Biomedical subjects

P Bischof

Publications and source records attributed to P Bischof.

At least 37 records · Page 2Linked to original sources

PubMed2000

Molecular mediators of implantation.

Because cytotrophoblastic cells (CTB) from first-trimester placenta form columns of invasive CTB they have been considered as a model for blastocyst implantation. This invasive behaviour is due to the ability of CTB to secret matrix metalloproteinases (MMPs) because tissue inhibitor of MMP (TIMP) inhibits their invasiveness. Although CTB behave like metastatic cells, in vivo they are only transiently invasive (first trimester) and their invasion is normally limited only to the endometrium and to the proximal third of the myometrium. This temporal and spatial regulation of trophoblast invasion is believed to be mediated in an autocrine way by trophoblastic factors and in a paracrine way by uterine factors. Several types of regulators have been investigated: hormones, extracellular matrix (ECM) glycoproteins and cytokines or growth factors. This review is not intended to be an exhaustive catalogue of all the potential regulators but is aimed at describing the mechanism of action of certain factors relevant in trophoblast-endometrial interactions.

Cytokines↗

PubMed2000

CA125 production by the peritoneum: in-vitro and in-vivo studies.

The source of CA125 synthesis is still debated. Endometrial, peritoneal, ovarian and amniotic cells have been demonstrated to produce and secrete CA125. Different studies show that the peritoneum is a source of CA125. The present study aimed at investigating in vivo and in vitro the peritoneal contribution to circulating CA125. Cultures of uterine peritoneum, abdominal peritoneum and myometrium explants were performed and CA125 measured in the culture medium. To modulate the potential production of CA125, the explants were cultured with or without cycloheximide, bacterial lipopolysaccharide (LPS) or ascitic fluid. In a prospective study, we compared a group of patients after abdominal surgery (n = 19; nine men, 10 women) with a group after extra-abdominal surgery (n = 21; 11 men, 10 women), in order to detect a postoperative increase of serum CA125. De-novo synthesis of CA125 could not be demonstrated in the cultures of uterine and abdominal peritoneum and in myometrium, but CA125 concentrations were detectable in the culture medium without being modulated by cycloheximide, LPS or ascitic fluid. After peritoneal surgery, the proportion of patients with increased serum CA125 was significantly higher (P < 0.03) after abdominal surgery as compared with extra-abdominal surgery. This is considered as indirect evidence for in-vivo production of CA125 by the peritoneum.

Abdomen↗

PubMed2000

Leptin modulates extracellular matrix molecules and metalloproteinases: possible implications for trophoblast invasion.

Leptin is a circulating hormone which plays an important role in the regulation of energy balance, haemopoiesis and reproduction. Leptin and its receptor (leptin-R) are localized in human placental tissue but their function is not known. In this study we have investigated the expression of leptin and leptin-R in the human placenta with particular attention to extravillous cytotrophoblastic cell islands and cell columns which play a pivotal role in trophoblast invasion and placental growth. We demonstrate that leptin-R immunoreactivity shows a strong expression in the distal extravillous cytotrophoblastic cells of cell columns invading the basal plate, whereas leptin expression is homogeneously expressed in all the cellular components of cell columns. Since the invasive ability of the distally located extravillous cytotrophoblast of cell columns is known to be regulated by a variety of proteases and some extracellular matrix molecules, we tested the influence of leptin on the in-vitro production of matrix metalloproteinase (MMP)-2, MMP-9 and fetal fibronectin (fFN) by cytotrophoblastic cells. We demonstrate that leptin increases, in a dose-dependent manner, the secretion of immunoreactive MMP-2 and fFN and enhances the activity of MMP-9 in cultured cytotrophoblastic cells. Our results suggest that leptin and leptin-R could have a role in the invasive processes of the extravillous cytotrophoblastic cells by modulating the expression of MMPs. In addition, these results provide a foundation for studying pathological conditions characterized by insufficient or excessive trophoblast invasion.

Carrier Proteins↗

PubMed2000

Expression of pro-EPIL peptides encoded by the insulin-like 4 (INSL4) gene in chromosomally abnormal pregnancies.

