Recombinant Gonadotropin Research Articles

Establishment of specific enzyme-linked immunosorbent assay (ELISA) for measuring Fsh and Lh levels in medaka (Oryzias latipes), using recombinant gonadotropins

The paucity of information on understanding the regulatory mechanisms that are involved in the control of piscine Fsh and Lh synthesis, secretion, and function, prompted the present work. Part of the problem is related to the molecular heterogeneity and the unavailability of Fsh and Lh assays for quantifying gonadotropins, in particular assays regarding the measurement of Fsh, and such assays are available today for only a few teleost species. The present study reports the development and validation of competitive ELISAs for quantitative determination of medaka Fsh and Lh by first producing medaka recombinant (md) gonadotropins mdFshβ, mdLhβ, mdFshβα, and mdLhβα by Pichia pastoris, generating specific antibodies against their respective β subunits, and their use within the development of ELISAs.The advantages of this protocol include:•The reproducibility of the ELISA demonstrated was relatively high, as shown by reasonably low intra- (Fsh 2.7%, Lh 3%) and interassay CVs (Fsh 5.3%, Lh 5.7%).•The high degree of parallelism between serial dilutions of the recombinant and native pituitary-derived Fsh and Lh, may be a sign of similar structures and immunologically similarity.•Two new competitive ELISAs for the quantification of medaka Fsh and Lh were established for the first time.

Medaka follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh): Developmental profiles of pituitary protein and gene expression levels

The two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are of particular importance within the hypothalamic-pituitary-gonadal (HPG) axis of vertebrates. In the current study, we demonstrate the production and validation of Japanese medaka (Oryzias latipes) recombinant (md) gonadotropins Fshβ (mdFshβ), Lhβ (mdLhβ), Fshβα (mdFshβα), and Lhβα (mdLhβα) by Pichia pastoris, the generation of specific rabbit antibodies against their respective β subunits, and their use within the development and validation of competitive enzyme-linked immunosorbent assays (ELISAs) for quantification of medaka Fsh and Lh. mdFsh and mdLh were produced as single-chain polypeptides by linking the α subunit with mdFshβ or mdLhβ mature protein coding sequences to produce a “tethered” polypeptide with the β-chain at the N-terminal and the α-chain at the C-terminal. The specificity of the antibodies raised against mdFshβ and mdLhβ was determined by immunofluorescence (IF) for Fshβ and Lhβ on medaka pituitary tissue, while comparison with fluorescence in situ hybridization (FISH) for fshb and lhb mRNA was used for validation. Competitive ELISAs were developed using antibodies against mdFshβ or mdLhβ, and the tethered proteins mdFshβα or mdLhβα for standard curves. The standard curve for the Fsh ELISA ranged from 97.6 pg/ml to 50 ng/ml, and for the Lh ELISA from 12.21 pg/ml to 6.25 ng/ml. The sensitivity of the assays for Fsh and Lh was 44.7 and 70.8 pg/ml, respectively. A profile of pituitary protein levels of medaka Fsh and Lh comparing juveniles with adults showed significant increase of protein amount from juvenile group (body length from 12 mm to 16.5 mm) to adult group (body length from 21 mm to 26.5 mm) for both hormones in male medaka. Comparing these data to a developmental profile of pituitary mRNA expression of medaka fshb and lhb, the mRNA expression of lhb also increased during male maturation and a linear regression analysis revealed a significant increase of lhb expression with increased body length that proposes a linear model. However, fshb mRNA expression did not change significantly during male development and therefore was not correlated with body length. In summary, we have developed and validated homologous ELISA assays for medaka Fsh and Lh based on proteins produced in P. pastoris, assays that will be used to study the functions and regulations of Fsh and Lh in more detail.

Serum levels of transforming growth factor β1 and C-reactive protein as possible markers of intra uterine insemination outcome.

