Karin Erb

Assisted reproductive technology in Europe, 2009: results generated from European registers by ESHRE

STUDY QUESTIONnThe 13th European in vitro fertilization (IVF)-monitoring (EIM) report presents the results of treatments involving assisted reproductive technology (ART) initiated in Europe during 2009: are there any changes in the trends compared with previous years?nnnSUMMARY ANSWERnDespite some fluctuations in the number of countries reporting data, the overall number of ART cycles has continued to increase year by year and, while pregnancy rates in 2009 remained similar to those reported in 2008, the number of transfers with multiple embryos (3+) and the multiple delivery rates declined.nnnWHAT IS KNOWN ALREADYnSince 1997, ART data in Europe have been collected and reported in 12 manuscripts, published in Human Reproduction.nnnSTUDY DESIGN, SIZE, DURATIONnRetrospective data collection of European ART data by the EIM Consortium for the European Society of Human Reproduction and Embryology (ESHRE); cycles started between 1st January and 31st December are collected on a yearly basis; the data are collected by the National Registers, when existing, or on a voluntary basis.nnnPARTICIPANTS/MATERIALS SETTING, METHODSnFrom 34 countries (-2 compared with 2008), 1005 clinics reported 537 463 treatment cycles including: IVF (135 621), intracytoplasmic sperm injection (ICSI, 266 084), frozen embryo replacement (FER, 104 153), egg donation (ED, 21 604), in vitro maturation (IVM, 1334), preimplantation genetic diagnosis/screening (PGD/PGS, 4389) and frozen oocyte replacements (FOR, 4278). European data on intrauterine insemination using husband/partners semen (IUI-H) and donor (IUI-D) semen were reported from 21 and 18 countries, respectively. A total of 162 843 IUI-H (+12.7%) and 29 235 IUI-D (+17.3%) cycles were included. Data available from each country are presented in the tables; total values (as numbers and percentages) refer to those countries where all data have been reported.nnnMAIN RESULTS AND THE ROLE OF CHANCEnIn 21 countries where all clinics reported to the ART register, a total of 399 020 ART cycles were performed in a population of 373.8 million, corresponding to 1067 cycles per million inhabitants. For IVF, the clinical pregnancy rates per aspiration and per transfer were 28.9 and 32.9%, respectively and for ICSI, the corresponding rates were 28.7 and 32.0%. In FER cycles, the pregnancy rate per thawing was 20.9%; in ED cycles, the pregnancy rate per transfer was 42.3%. The delivery rate after IUI-H was 8.3 and 13.4% after IUI-D. In IVF and ICSI cycles, 1, 2, 3 and 4+ embryos were transferred in 24.2, 57.7, 16.9 and 1.2%, respectively. The proportions of singleton, twin and triplet deliveries after IVF and ICSI (combined) were 79.8, 19.4 and 0.8%, respectively, resulting in a total multiple delivery rate of 20.2%, compared with 21.7% in 2008, 22.3% in 2007, 20.8% in 2006 and 21.8% in 2005. In FER cycles, the multiple delivery rate was 13.0% (12.7% twins and 0.3% triplets). Twin and triplet delivery rates associated with IUI cycles were 10.4/0.7% and 10.3/0.5%, following treatment with husband and donor semen, respectively.nnnLIMITATIONS, REASONS FOR CAUTIONnThe method of reporting varies among countries, and registers from a number of countries have been unable to provide some of the relevant data such as initiated cycles and deliveries. As long as data are incomplete and generated through different methods of collection, results should be interpreted with caution.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe 13th ESHRE report on ART shows a continuing expansion of the number of treatment cycles in Europe, with more than half a million of cycles reported in 2009. The use of ICSI has reached a plateau. Pregnancy and delivery rates after IVF and ICSI remained relatively stable compared with 2008 and 2007. The number of multiple embryo transfers (3+ embryos) and the multiple delivery rate have shown a clear decline.