The recent development of a specific immunoassay based on monoclonal antibodies directed to chain C and chain A of early placenta insulin-like peptide (EPIL) encoded by the INSL4 gene, has made it possible to demonstrate pro-EPIL peptide expression during normal pregnancy. In the present study, we report on the expression of pro-EPIL peptides in chromosomally abnormal pregnancies, namely trisomy 21 and 18. EPIL peptide levels were measured in amniotic fluid (AF) and maternal serum (MS) from pregnancies with trisomy 21 (n=16) or 18 (n=14) and compared to levels detected in AF and MS from 33 chromosomally normal pregnancies between 12 and 32 weeks of gestation. Pro-EPIL peptide levels were significantly higher in amniotic fluids from T21 than in AF from chromosomally normal pregnancies (mean pro-EPIL levels +/- SEM, 449+/-129.2 ng/mL vs 137+/-29.6 ng/mL, P = 0.0195), whereas there was only a trend towards an increase in pro-EPIL peptide levels in maternal serum. In a limited matched gestational age range (15 to 17 weeks), it was confirmed that pro-EPIL peptide levels were significantly higher in AF from T21 pregnancies (644.0+/-155.9 ng/mL, n = 11) than in AF from normal pregnancies (177.8+/-39.0 ng/mL, n = 12; P < 0.0001). Interestingly, the expression patterns of pro-EPIL peptides, human chorionic gonadotropin (hCG) and its free subunits were parallel in T21 pregnancies as recently observed in normal pregnancies. These results are in line with previous observations suggesting that the biosynthesis of both hCG and EPIL follows common regulation pathways.

Adult↗

PubMed2000

Mechanisms of endometrial control of trophoblast invasion.

Tumour invasion and trophoblastic invasion share the same biochemical mediators: the matrix metalloproteinases (MMPs) and their inhibitors. MMPs are a family of enzymes capable of digesting the extracellular matrices of the host tissues. Human cytotrophoblastic cells are constitutively invasive and produce MMPs. Tissue inhibitors of metalloproteinases inhibit cytotrophoblastic invasion in vitro, indicating that MMPs are causally related to trophoblast invasion in the endometrium. In contrast to tumour invasion of a host tissue, trophoblastic invasion during implantation and placentation is controlled stringently in both space and time. The factors responsible for these important regulatory processes are unknown but studies in vitro indicate that endometrial cytokines and growth factors are possible candidates. Insulin-like growth factor binding protein 1, the major secretory product of the decidua, interleukin 1, interleukin 6, leptin and tumour necrosis factor alpha, all of endometrial origin, are stimulators of MMPs, whereas transforming growth factor beta inhibits the proteolytic activity of cytotrophoblastic cells. Unfortunately, the ways in which these individual factors interact to regulate trophoblast invasion are far from being understood.

Animals↗

PubMed2000

A putative role for oncogenes in trophoblast invasion?

Tumour invasion and trophoblastic invasion share the same biochemical mediators: the matrix metalloproteinases (MMP) and their inhibitors (TIMP). MMP are a family of enzymes capable of digesting the extracellular matrices of the host tissues. Human cytotrophoblastic cells are constitutively invasive and produce MMP. That MMP are causally related to trophoblast invasion in the endometrium is shown by the fact that TIMPs inhibit cytotrophoblastic invasion in vitro. In contrast to tumour invasion of a host tissue, trophoblastic invasion during implantation and placentation is stringently controlled both in space and time. The factors responsible for these important regulatory processes are unknown, but in-vitro studies point to autocrine (trophoblastic) and paracrine (endometrial) controls by cytokines and growth factors. These regulators exert their effects directly or indirectly by activating nuclear transcription factors. Transcription factors are proteins or protein complexes (often the products of oncogenes) that activate genes by binding to specific sites of the DNA located in the regulatory (5' flanking) region of genes. This review describes the different DNA binding domains of the MMP-9 gene, summarizes our knowledge about the transcription factors involved and speculates about a potential role of these transcription factors (particularly the oncogenes Jun and Fos) in regulating trophoblast invasion.

Base Sequence↗

PubMed1999

Evidence for infection of the human embryo with adeno-associated virus in pregnancy.