Maternal immunity is important for the implantation phase, and exaggerated inflammatory responses may reduce the chance of implantation and pregnancy. Transforming growth factor β1 (TGF-β1) plays a role in the modulation of cellular growth, maturation and differentiation, extracellular matrix formation, immunoregulation, and apoptosis. In this study, we aimed to evaluate the changes in serum TGF-β1 and C-reactive protein (CRP) levels in infertile women following intrauterine insemination (IUI) according to the presence of pregnancy. Sixty-three infertile patients were selected for the study in a nine-month period. Clomiphene citrate or recombinant gonadotropins were used for ovulation induction, and all patients underwent IUI following human chorionic gonadotropin (hCG) trigger. The pregnant and non-pregnant groups' TGF-β1 and CRP levels were measured. The CRP levels increased significantly from the day of the hCG trigger to the 8th day after hCG trigger in the non-pregnant group (P = 0.003) whereas TGF-β1 levels decreased in the pregnant group (P = 0.001). Maternal inflammatory responses play an important role in the occurrence of pregnancy. Changes in the levels of TGF-β1 and CRP may have a role in the outcome of IUI. Serial measurements of TGF-β1 and C-reactive protein, if confirmed by larger studies, may become valuable in predicting the outcome of IUI.

Seasonal-and dose-dependent effects of recombinant gonadotropins on sperm production and quality in the flatfish Solea senegalensis

Consecutive treatments with recombinant follicle-stimulating and luteinizing hormones (rFsh and rLh, respectively) stimulate spermatogenesis and potentiate sperm production in pubescent specimens of the oligospermic Senegalese sole (Solea senegalensis). However, sperm production in response to the hormones is highly variable, and the steroidogenic potential of the testis may be diminished due to sustained hormone supply. Here, we compared the effectiveness of low (9 μg/kg) and high (18 μg/kg) doses of rFsh and rLh to improve sperm production in adult sole during late winter-early spring (onset of the natural spawning period), and in autumn under a controlled temperature of 12 °C (period of testicular recrudescence). Treatment with rFsh over six weeks during spring, followed by a single rLh injection, did not enhance sperm production, possibly because of an advanced stage of sexual maturation of the males, as reflected by high Lh plasma levels (~17 ng/ml) before rFsh treatment. In contrast, in autumn, when the Lh circulating levels were much lower (~3 ng/ml), the low doses of rFsh and rLh generated a four-times increase in sperm production, whereas the high doses of the hormones were ineffective. However, treatment with rLh, regardless of the effect of rFsh, improved the motility of spermatozoa during both spring and autumn. These data confirm that consecutive rFsh and rLh treatments increase sperm production and quality in adult sole males, although they seem to be highly sensitive to the rFsh dose. The efficiency of recombinant gonadotropins also appears to be season-dependent despite the asynchronous nature of the sole testis.

Expression and characterization of recombinant single chain beta-alpha equine chorionic gonadotropin in prokaryotic host

The aim of present study was to produce recombinant eCG/pituitary glycoprotein in the Escherichia coli (E. coli) BL21C host cells and to test its diagnostic efficacy. This aim was achieved by optimizing its expression, purification as well as its characterization through the immunoassays and bioassays. A bacterial protein expression vector system based on the phage T7 promoter and histidine tag was used for the expression and purification. The recombinant single chain beta-alpha equine chorionic gonadotropin (rbaeCG) encoding gene was constructed with beta and alpha sequences according to its biologically active counterpart. It was successfully cloned and when expressed in E.coli BL21C host, the purified recombinant protein was found to be active as revealed by enzyme linked immunosorbent assay (ELISA) and Western blotting. However, it was not found to exhibit any significant activity in vivo when tested in the mice

ЕФЕКТИВНІСТЬ ЗАСТОСУВАННЯ ПРЕГРАВІДАРНОЇ ПІДГОТОВКИ У ПАЦІЄНТОК ІЗ СИНДРОМОМ ПОЛІКІСТОЗНИХ ЯЄЧНИКІВ В ПРОГРАМАХ ДОПОМІЖНИХ РЕПРОДУКТИВНИХ ТЕХНОЛОГІЙ ПРИ ЗАСТОСУВАННІ ПРОТОКОЛІВ КОНТРОЛЬОВАНОЇ ОВАРІАЛЬНОЇ СТИМУЛЯЦІЇ З АНТАГОНІСТАМИ ТА ТРИГЕРОМ АГОНІСТОМ