Human menopausal gonadotropin versus recombinant follicle-stimulating hormone in normogonadotropic women down-regulated with a gonadotropin-releasing hormone agonist who were undergoing in vitro fertilization and intracytoplasmic sperm injection: a prospective randomized study

OBJECTIVEnTo evaluate clinical and endocrinological effects of intranasal (IN) vs. subcutaneous (SC) GnRH-a for pituitary down-regulation combined with hMG vs. rFSH.nnnDESIGNnProspective, randomized study.nnnSETTINGnUniversity hospital, IVF unit.nnnPATIENT(S)nThree hundred seventy-nine normogonadotropic women eligible for IVF or ICSI.nnnINTERVENTION(S)nRandomization to intranasal (IN) or SC GnRH-a and to hMG or rFSH.nnnMAIN OUTCOME MEASURE(S)nOocytes retrieved, embryos developed, clinical pregnancy, and delivery rates. Serum hormone concentrations on stimulation days 1 (S1) and 8 (S8), and oocyte pick-up (OPU) day.nnnRESULT(S)nAfter randomization, four groups were formed: IN/hMG (n = 100), IN/FSH (n = 98), SC/hMG (n = 89), and SC/FSH (n = 92). Mean number of oocytes retrieved and of transferable and transferred embryos were similar in the four groups. Clinical pregnancy rate per started cycle was significantly higher in the IN/HMG group than in the SC/FSH group (P<.05) and was intermediate in the two remaining groups. Se-LH on S8 in the two SC groups was significantly lower than in the two IN groups. Se-E2 on S8 in the SC/FSH group was significantly lower than in the other three groups.nnnCONCLUSION(S)nThe clinical and endocrinological outcome in IVF and ICSI-treated normogonadotropic women is significantly influenced by mode of down-regulation as well as gonadotropin formulation.

Assisted reproductive technology in Europe, 2012: results generated from European registers by ESHRE.

STUDY QUESTIONnThe 16th European IVF-monitoring (EIM) report presents the data of the treatments involving assisted reproductive technology (ART) and intrauterine insemination (IUI) initiated in Europe during 2012: are there any changes compared with previous years?nnnSUMMARY ANSWERnDespite some fluctuations in the number of countries reporting data, the overall number of ART cycles has continued to increase year by year, the pregnancy rates (PRs) in 2012 remained stable compared with those reported in 2011, and the number of transfers with multiple embryos (3+) and the multiple delivery rates were lower than ever before.nnnWHAT IS KNOWN ALREADYnSince 1997, ART data in Europe have been collected and re-ported in 15 manuscripts, published in Human Reproduction.nnnSTUDY DESIGN, SIZE, DURATIONnRetrospective data collection of European ART data by the EIM Consortium for the European Society of Human Reproduction and Embryology (ESHRE). Data for cycles between 1 January and 31 December 2012 were collected from National Registers, when existing, or on a voluntary basis by personal information.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnFrom 34 countries (+1 compared with 2011), 1111 clinics reported 640 144 treatment cycles including 139 978 of IVF, 312 600 of ICSI, 139 558 of frozen embryo replacement (FER), 33 605 of egg donation (ED), 421 of in vitro maturation, 8433 of preimplantation genetic diagnosis/preimplantation genetic screening and 5549 of frozen oocyte replacements (FOR). European data on intrauterine insemination using husband/partners semen (IUI-H) and donor semen (IUI-D) were reported from 1126 IUI labs in 24 countries. A total of 175 028 IUI-H and 43 497 IUI-D cycles were included.nnnMAIN RESULTS AND THE ROLE OF CHANCEnIn 18 countries where all clinics reported to their ART register, a total of 369 081 ART cycles were performed in a population of around 295 million inhabitants, corresponding to 1252 cycles per million inhabitants (range 325-2732 cycles per million inhabitants). For all IVF cycles, the clinical PRs per aspiration and per transfer were stable with 29.4 (29.1% in 2011) and 33.8% (33.2% in 2011), respectively. For ICSI, the corresponding rates also were stable with 27.8 (27.9% in 2011) and 32.3% (31.8% in 2011). In FER cycles, the PR per thawing/warming increased to 23.1% (21.3% in 2011). In ED cycles, the PR per fresh transfer increased to 48.4% (45.8% in 2011) and to 35.9% (33.6% in 2011) per thawed transfer, while it was 45.1% for transfers after FOR. The delivery rate after IUI remained stable, at 8.5% (8.3% in 2011) after IUI-H and 12.0% (12.2% in 2011) after IUI-D. In IVF and ICSI cycles, 1, 2, 3 and 4+ embryos were transferred in 30.2, 55.4, 13.3 and 1.1% of the cycles, respectively. The proportions of singleton, twin and triplet deliveries after IVF and ICSI (added together) were 82.1, 17.3 and 0.6%, respectively, resulting in a total multiple delivery rate of 17.9% compared with 19.2% in 2011 and 20.6% in 2010. In FER cycles, the multiple delivery rate was 12.5% (12.2% twins and 0.3% triplets). Twin and triplet delivery rates associated with IUI cycles were 9.0%/0.4% and 7.2%/0.5%, following treatment with husband and donor semen, respectively.nnnLIMITATIONS, REASONS FOR CAUTIONnThe method of reporting varies among countries, and registers from a number of countries have been unable to provide some of the relevant data such as initiated cycles and deliveries. As long as data are incomplete and generated through different methods of collection, results should be interpreted with caution.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe 16th ESHRE report on ART shows a continuing expansion of the number of treatment cycles in Europe, with more than 640 000 cycles reported in 2012 with an increasing contribution to birthrate in many countries. However, the need to improve and standardize the national registries, and to establish validation methodologies remains manifest.nnnSTUDY FUNDING/COMPETING INTERESTSnThe study has no external funding; all costs are covered by ESHRE. There are no competing interests.