Previous reports have demonstrated the presence of DNA of the human helper virus-dependent adeno-associated parvovirus (AAV) in uterine tissue and curettage material from early miscarriage. To examine infection of embryonic tissue during pregnancy, amnion fluids were analysed for the presence of AAV. Using polymerase chain reaction, AAV DNA was detected in 64 out of 238 DNA samples extracted from amnion cells. DNA of helper viruses were found in 12% (papillomavirus) and 18% (cytomegalovirus) of the samples (double infections with AAV in eight and nine cases, respectively). Furthermore, infectious AAV virions were found in 13 out of 43 AAV DNA-containing samples. In mothers with AAV DNA-positive amnion fluids, premature amniorrhexis and premature labour occurred significantly more frequently (P < 0.001). Using an immunofluorescence assay, 24% of newborn sera (unrelated to the amnion fluid samples) were found to contain IgM antibodies to AAV, in most cases paralleled by IgM antibodies in the mother's sera. The data demonstrate that AAV infection can occur in utero at early and at late stages of pregnancy. The observed complications at delivery should encourage studies to clarify possible pathological consequences of AAV infection in pregnancy and a possible latent infection of the fetus.

Amniotic Fluid↗

PubMed1999

Effects of interleukin-6 (IL-6) on cytotrophoblastic cells.

Tumour invasion and trophoblastic invasion share the same biochemical mediators: the matrix metalloproteinases (MMP) and their inhibitors. In contrast to tumour invasion of a host tissue, trophoblastic invasion during implantation and placentation is stringently controlled both in tissue localization and developmental stage. The factors responsible for these important regulatory processes are unknown, but in-vitro studies point to endometrial cytokines and growth factors as possible candidates. Here we examined the possibility that interleukin-6 (IL-6), a trophoblastic and endometrial cytokine, represents such a regulatory factor. Purified first trimester cytotrophoblastic cells (CTB) were cultured for 4 days in presence or absence of increasing concentrations of IL-6. MMP-2 and MMP-9 bioactivity (zymography) and immunoactivity were measured in the culture supernatants together with total human chorionic gonadotrophin (HCG), fetal fibronectin (FFN) and leptin. IL-6 did not change the cytotrophoblastic secretion of FFN or total HCG. In contrast, this cytokine induced a dose-dependent stimulation of the leptin secretion and increased the activity, but not the immunoreactivity, of MMP-9 and MMP-2. These results indicate that IL-6 could be considered as an endometrio-trophoblastic regulator of cytotrophoblastic gelatinases.

Abortion, Induced↗

PubMed1999

Modulation of human cytotrophoblastic leptin secretion by interleukin-1alpha and 17beta-oestradiol and its effect on HCG secretion.

To investigate the role of leptin during pregnancy, we assessed leptin production by pure cultured human cytotrophoblastic cells (CTB), its regulation by cytokines and 17beta-oestradiol and its effects on human chorionic gonadotrophin (HCG) secretion. Purified CTB from first trimester placenta were incubated in duplicates in the presence or absence of cytokines or 17beta-oestradiol. Medium was harvested on day 2 and the culture stopped on day 4. Results were corrected for protein content of each individual well and expressed as percent of controls per day (mean +/- SEM). Basal CTB leptin production was 25.2 +/- 2.6 (ng/mg prot). In comparison with controls, leptin production was stimulated to 320 +/- 16% (P < 0.0001) and 195 +/- 3.2% (P < 0.0004) by 3 and 10 ng/ml of interleukin-1alpha respectively. 17beta-oestradiol 10(-6) to 10(-9) mol/l increased basal leptin production 5-9-fold, while 10(-5) mol/l had no such effect. Basal CTB HCG secretion was 5722 +/- 1055 (mIU/mg prot). There was a dose-dependent leptin-induced increase in HCG secretion (P = 0.0039) reaching a 5-fold increase with a leptin concentration of 1 microg/ml (P < 0.006). Gonadotrophin-releasing hormone (GnRH) 8.5 x 10(-8) mol/l significantly increased HCG secretion to 140 +/- 21% of controls (P = 0.031). Cetrorelix (0.1 microg/ml) inhibited leptin-induced HCG secretion (P = 0.0028).

Cells, Cultured↗

PubMed1999

Effects of tumour necrosis factor-alpha, interleukin-1 alpha, macrophage colony stimulating factor and transforming growth factor beta on trophoblastic matrix metalloproteinases.