Синдром полікістозних яєчників (СПКЯ) – полісиндромне захворювання, що часто асоціюється з безпліддям, і є найчастішою патологією у жінок репродуктивного віку як в Україні, так і за кордоном. За даними літератури, СПКЯ діагностують майже у 50 % жінок із порушеннями менструальної та овуляторної функцій, та у 40 % пацієнток, які страждають від ендокринного безпліддя.Мета дослідження – оцінити ефективність прегравідарної терапії перед проведенням контрольованої стимуляції овуляції в протоколах ЕКЗ у пацієнток із СПКЯ.Матеріали і методи. Рандомізоване клініко-лабораторне дослідження було проведено серед 146 жінок із верифікованим діагнозом СПКЯ та наступними характеристиками: вік 29,8 (25–38) років, ІМТ – (27,2±4,9) кг/м², АМГ – 6,3 ng/ml (4,1–11,6 ng/ml). Тривалість безпліддя – від 2 до 19 років у середньому (6,8±0,5) року. Первинне безпліддя було у 102 пацієнток (69,9 %), а вторинне – в 44 жінок (30,1 %). Середній вік жінок, у яких починалось менархе, складав (14,1±0,3) року, в усіх жінок цикл був не регулярним, у 118 жінок (80,8 %) менструальний цикл тривав від 30 до 150 днів. Обстеження та лікування проводили відповідно до наказу Міністерства охорони здоров’я України № 787 від 09.09.2013р. “Про затвердження Порядку застосування допоміжних репродуктивних технологій в Україні” та наказу Міністерства охорони здоров’я України від 29.11.2013р. № 1030/102 “Про удосконалення системи планування сім’ї та охорони репродуктивного здоров’я в Україні”. Перед програмою ДРТ було проведено збір даних щодо попереднього обстеження та лікування подружніх пар із подальшим обов’язковим стандартним протоколом обстеження (ОСПО).Результати досліджень та їх обговорення. У статті показано ефективність терапії за допомогою препаратів “FT-500 Plus” (інозитол, фолієва кислота, глутатіон, лютеїн, цинк, вітамін С, вітамін Е, селен) та “Аквадетрин” вітаміну D3 в пацієнток із безпліддям при синдромі полікістозних яєчників (СПКЯ) перед проведенням контрольованої стимуляції овуляції (КОС) в протоколі екстракорпорального запліднення. Стимуляцію здійснювали при рекомбінантному гонадотропіні корифолітропіні-альфа “ЕЛ ОНВА” на 5–7 дні після протоколу залежно від реакції яєчників. Коли розмір фолікула досягав 14–15 мм, призначали щоденно антагоніст ГнРГ “Оргалутран” по 0,25 мг. На 8 день від введення препарату “ЕЛ ОНВА” продовжили стимуляцію рекомбінантним ФСГ “Пурегон” до кінцевого дозрівання ооцитів (3 фолікули>17 мм), в якості тригера використовували агоніст релізинг-гормону “Диферелін”. Забір ооцитів здійснювали через 36 год від введення тригера овуляції із подальшою вітрифікацією ембріонів на 5–6 доби (стадія бластоцисти).Висновки. Отримані результати показали, що у пацієнток, які отримували прегравідарну терапію, сумарний коефіцієнт ефективності отриманих ооцитів, ступінь їх зрілості, частота запліднення, дроблення, вихід бластоцист та, відповідно, настання вагітності були вищими порівняно з контрольною групою.

Urinary Versus Recombinant Gonadotropins for Ovarian Stimulation in Women Undergoing Treatment with Assisted Reproductive Technology.