Pilot scale purification of human monoclonal IgM (COU-1) for clinical trials

No standard procedure is available for the purification of human monoclonal antibodies for human i.v. administration. Here we describe the procedure developed for pilot scale purification of the human IgM monoclonal antibody COU-1 directed against a cancer-associated antigen. The hybridoma cells were grown in protein-free medium and purification from the clarified culture supernatant was carried out in 4 simple chromatographic steps: (1) hydroxylapatite chromatography; (2) hydrophobic interaction chromatography on phenyl-Sepharose: (3) cation-exchange chromatography on sulphonyl-Sepharose; and (4) anion-exchange chromatography on tetraethylamino-Sepharose. The product was substantially pure with regard to protein after step 3, but contained DNA which was removed in step 4. The average recovery of the IgM was 54% with a range of 40-65%. Importantly, the ability of the antibody to bind to its antigen in ELISA was fully maintained during the purification. Subsequently, the purified antibody was isotope labelled and successfully used for in vivo detection of colon, rectal and pancreas carcinomas in patients. The purification procedure described appears to compare favourably with previously published methods, but a critical comparison is not possible due to the lack of necessary information in the available literature.

Human‐human hybridoma producing monoclonal antibodies against colorectal cancer‐associated antigens

Lymphocytes from lymph nodes draining the tumor region in patients with colorectal cancer were fused with two different human B‐lymphoblastoid cell lines, LICR‐LON‐HMy‐2 (HMy‐2) and WI‐L2–729‐HF2 (729‐HF2), to generate hybridomas synthesizing antibodies reacting with tumor‐associated antigens. In this way 220 hybridomas were obtained which produce antibody reacting with colon cancer cells. All established clones produced IgM. Four human monoclonal antibodies have been furhter analyzed. The cell lines producing these antibodies are all hybrids based on DNA analysis. Three of the antibodies (G4146, B9165 and D4213) showed binding to differentiation antigens by immunocytochemical analysis on different cancer cell lines and normal human leucocytes and by immunohistochemical analysis on sections of frozen malignant and normal tissues, while the fourth (F11348) showed a reaction with all cells and tissues tested. Western blots of tumor extracts showed binding of G4146 to two components from colon cancer cells with Mr of 59 K. and 61 K, while B9165 bound to a 43 K component and F11348 to several components with Mr from 30 to 200K. D4213 showd no binding in this analysis. The results obtained demonstrate the successful application of hybridoma technology to produce human monoclonals with reactivity to differentiation antigens.

Measurement of C3 conversion by ELISA estimation of neo-determinants on the C3d moiety

An ELISA assay estimating neo-determinants on the C3d moiety is described. The C3d neo-determinants were detected by incubating the sample on F(ab)2 anti-C3d-coated plates followed by development with biotin-labelled anti-C3d and enzyme-labelled avidin. The assay was compared with rocket immunoelectrophoresis (IE) and crossed IE. Serum from a factor I deficient patient showed no detectable C3d when analysed by rocket IE, but activation was evident when analysing by crossed IE or ELISA. The results suggest that the neo-determinants detected by ELISA become exposed when C3 is split into C3a and C3b and this interpretation was supported by kinetic analysis of C3 activation by the three methods.

Immunoscintigraphy of colon cancers with the human monoclonal antibody COU-1.

