Randomized, assessor-blinded trial comparing highly purified human menotropin and recombinant follicle-stimulating hormone in high responders undergoing intracytoplasmic sperm injection.

OBJECTIVE: To evaluate the efficacy and safety of highly purified human menotropin (HP-hMG) and recombinant follicle-stimulating hormone (rFSH) for controlled ovarian stimulation in a population of patients predicted to be high responders. DESIGN: Randomized, open-label, assessor-blinded, parallel-group, noninferiority trial. SETTING: Fertility centers. PATIENT(S): A total of 620 women with serum antimüllerian hormone (AMH) ≥5 ng/mL. INTERVENTION(S): Controlled ovarian stimulation with HP-hMG or rFSH in a GnRH antagonist assisted reproductive technology (ART) cycle. Fresh transfer of a single blastocyst was performed unless ovarian response was excessive, in which all embryos were cryopreserved. Subjects could undergo subsequent frozen blastocyst transfer within 6 months of randomization. MAIN OUTCOME MEASURE(S): Ongoing pregnancy rate (OPR) after fresh transfer (primary endpoint), as well as cumulative live birth, ovarian hyperstimulation syndrome (OHSS), and pregnancy loss rates. RESULTS: OPR/cycle start after fresh transfer was 35.5% with HP-hMG and 30.7% with rFSH (difference: 4.7%, 95% CI -2.7%, 12.1%); noninferiority was established. Compared to rFSH, HP-hMG was associated with significantly lower OHSS (21.4% vs. 9.7% respectively; difference: -11.7%, 95% CI -17.3%, -6.1%) and cumulative early pregnancy loss rates (25.5% vs. 14.5% respectively; difference: -11.0%, 95% CI -18.8%, -3.14%). Despite 43 more transfers in the rFSH group, cumulative live birth rates were similar with HP-hMG and rFSH at 50.6% and 51.5% respectively (difference: -0.8%, 95% CI -8.7%, 7.1%). CONCLUSION(S): In high responders, HP-hMG provided comparable efficacy to rFSH with fewer adverse events, including pregnancy loss, suggesting its optimized risk/benefit profile in this population. CLINICAL TRIAL REGISTRATION NUMBER: NCT02554279 (clinicaltrials.gov).

Comparison of Human Oocyte Activation Between Round-Headed Sperm Injection Followed by Calcium Ionophore Treatment and Normal Sperm Injection in a Patient With Globozoospermia.

Fertilization failure is common in patients with round-headed sperm, a form of globozoospermia. Artificial oocyte activation is able to assist oocyte fertilization after sperm injection in these patients. Comparisons between oocyte fertilization with or without calcium ionophore have been reported in patients with round-headed sperm. However, no comparison has been reported between round-headed sperm injection followed by calcium ionophone activation and normal sperm injection. In this case report, half of oocytes from a patient were injected with her partner's round-headed sperm followed by calcium ionophore activation, and the other half of oocytes were injected with a donor sperm without calcium ionophore activation. The injected oocytes were cultured to examine fertilization, embryo development, and embryonic aneuploidies in the resulting blastocysts. The fertilization rate was lower in round-headed sperm injected oocytes (3/6) than that in donor sperm injected oocytes (5/6), but rates of blastocyst and aneuploidies were similar in the resulting embryos between the two groups. A euploid blastocyst resulted from round-headed sperm injection was transferred, and a healthy baby was delivered. These results indicate that calcium ionophore treatment can assist oocyte activation in patients with round-headed sperm, but its efficiency to activate oocytes is lower than that induced by a normal sperm injection. However, embryo development and chromosome integrity may not be affected by calcium ionophore treatment.

Cetrorelix lowers premature luteinization rate in gonadotropin ovulation induction-intrauterine insemination cycles: a randomized-controlled clinical trial.

Attempting to compare the rates of premature luteinization (PL), clinical pregnancy, and cycle cancellation in ovulation induction-intrauterine insemination (OI-IUI) cycles with and without the GnRH antagonist, cetrorelix, a randomized-controlled trial was undertaken in which patients were randomized to one of two OI-IUI protocols. Those in the cetrorelix arm showed a significantly reduced rate of PL and no change in clinical pregnancy or cycle cancellation rate, leading to the conclusion that GnRH antagonists can decrease the rate of PL, but appear to have no effect on pregnancy or cycle cancellation in gonadotropin OI-IUI cycles.