Globally, about 10%–15% couples are affected by infertility, with major role being played by the couple's lifestyle. Several gonadotropin preparations (urinary, purified urinary, recombinant, and biosimilars) are available for use. Purified urinary formulations offer numerous advantages over their predecessor, including lesser injection dose required, ability to be administered subcutaneously, less batch-to-batch variability, better efficacy, ability to individualize protocols as per patient's need, better control of developing follicles, less risk of multiple pregnancies, and hyperstimulation. Published results of Cochrane reviews and meta-analysis show no difference in efficacy or safety between urinary and recombinant gonadotropins. In the absence of any significant difference, cost plays an important role in deciding choice of gonadotropins. In this article, we have reviewed the results of comparative clinical trials, Cochrane analysis, and meta-analysis to derive consensus statements regarding efficacy, safety, and cost implications of urinary versus recombinant gonadotropin preparations.

Using specific recombinant gonadotropins to induce spermatogenesis and spermiation in the European eel (Anguilla anguilla)

New specific European eel (Anguilla anguilla) recombinant gonadotropins (aarGths) produced in the ovarian cells of Chinese hamsters (CHO) were used to induce maturation in captive male eels. In the first experiment, five different hormonal treatments were assayed: one group was given a constant dose of recombinant European eel follicle-stimulating hormone (aarFsh; 4 μg/fish) for 9 weeks, and the second group received a constant dose of recombinant European eel luteinizing hormone (aarLh; 2 μg/fish) also for 9 weeks. The other three groups were injected with different combinations of both aarGths (some doses constant, some variable). All five treatments stimulated androgen synthesis, but the increase was more pronounced in the fish treated with a combination of both aarGths. Unlike aarLh, aarFsh alone was able to induce spermiation, the best results were achieved in the fish that were treated with a constant dose of aarFSH and an increasing dose of aarLH, with spermiation being induced (20% motile cells) despite the fact that these fish were immature at the start of the experiment. In order to improve sperm quality, a second experiment was performed. Immature males received three constant doses of aarFsh (2.8, 1.4 or 0.7 μg/fish) and increasing doses of aarLh (every 3 weeks; 1, 2, 6 μg/fish). All the treatments induced spermiation, however the best sperm quality (with ≥50% motile cells) was observed in the males treated with the highest dose of aarFsh. In conclusion, these specific recombinant gonadotropins have demonstrated their capacity to induce spermatogenesis and spermiation in vivo in a teleost fish, the European eel.

Finding a place for corifollitropin within the PIVET FSH dosing algorithms

PIVET recombinant FSH (rFSH) dosing algorithms have been designed for rFSH injection pens, providing optimal pregnancy and live birth productivity rates whilst minimizing risk and occurrence of ovarian hyperstimulation syndrome (OHSS). Recently, long-acting recombinant gonadotrophin corifollitropin (Elonva) was approved for use in assisted reproduction, and welcomed by patients as the single injection allowed ovarian stimulation over 7 days without need for multiple injections. Consequently, another rFSH dosing algorithm was devised to incorporate Elonva, and these cycles were compared to standard rFSH agents, Gonal-f and Puregon. Initiated Elonva cycles (n = 165) were compared with 972 cycles initiated with standard rFSH. Elonva replaced standard rFSH dosages across the 200–400 IU range, but provided equivalent oocyte retrieval numbers and live birth outcomes. Elonva is considered risky for women whose antral follicle count is ≥20 follicles, and was inadvertently administered contra-protocol in 19 cycles with ≥20 follicles. However, while oocyte retrieval numbers were higher, raising risk for OHSS, no actual cases ensued. Taken together, this indicated that Elonva was equivalent to standard rFSH stimulation, and consequently has been added to the rFSH algorithms for medium to lower antral follicle counts and represented by green colour coding in the existing PIVET algorithmic charts.

Use of various gonadotropin and biosimilar formulations for in vitro fertilization cycles: results of a worldwide Web-based survey.