The aim of this study was to determine the effects of tumour necrosis factor alpha (TNF), interleukin-1 alpha (IL-1alpha), macrophage colony-stimulating factor (MCSF) and transforming growth factor beta (TGFbeta) on the secretion of matrix metalloproteinases (MMP), human chorionic gonadotrophin (HCG) and fetal fibronectin (fFN) by purified first trimester cytotrophoblastic cells (CTB) in vitro. CTB were obtained from legal abortions and cultured in vitro in the presence or absence of the different cytokines. Secreted gelatinases were analysed in the culture supernatants by zymography, by measurements of the total gelatinolytic activity and by enzyme immunoassays. HCG and fFN were measured by commercially available immunoassays. TNF increased the total gelatinolytic activity by increasing MMP-9 activity (P = 0.025-0.0177) but decreased MMP-2 activity (P < 0.03) and immunoreactivity (P < 0.05), fFN (P < 0.02) and HCG (P < 0.01). IL-1alpha significantly increased the secretion of fFN (P < 0.02), the activity (P < 0.02) and immunoreactivity (P < 0.05) of MMP-9 but had no effect on the other parameters. MCSF increased MMP-9 immunoreactivity (P < 0.05) and moderately decreased HCG. TGFbeta inhibited total gelatinolytic activity, MMP-9 activity and immunoreactivity, but was without effect on MMP-2 concentrations and activity. TGFbeta decreased HCG (P < 0.041) and increased fFN (P < 0.042). Our results indicate that TGFbeta, TNF and IL-1alpha are important regulators of trophoblastic MMP secretion.

Cells, Cultured↗

PubMed1999

Production and characterization of monoclonal antibodies against pregnancy-associated plasma protein A.

Pregnancy-associated plasma protein A (PAPP-A) was found to be a good first trimester maternal serum marker, together with free beta-human chorionic gonadotrophin (HCG) subunits, for the biochemical screening of fetal trisomy 21 (Down's syndrome). We have raised monoclonal antibodies (mAbs) against PAPP-A purified from human pregnancy serum. The different antibodies were characterized biochemically by Western blot analysis and in terms of specificity (reaction with non-pregnant and male serum). Their performance in Down's syndrome screening was assessed in comparison with an existing enzyme-linked immunosorbent assay method after labelling of the different mAbs with biotin or horseradish peroxidase. A pair of mAbs was eventually chosen for a double-antibody sandwich protocol. The selected combination was found to have a significantly increased specificity (P = 0.0116) over the method using (purified) polyclonal antibodies, together with slightly increased sensitivity. In our limited number of Down's syndrome pregnancy samples (n = 17) and controls (n = 18), the medians as well as the multiples of the median values (for the affected cases) were comparable between the two methods described.

Antibodies, Monoclonal↗

PubMed1998

Invasion of cytotrophoblastic JEG-3 cells is stimulated by hCG in vitro.

Trophoblast invasion into the uterine wall is controlled by many factors. Previously, a human chorionic gonadotropin (hCG) receptor has been found to be expressed on invasive trophoblast as well as on choriocarcinoma cells implying a possible role for the hormone in trophoblast invasion. Therefore, this study examined the role of hCG in the invasion of trophoblastic (JEG-3) cells. Increasing hCG concentrations were applied in a trophoblast invasion model, JEG-3, through matrigel-coated filters. The proliferation was quantified by WST-1 cleavage assay. Cell migration was studied by examining the number of cells that had passed the uncoated porous (8-microm pore size) filters. After staining, filters were examined microscopically for cells on the underside of the membrane. A quantitative protease assay was also performed. Flow cytometric analysis of alpha5 and alpha6 integrin subunits, which are essential for interactions between cells and extracellular matrix, was performed. hCG increased significantly (P<0.01) the in vitro invasion of trophoblastic JEG-3 cells in a dose-dependent manner. Migration was also increased by hCG (P<0.01). However, cell proliferation remained unchanged. The second messenger analogue dibutyryl cAMP (db cAMP) and the cAMP elevating factor (forskolin) mimicked the effects of hCG by stimulating a dose-dependent increase of trophoblastic cell UEG-3) invasion. The collagenolytic activity of trophoblastic cells (EG-3) was increased by hCG stimulation. No changes were shown in the expression of alpha5 and alpha6 integrin subunits on JEG-3 cells. In vitro hCG is a regulatory factor of invasion and migration in trophoblastic JEG-3 cells, whereas proliferation is not influenced. The endogenous production of hCG by the trophoblast in vivo implies an autocrine control of invasion processes by hCG.

Bucladesine↗

PubMed1998

Effects of decidua-conditioned medium and insulin-like growth factor binding protein-1 on trophoblastic matrix metalloproteinases and their inhibitors.

