Recombinant vs urinary gonadotropins – An update

Introduction

Human gonadotropins play an important role in the reproductive process. Hence, their pharmacological preparations have been extensively used to stimulate follicular development in infertile women. During the 1970s, urinary hMG (derived from the urine of menopausal women) was the only gonadotropin used in infertility treatment. It contained both FSH and LH. Later, a wide range of subproducts of urinary hMG were introduced, from which most or all of the LH content had been removed.¹   During the mid-1990s, modern technology was used to produce recombinant FSH (rFSH) in vitro   from hamster ovarian cell cultures.²

rFSH rapidly proceeded to replace the urine-derived gonadotropins. However, these newer gonadotropins are considerably more expensive, especially since urinary gonadotropins are no longer protected by patents and can now be offered under generic pricing structures.³

Moreover, there is as yet no significant evidence that recombinant gonadotropins are superior to urinary gonadotropins in clinical pregnancy rates. At best, they have been shown to be at least as effective as the urinary products.⁴

Further, urinary gonadotropins score over their recombinant counterparts by being easily available. The highly purified gonadotropins are devoid of impurities, and are as good as recombinant gonadotropins.

Thus, when choosing a gonadotropin regimen, the cost, patient acceptability, safety and drug availability should also be taken into account.⁵

rFSH vs uFSH – no evidence of clinical superiority

The pharmacokinetics of rFSH and uFSH were compared in a study by le Cotonnec et al.⁶   For this, both preparations were administered to 12 pituitary downregulated, healthy female volunteers in a balanced, random order, crossover sequence in a single intravenous dose of 150 or 300 IU, separated by 1 week of washout.

The total clearance of the preparations was comparable, judging from immunoassay and bioassay data (0.5 and 0.15 L/h, respectively). Based on the immunoassay, renal clearance of uFSH was 0.1 L/h, whereas for rFSH it was slightly lower at 0.07 L/h, indicating that less than one fifth of the administered dose was excreted in the urine. The initial and terminal half-lives of the two preparations were similar (2 and 17 hours, respectively). The volumes of distribution at steady state (11 L) were similar. The results of the in vitro bioassay confirmed this pharmacokinetic analysis. Just after i.v. administration, there was an initial decrease in the serum bioassay:immunoassay ratio, which could be ascribed to dilution of uFSH or of rFSH in the residual endogenous FSH pool. Then the ratio increased progressively with time, suggesting either metabolic selection or activation of both types of injected human FSH toward forms with greater in vitro bioactivity. The bioassay:immunoassay ratio returned to baseline by day 7.

These results demonstrate that the pharmacokinetic characteristics of rFSH and uFSH are similar.

To obtain clinical evidence, a meta-analysis by Al-Inany and colleagues⁵   analyzed the results of randomized controlled trials comparing recombinant FSH and urinary-derived FSH gonadotropins [hMG, urinary purified FSH (FSH-P) and highly purified FSH (FSH-HP)] in an IVF/ICSI program.

Results showed no statistically significant difference in the pregnancy rate per started cycle between rFSH and urinary-derived FSH gonadotropins. Subgroup analysis revealed no statistically significant difference in the pregnancy rate per started cycle between rFSH versus hMG, rFSH versus FSH-P and rFSH versus FSH-HP. There was no significant heterogeneity of treatment effect across the trials.

Thus, no evidence was found of clinical superiority of rFSH over different urinary-derived FSH gonadotropins as regards clinical pregnancy rate.

Apart from efficacy, there are certain concerns regarding rFSH which go against its use. For instance, GnRH agonist downregulation in some normogonadotrophic women may cause profound suppression of LH concentration, impairing adequate estradiol synthesis.⁷   Therefore, in such cases when rFSH is used for ovarian stimulation after GnRH agonist downregulation, very low serum LH concentrations may adversely affect IVF outcome.⁵

It would be pertinent to point out here that like rFSH, other biotech substitutes, such as insulin and Factor VIII, have also been found to offer no significant medical benefits in clinical studies.⁸

In another study, Selman et al ⁹   compared highly purified (HP) uFSH with rFSH to evaluate possible differences in oocyte and embryo quality and in implantation and pregnancy rates. The mean number of retrieved oocytes and the number of morphologically mature oocytes did not differ between the uFSH group and the rFSH group. Pregnancy and implantation rates were nonsignificantly higher in the uFSH group than in the rFSH group. The grade 1 embryo score was significantly higher in the uFSH group than the rFSH, and the live birth rate was nonsignificantly higher in the uFSH group. Thus, HP uFSH was found to be as effective, efficient, and safe for clinical use as rFSH.