The purpose of this study was to identify trends in gonadotropin therapy in patients undergoing in vitro fertilization (IVF) treatment worldwide. Retrospective evaluation utilizing the results of a Web-based survey, IVF-Worldwide ( www.IVF-worldwide.com ) was performed. Three hundred fourteen centers performing a total of 218,300 annual IVF cycles were evaluated. Respondents representing 62.2% of cycles (n=135,800) did not believe there was a difference between urinary and recombinant gonadotropins in terms of efficacy and live birth rate. Of the respondents, 67.3% (n=146,800) reported no difference between recombinant and urinary formulations in terms of short-term safety and risk of ovarian hyperstimulation syndrome. In terms of long-term safety using human urinary gonadotropins, 50.6% (n=110,400) of respondents believe there are potential long-term risks including prion disease. For 95.3% of units (n=208,000), the clinician was the decision maker determining which specific gonadotropins are used for IVF. Of the units, 62.6% (n=136,700) identified efficacy as the most important factor in deciding which gonadotropin to prescribe. While most (67.3%, n=146,800) were aware of new biosimilar recombinant FSH products entering the market, 92% (n=201,000) reported they would like more information. A fraction of respondents (25.6%, n=55,900) reported having experience with these new products, and of these, 80.3% (n=46,200) reported that they were similar in efficacy as previously used gonadotropins in a similar patient group. Respondents representing the majority of centers do not believe a difference exists between urinary and recombinant gonadotropins with respect to efficacy and live birth rates. While many are aware of new biosimilar recombinant FSH products entering the market, over 90% desire more information on these products.

Superovulatory responses in cynomolgus monkeys (Macaca fascicularis) depend on the interaction between donor status and superovulation method used.

The current study was performed to investigate the effect of oocyte donor status, including age and body weight, on metaphase II (MII) oocyte recovery using two superovulation methods in cynomolgus monkeys. The use of Method A [recombinant gonadotrophin (75 IU/kg, 3 ×, 3-day intervals) and human chorionic gonadotropin (hCG)] led to great increases in ovary size and the mean number of MII oocytes retrieved in age- and body-weight-dependent manner; in contrast, both the parameters were similar in Method B [recombinant gonadotrophin (60 IU, twice daily, 6 days), recombinant gonadotropin and recombinant human luteinizing hormone (rhLH) (60 IU, twice daily, 3 days), and hCG]. Importantly, Method A showed maximal MII oocyte recovery rate in > 60-month-old or 4.5–5.0-kg female monkeys, whereas Method B was equally effective regardless of the donor age and body weight. These results indicate that superovulatory responses depend on the interaction between oocyte donor status and the superovulation method used in cynomolgus monkeys.

Biologically active recombinant carp LH as a spawning-inducing agent for carp.

Currently, spawning is induced in carp species by carp pituitary extract (CPE) and a combination of synthetic agonist of GnRH combined with a dopamine antagonist. The main goal of this study was the production of recombinant gonadotropins (GtHs) on a large scale to serve as an alternative to currently used agents. We produced carp (c) recombinant (r) Lh as a single chain in the methylotrophic yeast Pichia pastoris Lha subunit was joined with Lhb subunit with a flexible linker of three glycine-serine repeats and six Histidines to form a mature protein, the β-subunit formed the N-terminal part and the α-subunit formed the C-terminal part. The ability of the rcLh to elicit biological response was tested by in vivo stimulation of estradiol (E2) and 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and by its in vivo potency to induce ovulation and spawning induction. rcLh tested in this work significantly enhanced both E2 and DHP secretion in a dose-dependent manner similar to the results obtained with CPE. E2 levels showed a moderate rise following the priming injection and a subsequent decrease during the rest of the trial. DHP levels were only increased after the resolving injection, approximately 5 h before spawning. At the highest dose of rcLh (350 µg/kg BW), the recombinant protein was more efficient than CPE in terms of both spawning success and fertilization rate. It is shown here that rcLh can elicit the secretion of DHP in vivo and actually trigger spawning. These novel findings introduce the potential of utilizing recombinant gonadotropins in aquaculture.