The regulatory role of in vitro decidualized stromal cells (DESCM) and their main secretory product insulin-like growth factor binding protein-1 (IGFBP-1) was studied on the secretion of trophoblastic gelatinases and tissue inhibitor of metalloproteinase (TIMP-1). First trimester cytotrophoblastic cells (CTB) were obtained from abortions and cultured in vitro in presence or absence of DESCM or IGFBP-1. Secreted gelatinases were analysed in the culture supernatants by zymography and by measurements of the total gelatinolytic activity. TIMP-1, hCG, and fetal fibronectin (fFN) were measured by commercially available immunoassays. DESCM inhibited the total gelatinolytic activity of CTB but increased trophoblastic MMP-9, TIMP-1 and fFN. In contrast, IGFBP-1 increased the total gelatinolytic activity and TIMP-1, had no effect on MMP-2 , MMP-9 or fFN but inhibited hCG. It is concluded that a factor secreted by decidual cells inhibits the gelatinolytic property of trophoblast by increasing TIMP-1. Other decidual factors, as yet unidentified, increase MMP-2 and MMP-9 to an extent which does override the inhibitory effect of TIMP-1. Since in contrast to DESCM, IGFBP-1 increases the total gelatinolytic activity of CTB, it cannot be the primary active decidual factor regulating the proteolytic activity of CTB. The possibility of an integrin-mediated effect of IGFBP-1 on CTB is discussed.

Adult↗

PubMed1998

Involvement of trophoblast in embryo implantation: regulation by paracrine factors.

In order to investigate the regulatory role of only one endometrial cell type on trophoblastic invasion, we explored the effects of culture medium conditioned by in vitro decidualised stromal cells (DCM) and of insulin-like growth factor binding protein-1 (IGFBP-1, the main secretory product of decidual cells) on the trophoblastic secretion of gelatinases and tissue inhibitor of metalloproteinases (TIMP-1). First trimester cytotrophoblastic cells (CTB) were obtained from abortions and cultured in vitro in presence or absence of DCM or IGFBP-1. Secreted gelatinases were analysed in the culture supernatants by zymography and by measurements of the total gelatinolytic activity. Tissue inhibitor of metalloproteinases (TIMP-1) was measured by a commercially available immunoassay. DCM inhibited the total gelatinolytic activity of CTB but increased trophoblastic MMP-9 and TIMP-1. In contrast, IGFBP-1 increased the total gelatinolytic activity and TIMP-1 and had no effect on MMP-2 and MMP-9. We conclude that a factor secreted by decidual cells (possibly TGFbeta) inhibits the total gelatinolytic activity of trophoblast by increasing TIMP-1 but other factors, as yet unidentified, increase MMP-2 and MMP-9 to an extent which does not shift the equilibrium between the gelatinases and TIMP-1 in favour of the gelatinases. In contrast to DCM, IGFBP-1 increases the total gelatinolytic activity probably by stimulating another gelatinase (stromelysin-1?) as MMP-2 and MMP 9 are unchanged by IGFBP-1. The possibility of an integrin mediated effect of IGFBP-1 on CTB is discussed.

Collagenases↗

PubMed1998

The apparent late half-life of human chorionic gonadotropin (hCG) after surgical treatment for ectopic pregnancy. A new approach to diagnose persistent trophoblastic activity.

OBJECTIVE: The disappearance kinetic of human chorionic gonadotropin (hCG) follows a biexponential decay with a rapid initial fall followed later by a slow disappearance. This kinetic is characterised by two half-lives: an early and a late. The objective of this study was to determine if and which half-life could be used clinically to detect persistent trophoblast after conservative surgery in patients with ectopic pregnancy. DESIGN: Retrospective analysis of patients having undergone salpingostomy by laparoscopy for an ectopic tubal pregnancy between January 1990 and October 1993. SETTING: Gynaecology Department of an University Hospital. PATIENTS: 104 women with diagnosed tubal ectopic pregnancy were treated by salpingostomy performed under laparoscopy. In seven cases, persistent trophoblast was diagnosed on the basis of plateauing or increasing peripheral hCG values. MAIN RESULTS: From the individual disappearance curves of hCG we calculated the early half-life (early T0.5, from samples obtained between 0 and 48 h postsurgery) and the late half-life (late T0.5, from samples obtained between 2 and 7 days postsurgery). Late T0.5 but not early T0.5 were significantly (P<0.0001 and P=0.416 respectively) longer in women (n =7) in whom a persistent trophoblast was diagnosed. Early T0.5 was dependant on the preoperative value of hCG, whereas late T0.5 was independent. We propose to use late T0.5 as a parameter to follow ectopic pregnancies after treatment.