These results can be explained by the fact that rFSH contains a higher proportion of the less acidic isoforms, whereas uFSH contains both acidic and mid-acidic isoforms. The less acidic isoforms are supposed to have faster circulatory clearance and, thus, a shorter circulatory half-life¹⁰   than the acidic isoforms.^11,12     It has been shown that the slow clearance of the acidic isoform results in better follicular maturation and estradiol secretion than the less acidic isoform.¹³

A prospective randomized comparative study by Meden-Vrtovec and colleagues¹⁴   analyzed ovarian stimulation with rFSH vs. uFSH in terms of hormonal events within ovarian follicles and the outcome of in vitro   fertilization.

The study involved 131 patients, of whom 70 received rFSH and 61, uFSH. The hormone measured were serum estradiol (E²  ) on the day of human chorionic gonadotropin (hCG) administration, and E²  , androstenedione (A) and testosterone (T) at the time of follicular aspiration in the follicular fluid and serum.

The study yielded the following results:

• The total dose of gonadotropins required and the length of ovarian stimulation were the same in the 2 groups
• In follicular fluid the E²   and the A levels were significantly higher in the rFSH group, whereas A:E²   and T:E²   ratios were significantly lower (Table 1)
• Serum hormonal levels, number of oocytes retrieved and pregnancy rates did not differ significantly between the groups

The authors concluded that results with rFSH are similar to those with uFSH.

In another study¹⁵   that compared the efficacy and safety of uFSH and rFSH for ovulation induction in patients with clomiphene citrate-resistant, normogonadotropic, chronic anovulation, 51 women were randomized to receive either uFSH (n=35, 64 cycles) or rFSH (n=16, 32 cycles). A maximum of three cycles using the low-dose step-up protocol with the same gonadotropin were undertaken.

Both urinary and recombinant FSH were found to be efficient and safe for inducing ovulation with the low-dose step-up protocol.

uFSH vs rFSH in older women

The number of infertile women in their advanced reproductive age (over 39 years) is increasing throughout the world. These women are potentially low responders to controlled ovarian hyperstimulation (COH) and have a poor reproductive outcome, posing a therapeutic challenge to fertility experts. It has been reported that in COH for IVF the prevalence of poor responder women, estimated to be 10% in the general population, is significantly higher in patients who are 40 years or older.

A study by Mohamed and colleagues¹⁶   evaluated the effectiveness of urinary vs recombinant FSH in women belonging to the late reproductive age group. This randomized controlled study recruited 257 women older than 39 years undergoing their first IVF cycle.

At the end of the study period, women treated with urinary FSH required a significantly lower total amount of FSH, and a lower amount of FSH per oocyte than women treated with recombinant FSH. This too can be explained by the fact of higher acidic isoforms in uFSH showing a decreased clearance rate and being produced when estrogen levels are low, such as in the early follicular and late luteal phase when primordial follicles are recruited.

In older women, the menstrual cycle is shortened due to a reduced follicular phase; this has been shown to depend on an earlier start of follicular growth, defined as “advanced growth.” Therefore, in these patients the available cohort of antral follicles starting the growth in each cycle is significantly smaller than in younger women. In older women, the recruitment of a larger follicle cohort is the primary problem in order to achieve a higher number of oocytes, and the use of uFSH could help in the follicle recruitment.

The reduction in the amount of FSH used in COH also led to a reduction in the cost of the IVF cycle and of the babies born. Moreover, the cost of uFSH was much lower than that of rFSH, resulting in considerable savings.

Based on the results of this study, it can be concluded that uFSH should be preferred in older women undergoing COH because it allows the use of smaller amounts of gonadotropins with similar results for the number of oocytes retrieved, embryos obtained, and pregnancy rate with a more favorable cost-effective ratio than rFSH. Further studies are required to confirm these findings and ascertain the best protocol for different patient groups.

hMG vs rFSH

van Wely and colleagues¹⁷   conducted a meta-analysis of 6 randomized controlled trials involving 2030 women undergoing IVF or ICSI. Ovarian hyperstimulation was done with either hMG or recombinant FSH after downregulation. In all trials, the group of women treated with hMG had higher pregnancy rates. Pooling the five trials that used a long GnRH agonist protocol revealed a higher clinical pregnancy rate for hMG compared with recombinant FSH. However, there was no evidence of a difference in rates of ongoing pregnancy or live birth per woman between hMG recipients and recombinant FSH recipients. No differences were found in gonadotropin dose used, oocytes retrieved, miscarriage rate, or multiple pregnancy rate.