Reproductive outcomes in recipients are not associated with oocyte donor body mass index (BMI): a cohort study of 2927 cycles

Increased BMI is associated with negative impact on reproductive outcomes in Assisted Reproductive Technology (ART), implying a role of defective endometrial receptivity. Recently, it has been suggested that the oocyte quality contributes to a reduction in clinical pregnancy rates with increasing donor BMI, since the odds of clinical pregnancy and live birth declined with increasing single units of BMI( 1 kg/m2). The aim of the present study is to assess the impact of donor BMI in live birth rates (LBR) in a large cohort of recipients of donated oocytes. A retrospective cohort study of 2927 consecutive donor oocyte IVF cycles performed between January 2011 - December 2014 at Salud de la Mujer Dexeus. Oocyte donor cycles. Non polycystic ovarian syndrome donors were treated with recombinant gonadotropins and antagonist protocol. Recipients primed with oral estrogens and vaginal progesterone. Following adjustments for recipient BMI, the relationship between donor BMI (categorized in quartiles) and LBR was assessed. In the population of 2927 donors, no differences were observed in LBR with increasing BMI. When comparing the population of recipients, no significant differences were observed in LBR and cumulative live birth rates (CLBR) with increasing donor BMI. When the analysis was performed according to recipient BMI quartiles a significant decrease in LBR and CLBR was observed. We did not observe a deleterious impact of donor BMI in LBR. Moreover, we could not confirm the findings reported in another recent published study that presented opposite results. We observe that increase BMI in the recipient population is associated with a significant decrease in LBR and CLBR most probably due to a defective endometrial receptivity. Therefore, any deleterious effect of the BMI in the reproductive outcomes could be exerted through a defective endometrial receptivity and probably not due to a negative impact at the oocyte level. Main strength: The big sample size in a single center. Limitations: The retrospective design and characteristics of the population of mainly non-obese donors and recipients.

Profile of follitropin alpha/lutropin alpha combination for the stimulation of follicular development in women with severe luteinizing hormone and follicle-stimulating hormone deficiency.

A severe gonadotropin deficiency together with chronic estradiol deficiency leading to amenorrhea characterizes patients suffering from hypogonadotropic hypogonadism. Administration of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to these patients has been shown to be essential in achieving successful stimulation of follicular development, ovulation, and rescue of fertility. In recent years, the availability of both recombinant FSH (rFSH) and recombinant LH (rLH) has provided a new therapeutic option for the stimulation of follicular growth in hypopituitary–hypogonadotropic women (World Health Organization Group I). In this article, we review the data reported in the literature to highlight the role and the efficacy of using recombinant gonadotropins, rFSH and rLH, in the treatment of women with severe LH/FSH deficiency. Although the studies on this issue are limited and the experiences available in the literature are few due to the small number of such patients, it is clearly evident that the recombinant gonadotropins rFSH and rLH are efficient in treating patients affected by hypogonadotropic hypogonadism. The results observed in the studies reported in this review suggest that recombinant gonadotropins are able to induce proper follicular growth, oocyte maturation, and eventually pregnancy in this group of women. Moreover, the clinical use of recombinant gonadotropins in this type of patients has given more insight into some endocrinological aspects of ovarian function that have not yet been fully understood.

Use of recombinant luteinizing hormone for controlled ovarian hyperstimulation in infertile patients.

Controlled ovarian stimulation has become an integral part of a high complexity infertility treatment. Treatment options with recombinant gonadotrophins add more to knowledge on folliculogenesis and ovarian steroidogenesis. Therefore, a literature search was conducted in the following data bases: Medline, Scielo and PubMed. The descriptors/ key words used were ovarian stimulation, in vitro fertilization, recombinant luteinizing hormone, supplementation LH. The aim of this study was to review the available literature and to assess the benefits of using recombinant luteinizing hormone associated with recombinant follicle stimulating hormone in different populations who have undergone assisted reproduction procedures.

