groups. The endometrial thickness appears greater in Groups II and III. The number of blastocysts and blastocyst formation rate were similar among the three groups. Group III showed the highest biochemical pregnancy rate, clinical pregnancy rate, and live birth rate (P=0.063); however, the cumulative live birth rate was similar between the three groups. Table 3 shows the direct medical costs for oocyte retrieval. There were statistical differences of the direct medical costs among the three groups in per oocyte retrieval cycle, per embryo and per blastocyst ( P <0.05). Compared with the Groups II, the costs of per oocyte retrieval cycle (17,052 vs 14,940), per embryo (2264 vs 1826) and per blastocyst (5304 vs 3874) were higher in the Group III. Table 3 Direct Medical Costs for Oocyte Retrieval Variables Group I (n=115) Group II (n=244) Group III (n=97) P Oocyte retrieval cycle (CNY/per), mean±SD 14940±1983 15,524±1996 a * 17,052±2325 ab ** <0.001 Embryo (CNY/per), mean±SD 1826±1099 1860±1018 2264±1654 ab * 0.011 Blastocyst (CNY/per), mean±SD 3874±3314 4610±3685 5304±4467 a * 0.026 * P <0.05, ** P <0.001. a vs. Group I. b vs. Group II. Group I: FSH; group II: FSH+HMG/r-LH; group II: FSH+HMG+r-LH. Abbreviation : SD, standard deviation. Direct Medical Costs for Oocyte Retrieval * P <0.05, ** P <0.001. a vs. Group I. b vs. Group II. Group I: FSH; group II: FSH+HMG/r-LH; group II: FSH+HMG+r-LH. Abbreviation : SD, standard deviation.

In the retrospective observational study, women who received IVF or ICSI treatments with early-follicular phase prolonged GnRH-a protocols were included from the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University between February 1, 2021 and March 31, 2022. This research was conducted in accordance with the Declaration of Helsinki, and approved by the Medical Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University (2022-K-53-01). Informed consent was obtained from all women. The inclusion criteria were as follows: (1) 18–40 years old; (2) infertility due to tubal dysfunction or unexplained factor in women with normal ovarian response; (3) the FSH ≤10 U/L, FSH/LH ratio ≤3, anti-Müllerian hormone (AMH) ≥1.5 ng/mL, and antral follicular count (AFC) ≥7 on cycle days 2–5. The exclusion criteria were: (1) women with obesity [body mass index (BMI) ≥30 kg/m 2 ] (greater difficulties in IVF success in all populations 15 , 16 ); (2) only one ovary; (3) polycystic ovary syndrome; (4) grade 3–4 endometriosis; or (5) metabolic or autoimmune diseases. According to the treatments, women were divided into the FSH (Group I), FSH+HMG/r-LH (Group II), and FSH+HMG+r-LH (Group III). GnRH-a (3.75mg once, Triptorelin, Germany) was administered on days 2–5 of the menstrual cycle. 17 After 30–37 days, pituitary downregulation was achieved, reflected by antral follicles ≤5mm, serum estradiol <50pg/mL, serum LH=5U/L, and the endometrial thickness <5mm. Then, gonadotropin treatment started, using the FSH (Gonal-F; Merck Serono, Switzerland), HMG (hMG; Lizhu Pharma, China), and r-LH (Merck Serono). The preliminary dose was based on age, antral follicular count, and BMI. Follicle development was monitored. Gonadotropin dose was titrated according to follicle growth by transvaginal ultrasound and serum levels of estradiol, LH, FSH, and progesterone. When one dominant follicle reached 18 mm in diameter or two leading follicles reached ≥17mm in diameter, HMG (4000–10,000U) was administered to trigger final oocyte maturation, and oocytes were retrieved 36–38h later. One day later, luteal phase support started using dydrogesterone (40mg twice daily, Solvay, Netherlands) and progesterone (200mg daily, Cyndea Pharma, Spain). At 4–6h after retrieval, the oocytes were fertilized by IVF or ICSI. Embryos were cultured at 37°C under standard condition and scored by the Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. 18 Following fertilization, two prokaryotes and two polar bodies were detected 16–18h later as a sign of normal fertilization. Embryo were scored by the number, size, and shape of blastomeres and the proportion of fragments at the cleavage stage. 18 , 19 The high-quality embryo was characterized by four blastomeres on day 2, ≥7 blastomeres on day 3 with a normal zona pellucida, <20% of fragments, and a complete absence of multinucleated blastomeres at any stage of early cleavage. 