Another meta-analysis by the same authors¹⁸   studied four randomized trials, which also showed an increase in ongoing pregnancies or live births with hMG compared with rFSH.

In yet another meta-analysis,¹⁹   the live-birth rate was significantly higher with hMG than with rFSH, but ovarian hyperstimulation syndrome (OHSS) rates were not significantly different. The clinical pregnancy rate also statistically significantly favored the hMG group. The other secondary outcomes were not different between the two groups. Thus, hMG was demonstrated to be superior to rFSH with regard to the clinical outcomes, but had equivalent patient safety during assisted reproduction.

Coomarasamy et al ²⁰   conducted a systematic review and meta-analysis of randomized trials comparing the effectiveness of hMG with that of rFSH following a long downregulation protocol in IVF-ICSI cycles, on the primary outcome of live birth per woman randomized, as well as several other secondary outcomes.

The investigators identified 7 randomized trials, consisting of a total of 2159 randomized women. A meta-analysis of these trials showed a pooled 4% increase in live birth rate with hMG when compared with rFSH, which was significant. hMG also led to an greater increase in clinical pregnancy rates as compared to rFSH. No significant differences were noted for gonadotropin use, spontaneous abortion, multiple pregnancy, cancellation and ovarian hyperstimulation syndrome rates.

HP hMG vs rFSH

In addition to the introduction of various types of recombinant gonadotropin preparations, better human-derived gonadotropins have also entered the market, such as highly purified (HP) hMG. The purification process of HP hMG allows its administration through the subcutaneous route with an incidence of local cutaneous reactions comparable to that with recombinant products.

The clinical profile and efficacy of HP hMG and recombinant FSH-alpha in assisted reproductive treatment were compared in a prospective, randomized, controlled study,²¹   in which 50 infertile women were treated with HP hMG and 50 with rFSH-alpha. Both drugs were given in the dose of 150 IU/day following GnRH agonist suppression (long regimen) until more than three follicles >17 mm and estradiol (E²  ) levels >600 pg/ml were reached.

The treatment duration and gonadotropin dose were lower in the HP hMG group, whereas peak pre-ovulatory E^2,   area under the curve of E²  , immunoreactive serum FSH and hCG were higher in the HP hMG group compared to the rFSH group (Table 2, Fig. 1).

There was no significant difference in the cycle cancellation rates, transferred embryo number, pregnancy rates per started cycle (30 versus 28%) and per embryo transfer (35 versus 35%) and miscarriage rates (6 versus 6%).

It was thus concluded that HP hMG treatment was associated with:

• Better patient response, as reflected by reduced treatment duration and gonadotropin requirements
• Increased serum levels of hCG, E²   and immunoreactive FSH during treatment
• ICSI outcome similar to that with rFSH-alpha

In a systematic review²²   of properly randomized trials comparing HP-hMG vs rFSH in women undergoing IVF and/or ICSI, 6 trials comprising 2371 participants were included. Pooled results showed that the probability of clinical pregnancy following HP-hMG administration was higher than with rFSH, but the ongoing pregnancy/live-birth rate was not statistically different between the two drugs, although it showed strong trends towards improvement with HP-hMG. On subgroup analysis comparing both drugs in IVF cycles too, a statistically significant better ongoing pregnancy/live-birth rate was observed in favor of HP-hMG.

In ICSI cycles, however, an almost equal ongoing pregnancy/live-birth rate was noted. Hence, the study authors concluded, HP-hMG should be preferred over rFSH in women undergoing assisted reproduction, especially if IVF is the intended method of fertilization.

A similar study²³   evaluated the live birth rates resulting from ovarian stimulation with HP hMG or rFSH in women undergoing IVF cycles following a long downregulation protocol. Raw data from two randomized controlled trials were examined, in which 491 subjects were given HP hMG and 495, rFSH. HP hMG was found to yield a live birth rate of 26.5% per cycle, whereas rFSH yielded 20.8%. The odds ratio in favor of HP hMG was 1.36 (95% confidence interval: 1.01-1.83). Thus, ovarian stimulation with HP hMG, following a long downregulation protocol, in IVF cycles results in significantly more live births than stimulation with rFSH alone.