Effect of Abdominal Massage before In Vitro Fertilization Injection on Alleviating Pain among Infertile Women.

The purpose of this study was to evaluate the effect of abdominal massage on alleviating pain caused by the injection of recombinant gonadotropin for In Vitro Fertilization (IVF) among infertile women. This study employed a nonequivalent control group non-synchronized design. A total sample of 149 infertile women who never experienced in vitro fertilization was recruited at C fertility center. Seventy women were assigned into experimental group and 79 into controls. The experimental group had been informed to do abdominal massage prior to the injection and to record their subjective pain using visual analogue scale. The control group had been informed to record their subjective pain in the same way just after the injection. Compared to the pre-tested pain scores, the pain scores in experimental group were significantly reduced by 0.7 points, whereas the control group increased by 0.9 points (t=-4.55, p=.001). This study confirms that the use of abdominal massage prior to the injection is an effective way to alleviate pain on injection site. This massage may be a useful intervention for infertile women about pain alleviation.

Impact of quantitative parameters, such as number of retrieved oocytes, number of transferred embryos and availability of surplus embryos for cryopreservation, on clinical pregnancy rates in ART

Objective: To estimate the impact of quantitative parameters such as number of retrieved oocytes, number of transferred embryos and availability of surplus embryos for cryopreservation on clinical pregnancies in assisted reproductive technology (ART).Design: We used the database of fertility clinic on in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles from year 2000 to 2010. Follicular fluids containing oocyte–cumulus complexes were recovered by a single lumen follicle aspiration needle, and IVF or ICSI procedure was used depending on infertility indications. Fertilization determination was performed on the second day. Zygotes were transferred into fresh medium and cultivated until the embryo transfer procedure. According to morphological criteria, the best quality embryos were transferred into uterus on the second or third day. Surplus embryos were frozen by slow freezing or by vitrification. We evaluated only clinical pregnancies that were diagnosed by ultrasonography.Patients: We allocated all 1161 women cohort into three age groups: 671 women were <35; 397 were 35–40 years old and 93 women were >40. Indications for IVF treatment were as follows: mechanical factor (436 cases), male infertility (361 cases), idiopathic infertility (129 cases), endometriosis (78 cases), immunological infertility (14 cases), anovulation (28 cases), and other indications (28 cases). There were no data about the cause of infertility of 87 patients. The stimulation protocol was composed from gonadotropin hormone stimulation with antagonist or agonist supplementation. The triggering of luteinizing hormone (LH) surge was performed by recombinant human chorionic gonadotropin (hCG). The clinical pregnancy rates were: 42.2% (283/671) for women <35, 31.2% (124/397) for women 35–40-year-old and 16.1% (15/93) for women >40.Interventions: All materials and methods that we used were based on results from our daily practice in IVF clinic and have no experimental design.Main outcome measures: We evaluated clinical pregnancies in terms of the number of obtained oocytes, the amount of transferred embryos and the availability of surplus embryos for freezing. The number of retrieved oocytes was divided as follows: 1–5 (group I); 6–10 (group II); ≥11 (group III). The number of transferred embryos was from 1 to 3. Cases at which cryopreservation of embryos was not performed were evaluated as well.Results: For women <35 and 35–40 years old, the highest percentage of pregnancies was achieved when 11 and more oocytes were obtained. Differences in women under 35 between groups were statistically significant. Statistically significant differences in clinical pregnancies depend on the number of transferred embryos in the age groups of <35 and 35–40. Women <35 and 35–40 years old, with the availability of surplus embryos for freezing, had better chances for pregnancy compared with women, who had no excess embryos for freezing.Conclusions: The number of obtained oocytes, the number of transferred embryos and the availability of surplus embryos for freezing may serve as predictors of pregnancy rates in ART.