Imatinib decreases endometrial stromal cell transmesothial migration and proliferation in the extracellular matrix of modeled peritoneum.

OBJECTIVE: To characterize imatinib's effect on endometrial stromal cell (ESC) attachment, proliferation, and invasion in modeled peritoneum. DESIGN: In vitro study. SETTING: Academic medical center. PATIENT(S): Twelve normally cycling women. INTERVENTION(S): Imatinib treatment in ESCs from women without endometriosis. MAIN OUTCOME MEASURE(S): Rate of ESC attachment, proliferation, and invasion. RESULT(S): Imatinib treatment at 10 µM had no effect on ESC attachment. Treatment with 0.5 µM, 2 µM, and 10 µM of imatinib reduced ESC proliferation by 30%, 72%, and 76%, respectively. The 0.1 µM dose of imatinib had no effect on proliferation. Treatment with 5 µM and 10 µM of imatinib reduced ESC invasion by 30% and 73%, respectively. The 2 µM dose had no effect on invasion. CONCLUSION(S): Imatinib treatment reduces ESC proliferation and invasion in modeled peritoneum without altering attachment. Imatinib may have a therapeutic role in endometriosis treatment.

A potential role for colony-stimulating factor 1 in the genesis of the early endometriotic lesion.

OBJECTIVE: To investigate the role(s) of colony-stimulating factor 1 (CSF-1) on the development of early endometriosis in a murine model by comparing rate of lesion formation in mice [1] homozygous for a CSF-1 mutation versus syngeneic controls and [2] after treatment with imatinib, a commercially available tyrosine kinase inhibitor that alters interaction(s) between CSF-1 and its receptor, c-fms. DESIGN: Prospective, placebo-controlled animal study. SETTING: Academic medical center. ANIMALS: Six- to 8-week old female FVB, wild-type C57BL/6, and CSF-1 op/op mice. INTERVENTION(S): Endometrial tissue from donor mice was used to induce endometriosis in murine recipients. In some experiments, mice homozygous for a CSF-1 mutation (CSF-1 op/op) were donors or recipients. In other experiments, donor and/or recipient mice received imatinib. MAIN OUTCOME MEASURE(S): Histologic confirmation of endometriosis, rate of lesion formation. RESULT(S): By 40 hours, recipient mice developed a mean of 7.2 +/- 0.9 endometriotic lesions that had invaded host surfaces, and mesothelial cells had proliferated over the entire surface of the implants. The CSF-1 op/op mice developed significantly fewer (mean 0.9 +/- 0.3) endometriotic lesions versus syngeneic controls. Imatinib treatment resulted in significantly fewer lesions when compared with sham-treated controls. CONCLUSION(S): Colony-stimulating factor 1 has a role in establishing early endometriotic lesions. Agents targeting CSF-1 or its actions have therapeutic potential for treating endometriosis.

Modeling the early endometriotic lesion: mesothelium-endometrial cell co-culture increases endometrial invasion and alters mesothelial and endometrial gene transcription.

OBJECTIVE: To determine the role of peritoneal mesothelial cells (PMCs) in the process of endometrial invasion into the peritoneum and to evaluate gene expression after endometrial-PMC co-culture. DESIGN: In vitro study. SETTING: University laboratory. PATIENT(S): Reproductive-age women without endometriosis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The rate of endometrial invasion through modeled peritoneum in the presence and absence of PMCs was evaluated. The influence of endometrial-PMC attachment on the expression of target genes, implicated in the pathogenesis of endometriosis, was examined by using reverse transcription polymerase chain reaction. RESULT(S): Endometrial stromal cell (ESC) invasion through invasion chambers coated with Matrigel (MTGL) and with growth factor-reduced Matrigel (GFR-MTGL) was increased 10-fold when a PMC monolayer was present. Endometrial epithelioid cell (EM42) invasion increased greater than threefold through the MTGL and GFR-MTGL-coated membranes when a PMC monolayer was present. Endometrial stromal cell, EM42, and PMC transcription of extracellular signal-related kinase, colony stimulating factor-1, c-fms, and c-Met was increased after endometrial-PMC attachment. Similar changes were not seen when endometrial cells were exposed to PMC-conditioned media and when PMCs were exposed to endometrial cell conditioned media. CONCLUSION(S): Peritoneal mesothelial cells increased invasion of ESCs and EM42s through modeled peritoneum. Endometrial-PMC co-culture led to alterations in gene transcription by endometrial cells and PMCs. This study suggests that PMCs contribute to the process of endometrial invasion into the peritoneum.