18 , 20 The blastocyst was evaluated by the Gardner scoring system, including the degree of blastocoel expansion, the size and density of inner cell masses, and the density and number of cells in the trophoblastic layer. 18 , 21 The blastocyst was transferred 5 days after incubation. The frozen-thawed embryo transfer was performed in women who failed fresh embryo transfer. Hormone replacement therapy was used to prepare the endometrium, starting with estradiol valerate from day 2–5 of the cycle (4 mg/day oral, Femoston; Abbott Biological, China) to day 14 after embryo transfer, titrated by endometrial thickness every 3–5 days. After 12 days of treatment, when the endometrial thickness ≥8 mm and the progesterone <1.5 ng/mL a transformation of the endometrium started with dydrogesterone (20 mg/day oral, Abbott Biological) and progestin (400 mg oral/vaginal, daily, Cyndea Pharma, Spain) or progesterone (90 mg sustained-release vaginal gel, daily, Fleet Laboratories, UK) daily for 3–5 days before embryo transfer. Pregnancy was confirmed when serum human chorionic gonadotropin (hCG) level 14 days after blastocyst transfer and a gestational sac and cardiac pulsation via ultrasound at 4–5 weeks following transfer. Data of women receiving IVF or ICSI treatments were collected in this current study. Before the treatments, the age (years), BMI (kg/m 2 ), infertility type (primary or secondary), baseline FSH (U/L), baseline LH (U/L), baseline FSH/LH ratio, baseline prolactin (ng/mL), baseline progesterone (ng/mL), baseline estradiol (pg/mL), baseline testosterone (ng/mL), AMH (ng/mL), and AFC were recorded. The LH (U/L), estradiol (pg/mL), progesterone (ng/mL), and number of follicles on the first day of GnRH-a were noted. During the ovarian stimulation, several parameters were recorded, including the preliminary GnRH-a dose (IU), maximum daily GnRH-a dose (IU), GnRH-a duration (days), total GnRH-a dose (IU), number of mature follicles ≥14 mm, estradiol (pg/mL), LH (U/L), progesterone (ng/mL), endometrial thickness (mm), retrieved oocytes, mature oocytes, fertilized oocytes, fertilization rate, number of total embryos, number of total high-quality embryos, high-quality embryo rate, blastocysts, blastocyst formation rate, and fresh embryo transfer cycles. The cost per embryo and blastocyst were also included. The pregnancy outcomes involved the rates of biochemical pregnancy, clinical pregnancy, live birth, and cumulative live birth. Biochemical pregnancy refers to pregnancy duration less than 12 weeks. The live birth rate was calculated as live births/fresh embryo transfer cycles. The cumulative live birth rate was the live births following the use of all fresh and frozen embryos derived from a single controlled ovarian stimulation cycle. 22 Cost is one of major issues for IVF treatment. 23 In China, it is an out-of-pocket expense, costing 17,000–20,000 Chinese yuan (USD 2459–28,931). In contrast, the per capita disposable income was 35,128 yuan with the expenditure 24,100 yuan in 2021. 24 Therefore, IVF/intracytoplasmic sperm injection (ICSI)-ET treatment can impose significant financial stress on the average family, especially those with a single income, adversely affecting pregnancy outcomes resulting in repeated treatments. Repeated ovarian hyperstimulation can markedly affect the patient’s health. 25 Therefore, patients prefer obtaining a decent number of embryos from one oocyte retrieval cycle. The direct medical costs were calculated based on the 2021 price in the two hospitals. The costs per oocyte retrieval cycle, per available embryo, and per blastocysts were reported. Continuous numerical variables are presented as mean±standard deviation and analyzed using one-way ANOVA with Tukey post hoc tests. Categorical variables are shown as the number (percentage) and analyzed using the Chi-square test (SPSS V24.0, IBM Corp., NY, USA). As increased age and BMI can increase the difficulties of conceiving, we also studied two sub-cohorts, those ≥32 years (when oocyte number decline accelerates) 26 and those with overweight (23 kg/m 2 ≤ BMI <30 kg/m 2 ). P <0.05 were considered statistically significant.

Adding HMG and/or r-LH to FSH can promote endometrial receptivity and pregnancy outcomes in women with normal ovarian responders, specifically those overweight and ≥32 years. Such practice may be cost-saving for couples aiming for multiple babies.