Chorionic Gonadotropin↗

PubMed1998

Triptorelin acetate administration in early pregnancy: case reports and review of the literature.

When using a long protocol with cycle day 23 gonadotrophin-releasing hormone agonists (GnRH-a) administration, an elevated estradiol level or a missed period 10-14 days after initiating pituitary downregulation should alert the physician to the possibility of a pregnancy. We report 4 pregnancies occurring during pituitary downregulation with Triptorelin acetate in 366 in-vitro fertilization (IVF) cycles resulting in 3 deliveries of 4 normal neonates at term and 1 first trimester abortion. This supports published data reporting a 1% spontaneous pregnancy incidence in women undergoing pituitary desensitization GnRH-a during the luteal phase prior to planned IVF treatment, a 15.9% abortion rate and a 1.7% malformation rate. Our cases together with other published data suggest that pregnancy outcome is not adversely affected by GnRH-a administration during the luteal phase of the conception cycle. However, long term follow-up of these babies is still lacking and the number of reported cases is too small adequately to rule out the possibility of any detrimental effect related GnRH-a administration in pregnancy.

Abortion, Spontaneous↗

PubMed1998

Second-trimester maternal serum screening for Down's syndrome: free beta-human chorionic gonadotrophin (HCG) and alpha-fetoprotein, with or without unconjugated oestriol, compared with total HCG, alpha-fetoprotein and unconjugated oestriol.

The aim of our study was to compare three protocols for second-trimester maternal serum screening for Down's syndrome in the same serum samples, using two triple tests [total human chorionic gonadotrophin (HCG), alpha-fetoprotein, unconjugated oestriol; and free beta-HCG, alpha-fetoprotein, unconjugated oestriol] and a double test (free beta-HCG and alpha-fetoprotein). The three protocols were compared in a series of 23 serum samples from Down's syndrome pregnancies and in a cohort of 2516 pregnant women receiving routine antenatal care between June 1992 and June 1993. Among the 23 affected cases, at a cut-off risk of 1:380, the detection rate of Down's syndrome was comparable with the double test (74%; 17/23) and the triple tests (65%; 15/23) (not significantly different). At the same cut-off risk, in the cohort of 2516 pregnant women screened between 15 and 18 weeks gestation, both protocols using free beta-HCG achieved a significant reduction of the number of false positive cases (P = 0.013 and 0.004 for double and triple tests respectively). We conclude that, compared to total HCG, alpha-fetoprotein and unconjugated oestriol, use of free beta-HCG and alpha-fetoprotein represents a better second-trimester screening test for Down's syndrome, because it significantly decreases the false positive rate at a lower running cost. The addition of unconjugated oestriol to the double test adds no further advantage.

Adult↗

PubMed1998

The rate of development and time of transfer play different roles in influencing the viability of human blastocysts.

Improved embryo culture protocols now render more feasible the possibility of obtaining human blastocysts after in-vitro fertilization. In this study we present: (i) results of blastocyst development from supernumerary embryos after co-culture on green monkey kidney epithelial cells and (ii) pregnancy rates after transfer of frozen blastocysts. In addition, we have examined the influence of the day of blastocyst freezing and the day of transfer after the luteinizing hormone (LH) peak on pregnancy and implantation rates. Of 423 supernumerary embryos, 200 developed to the blastocyst stage (47.3%). By days 5 and 6, 67% of the blastocysts had reached the blastocyst stage, and were frozen, compared to 28.5% by day 7. When we compared the cases where only blastocysts frozen on days 5 and 6 were transferred compared to those frozen and transferred on or after day 7 the pregnancy rates were 7/18 (38.9%) and 1/16 (6.2%) respectively. In contrast, when we examined the influence of the day of transfer we found that pregnancies were established from day 5 up to day 9 post LH peak. Based on these results, we suggest that every attempt should be made to increase the development rate of supernumerary embryos to the blastocyst stage, as it appears that the quality of blastocysts transferred, as shown in this study by rate of development, plays a more crucial role than the timing of transfer.

Animals↗