The human monoclonal immunoglobulin M (IgM) antibody, COU‐1 is obtained from a human‐human hybridoma, which is derived by fusion between a human B‐lymphoblastoid cell line and lymphocytes obtained from mesenteric lymph nodes from a patient with colorectal cancer. COU‐1 recognizes a 43 kilodaltons intracellulary located cytokeratin‐like protein, strongly expressed by adenocarcinoma tissue as compared to normal tissues. In tumor‐bearing nude mice, antibody COU‐1 labeled with 125I has been shown to accumulate in human colon cancer grafts when compared to human melanoma grafts and the normal mouse tissues. The observed accumulation was sufficient to be detected externally by immunoscintigraphy. Antigen‐binding fragments of the antibody were also prepared and were shown to accumulate in colon cancer grafts. Improved tumor to normal tissue ratio was seen with the half‐monomeric fragment, and the time required was reduced.

Semi-automatic analysis of proteins and protein complexes by automated enzyme immuno assay after separation by high-performance gel-permeation chromatography : Size distribution of C3—IgG complexes

Enzyme immunoassay (ELISA) was applied in the establishment of an immunological method for the characterization of column effluents. Serum samples, containing aggregated immunoglobulin (IgG), were separated by high-performance gel-permeation chromatography on a TSK column and fractions were collected directly in 96-well ELISA microplates. IgG was determined on anti-IgG-coated plates, followed by development with biotin-labelled anti-IgG and enzyme-labelled avidin. Complement factor C3 was determined on anti-C3-coated plates, developed with biotin-labelled anti-C3 and enzyme-labelled avidin. Complexes between IgG and complement factor C3 were determined on anti-IgG-coated plates, developed with biotin labelled anti-C3 antibody and enzyme-labelled avidin. Complex formation between C3 and IgG after incubation of serum with aggregated IgG was demonstrated. The methodology described is generally applicable to the analysis of biological components and is uniquely useful for the analysis of complexes between different components.

Tumour localization and pharmacokinetics of iodine-125 human monoclonal IgM antibody (COU-1) and its monomeric and half-monomeric fragments analysed in nude mice grafted with human tumour

Human monoclonal IgM antibodies reactive with cancer-associated antigens may not have the optimal imaging capability due to their large size. Fragmentation of human IgM is less than straight-forward due to the loss of immunoreactivity. From the human monoclonal IgM antibody COU-1 we have prepared monomeric and half-monomeric fragments, which retain the ability to bind to colon cancer cells in vitro. The pharmacokinetics and tumour localization were evaluated in nude mice bearing human colon adenocarcinoma and human melanoma grafts. Faster clearance from the circulation was seen for the smaller half-monomeric fragment with a half-life (rapid phase/slow phase) of 2 h/16 h compared with the intact antibody, 4 h/25 h, and the monomeric fragment, 3 h/27 h. Intact COU-1 as well as the fragments accumulated in the colon tumour graft. Higher amounts of radioactivity were found in the colon tumour as compared to normal organs for intact COU-I at days 4 and 6, for the monomeric fragment at day 4, and for the half-monomeric fragment at day 2 after injection. This investigation demonstrates the favourable biodistribution of the half monomeric COU-1 fragment. The fast clearance of this fragment resulted in a tumour-to-muscle ratio as high as 22 on day 2 after injection. Also, only this fragment gave a positive tumour-to-blood ratio. Normal IgM and its fragments were used as controls. Radioimmunoscintigraphy demonstrated the colon tumour discriminatory properties of each of the three iodine-labelled antibody preparations. The results compare favourably with previously reported investigations of the localization of human monoclonal antibodies and suggest that fragments of human monoclonal IgM antibodies may be useful tools for the immunodetection of cancer in patients.

Curing human hybridomas infected with Mycoplasma hyorhinis

Tiamuline and minocycline were evaluated for the treatment of an IgM producing human-human hybridoma cell line infected with Mycoplasma hyorhinis. Tiamuline was used at a concentration of 10 micrograms/ml culture medium and minocycline at a concentration of 5 micrograms/ml culture medium. Both antibiotics were found to eliminate mycoplasma infection over a treatment period of 3 weeks, and the hybridoma cell line remained mycoplasma-free for 6 months after treatment. Tiamuline had no effect on either cell growth or IgM secretion. Whereas treatment with minocycline alternated the cell proliferation and completely inhibited IgM secretion. This effect on cell function was found to be reversible since both cell growth and IgM secretion returned to normal 1 week after the minocycline had been removed. Tiamuline as well as minocycline may be recommended for the treatment of human hybridomas infected with mycoplasma.