In this current study, the endometrial thickness in women with normal ovarian response who receiving the FSH+HMG/r-LH or FSH+HMG+r-LH was thicker in comparison to those who receiving the FSH. Women with the two therapies had higher pregnancy and live birth rates, specifically among patients older than 32 years or overweight who normally face challenges in conceiving and sustaining a pregnancy. Here, the participants were unique, who had normal ovarian responses, by Bologna criteria 27 and POSEIDON classification. 28 The strength lies in the same controlled ovarian stimulation protocol used for all patients undergoing IVF/ICSI, ie early follicular phase prolonged GnRH-a protocol, allowing valid comparisons between the groups. The major benefit of adding both HMG and r-LH to FSH is the improvement in endometrial thickness. Endometrial receptivity is a major factor affecting IVF/ICSI success. 29 Poor endometrial receptivity and abnormal embryo-endometrial interaction account for two-thirds of implantation failures. 29 LH can increase the de novo ovarian progesterone synthesis to regulate ovarian growth and differentiation after ovulation, which is critical in endometrium preparation for implantation and maintenance of pregnancy. 30 Low LH levels can inhibit endometrial growth and even stagnation, impairing endometrium receptivity 31 and implantation rate. 32 Our findings are consistent with a meta-analysis 33 where in patients >30 years old, the FSH+HMG+r-LH improved endometrial thickness. Ata et al 34 reported no linear relationship of endometrial thickness with live birth rate, and no predictive value of endometrial thickness for live birth rate. Gursu et al 35 mentioned that the endometrial thickness measurement in patients undergoing the infertility treatment may provide little benefit to clinical outcomes. Previous studies showed that endometrial thickness on the day of hCG administration can be a potential prognostic tool for the outcomes of the assisted reproductive technology, which serve as an indicator of endometrial receptivity. 36 , 37 Further studies need to validate our findings. Sufficient LH is also required to support follicle development and oocyte maturation, 6 , 38–40 and facilitate the meiosis and extrusion of the first polar body. 41 The blastocyst formation rate is closely related to pregnancy outcomes. 42 Live birth is the ultimate goal for IVF/ICSI treatment. 43 Our results show that clinical pregnancy and live birth rates were highest in the FSH+HMG+r-LH group, albeit marginal impact on blastocyst formation. However, HMG and/or r-LH showed a marginal benefit in the cumulative live birth rate for those having multiple transfers, maybe related to the small sample size. It is still reasonable to postulate that HMG+r-LH promote optimal embryo and pregnancy success. The dosage and duration of gonadotropin needed can reflect ovarian response. Groups II and III required more gonadotropin, suggesting some impaired ovarian response albeit clinicaly classified as “normal”. Thus, additional hormone(s) (HMG and/or r-LH) is necessary to achieve optimal pregnancy outcomes. Age and BMI may play a role, both of which affect fertility and IVF/ICSI success. 44–46 Overweight/obese patients benefit from additional r-LH to improve embryo quality and ongoing pregnancy rates. 47 Here, adding HMG and/or r-LH appeared to improve the clinical pregnancy rate and live birth rate in the sub-cohort of obese/overweight women. The lack of statistical significance is primarily due to the small sample size. However, for clinicians, the physiological significance is more important than the statistical significance regarding the patient’s outcome. Age also affects IVF-ET success, 48 as oocyte quantity and number decline with age. 49 At 32 years, there is a marked decline in oocyte number. 26 Indeed, the sub-cohort (32–40 years old) had lower pregnancy and live birth rates compared with the whole cohort. Group III were also older and heavier than those in Groups I and II, yet without differences in the live birth rate or cumulative live birth rate. Thus, it was reasonable to postulate that in “normal” ovarian responders, adding HMG and rLH can compensate for the age effects on pregnancy outcomes, helping older women achieve comparable live birth and cumulative live birth rate to young women. Assisted reproductive treatment is costly. The extra embryos will be frozen between transfers or when ovarian hyperstimulation syndrome or excessive trigger day progesterone elevation occurs. For a second transfer, additional costs included embryo freezing (~1500–2000/freezing) and transfer (~6000/procedure), far cheaper than another IVF-ET (min 14,000 yuan). Therefore, an optimal protocol to maximize the live birth rate is more cost-saving than repeated IVF/ICSI procedures. We acknowledge that our group size was relatively small, especially when sub-cohorts were studied. Secondly, the study was retrospective when the treatment decision was made rather than a by randomization design, which can be addressed by future prospective studies to verify our results.