In a large, randomized, single-blind, multicentre, multinational study,²⁴   desensitization of 496 patients in a long GnRH-agonist protocol was followed by the administration of HP hMG (n=231) or rFSH (n=232) in a fixed dose of 150 IU subcutaneously, until there was adequate follicular development. Results showed the occurrence of moderate to severe OHSS in 2.8% patients on rFSH as against 1.2% patients on HP hMG (Fig. 2).

Association of rFSH with deep vein thrombosis

Use of rFSH is associated with subclavian deep vein thrombosis (DVT). Loret de Mola et al ²⁵   described the case of two women undergoing IVF with ICSI due to male factor infertility. Use of recombinant gonadotropins in these women during COH led to mild OHSS and left subclavian thrombosis. The possibility that recombinant gonadotropins increase the risk for this complication should be further studied, suggested the authors.

Soluble P-selectin is a marker of platelet activity, with high levels being found in patients with thrombotic disease. Orvieto and colleagues²⁶   observed that during COH, serum P-selectin increased until estradiol levels peaked, suggesting that COH may potentiate a state of platelet activation which is substantially attenuated after hCG administration.

Keeping this in mind, the same authors tried to compare the levels of serum P-selectin in patients undergoing COH cycles with urinary (uGn) versus recombinant gonadotropins (rGn) and determine their possible correlation with COH variables.

In this study,²⁷   14 consecutive patients underwent the routine COH protocol for unexplained infertility with either uGn or rGn. Blood was drawn three times during the COH cycle as per the following schedule:

• On day 2 or 3 of the menstrual cycle and before gonadotropin treatment (day-0)
• On the day of or prior to hCG administration (day-hCG)
• On the day of ovulation (day-OVU)

Levels of sex steroids and serum P-selectin were compared among the three time points between the uGn and rGn subgroups. P-selectin was measured with a commercial quantitative sandwich immunoassay technique.

In both Gn subgroups, there was a non-significant increase in P-selectin level between day-hCG and day-0. In the rGN subgroup, no significant difference was observed in P-selectin levels between day-OVU and day-hCG. In the uGn subgroup, P-selectin levels were significantly lower in day-OVU as compared to day-hCG and day-0. On day-OVU, P-selectin levels were significantly lower in the uGn than the rGn subgroups.

In this study, while both forms of gonadotropin potentiated a state of platelet activation during COH, the administration of hCG caused a significant decrease in platelet activation in cycles pretreated with uGn. On the other hand, the level of platelet activation remained unchanged in rGn cycles. Since thrombosis is known to occur long after gonadotropin administration – usually coinciding with pregnancy and under the effect of endogenous hCG – this study suggests that uGn may have a potential benefit over rGn in patients at high risk for thromboembolism (e.g., congenital or acquired thrombophilia).

The difference in the effect on platelets between uGn and rGn can be understood by considering the source of each product. While uGn is extracted from humans, rGn has an animal origin. The use of a cell product from a different species could contribute to the thrombogenic effect of the rGn preparations. Alternatively, the human-derived uGn preparation may contain an anti-thrombotic factor that inhibits platelet function.

Combination of uFSH and rFSH

Urinary and recombinant gonadotropins are sometimes combined to give better outcomes. In a study by Pacchiarotti and coworkers,²⁸   a combination of both urinary and recombinant FSH was used for ovarian stimulation in an IVF treatment program. The study enrolled 119 infertile women undergoing ICSI treatment, who, after a standard downregulation with GnRH analog, were randomized to receive either combined urinary and recombinant FSH (n=58), starting with uFSH and then rFSH, or only recombinant FSH (n=61, controls).

Pregnancy and implantation rates were significantly higher in the combined uFSH/rFSH group than the control (rFSH) group (Fig. 3). The number of metaphase II oocytes and grade 1 embryos was significantly higher in the combined uFSH/rFSH group than in the recombinant FSH group.

This study shows that using a combination of both urinary and recombinant FSH for ovarian stimulation improves oocyte maturity and embryo cleavage, and increases pregnancy and implantation rates.

Conclusion

Pharmacological preparations of human gonadotropins have been extensively used to stimulate follicular development in infertile women. Although recombinant gonadotropins have been introduced recently, there is no clear evidence of their superiority over the urinary gonadotropins. Moreover, rFSH has been found to be associated with deep vein thrombosis. Hence, issues of cost, patient acceptability, safety and drug availability should also be considered when choosing a gonadotropin.

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