Successful twin pregnancy in panhypopituitarism

Panhypopituitarism is characterized by inadequate or absent production of anterior pituitary hormones. This involves hypofunction of most of the major endocrine functions that affect fertility and reproduction. Deficiency of all six anterior pituitary hormones leads to deficiencies of gonadotrophins (FSH, LH), thyroid axis (TSH), adrenocortical hormones (ACTH), Growth Hormone (GH) and Prolactin (PRL). GH and gonadotrophin deficiency are commoner (1) than TSH and ACTH deficiency (2). Although pregnancy after complete loss of pituitary function is uncommon, advances in fertility treatment have led to increased pregnancy rates in these women. Problems related to pregnancy include infertility, lactation failure and increased risk of complications such as miscarriage, anaemia, pregnancy-induced hypertension, placental abruption, premature birth, and postpartum hemorrhage (3, 4, 5). Conception in this group of patients usually requires the support of assisted reproductive techniques. Induction of ovulation by means of human or recombinant gonadotrophins is widely used in current practice. Progestrogen support following conception, optimizing other hormonal replacement and careful monitoring throughout pregnancy play a vital role in successful pregnancy outcomes. We report a subject with panhypopituitarism who was managed in our Endocrine Antenatal clinic with successful outcome.

Characterization of the Biological Activities of Human Luteinizing Hormone and Chorionic Gonadotropin by a Förster Resonance Energy Transfer-Based Biosensor Assay

A Förster resonance energy transfer (FRET)-based cyclic adenosine monophosphate (cAMP) biosensor was used to study the activation of the mutual receptor for human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) in living cells stably expressing the shared recombinant receptor for these hormones. This work demonstrates the applicability of a cAMP biosensor-based assay for the study of receptor-mediated signal transduction and the characterization of the biological activities of gonadotropins and their isoforms. The importance of this approach lies in the fact that only the active portion of the total hormone concentration elicits a measurable response. No significant differences in early receptor activation profiles were detected for either of the tested recombinant gonadotropins. However, the subsequent physiological regulations mediated by these hormones may be more complex. The ability to detect biologically active hormones at low concentrations forms the basis for the future quantification of human chorionic gonadotropin in embryo culture medium and the use of this information for unbiased embryo selection during in vitro fertilization. Herein, a novel method for describing the biological activities of gonadotropins in various samples and preparations is reported.

Managing infertility with the follitropin alfa prefilled pen injector - patient considerations.

Gonadotropin treatment has been used in fertility treatment since the 1930s. First, preparations coming from animals were injected, then, gonadotropins prepared from the pituitary glands of human cadavers. A great step was achieved with the introduction of human menopausal gonadotropin extracted from the urine of postmenopausal women. When cases of Creutzfeld-Jacob disease were recognized after the use of human pituitary-derived hormone injections, urinary gonadotropins were increasingly purified and then produced by the use of recombinant DNA technology. Recombinant gonadotropins were characterized by the extreme high specificity and the nearly 100% purity. This allows for follitropin alfa, the first recombinant-human follicle stimulating hormone (r-hFSH) approved, to be quantified and filled by mass, with a small variance of only ±2% and no more with a bioassay with a variance of 45%. With recombinant preparations, it is also possible to cover the tremendous growing demand for gonadotropins. Ovarian stimulation has become a self-injecting procedure for the patients. Accurate and easy-to-use injection devices which minimize pain, difficulty, and stress are essential for patient compliance. So, two pen injectors adapted from the well-known insulin pen were introduced in fertility treatment, one as a multiple-use device rechargeable with premixed, prefilled cartridges with r-hFSH (follitropin β) and the other a disposable, prefilled drug delivery system with a liquid formulation of follitropin alfa filled by mass. The efficacy in comparison to the quite more cumbersome handling with ampoules and syringes has been proven very quickly. In several studies, it has been shown that patients had a preference to the prefilled follitropin alfa pen due to the faster preparation and were more confident of accurate dosing. The follitropin alfa (filled by mass [FbM]) prefilled pen is a move toward better quality of treatment and also better quality of life for the women within the stressful period of fertility treatment.