Prior fertility in the male partner does not predict a normal semen analysis.

A history of male fertility is not an accurate predictor of a normal semen analysis result. The semen analysis should remain part of the evaluation of the infertile couple even in cases where a history of male fertility is reported.

A novel in vitro model of the early endometriotic lesion demonstrates that attachment of endometrial cells to mesothelial cells is dependent on the source of endometrial cells.

OBJECTIVE: To characterize the source of variability in endometrial stromal cell (ESC) binding to peritoneal mesothelial cells (PMC). DESIGN: In vitro study. SETTING: University medical center. PATIENT(S): Reproductive-age women without endometriosis undergoing surgery for benign conditions. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Binding of ESCs (n = 9) to PMCs collected from the anterior abdominal wall (AAW) (n = 5), a commercially available mesothelial cell line (LP9) (three different passages) and normal ovarian surface epithelium (NOSE) (n = 5). RESULT(S): There were no differences in the binding of same-source ESCs to mesothelial cells obtained from the AAW of different women, to different passages of LP9s or to NOSE of different women. There was a trend toward increased binding of ESCs to NOSE compared to AAW PMCs. In contrast, there were significant differences in the ability of ESCs obtained from different women to bind to same-source PMCs. CONCLUSION(S): There is significant variability in ESC binding to PMCs. This variation is dependent primarily on the source of the ESCs. The ESC binding to LP9 PMCs was similar to AAW PMCs and NOSE.

Cell adhesion molecules and endometriosis.

The pathogenesis of endometriosis remains poorly defined. The interaction of endometrium with peritoneum is an important aspect of the disease process. Cell adhesion molecules (CAMs) are transmembrane receptors that facilitate intercellular binding and cellular interaction with the extracellular matrix (ECM). CAMs and components of the ECM are divided into large families based on sequence homology and similarity of tertiary structures. The function of eutopic and ectopic endometrial CAMs has been a focus of recent studies concerning the pathogenesis of endometriosis. Specific alterations in endometrial and peritoneal CAMs could facilitate binding of reflux menstruated endometrium at ectopic sites. In addition, the expression of CAMs by endometriotic lesions has been investigated to help understand mechanisms involved in the maintenance of endometrial tissue in ectopic locations. An understanding of the mechanisms involved in the interaction of endometrium with peritoneal tissues may provide new strategies to prevent endometriotic implants from forming and help treat existing lesions.

Pathogenesis of endometriosis--current research.

Proliferative, secretory and menstrual endometrial cells of both the stroma and epithelium adhere to intact peritoneal mesothelium and mesothelial monolayers. Endometrial attachment to the mesothelium appears to occur rapidly (within 1 h) and transmesothelial invasion occurs between 1 and 18-24 h. These results demonstrate that the mesothelium is not a 'no-stick' surface and indicates that molecules present at the surface of the mesothelium are involved in the pathogenesis of the early endometriotic lesion. The inhibition of endometrial cell adherence to peritoneal mesothelium by hyaluronidase indicates that CD44-hyaluronan binding is at least one of the mechanisms involved in the pathogenesis of endometriosis. We believe that investigation of mesothelial cell adhesion molecules is central to understanding the pathogenesis of endometriosis.

Time series analysis of transmesothelial invasion by endometrial stromal and epithelial cells using three-dimensional confocal microscopy.

OBJECTIVE: To evaluate endometrial adhesion and invasion of peritoneal mesothelium. DESIGN: Descriptive study using confocal laser-scanning microscopy. SETTING: University-based laboratory. PATIENT(S): Women undergoing surgery for benign conditions. INTERVENTION(S): Fluorescence-labeled peritoneal mesothelial cells (PMCs) were grown on coverslips. Fluorescence-labeled endometrial stromal cells (ESCs) and epithelial cells (EECs) and myometrial cells (Myos) were plated on the PMCs. Cultures were examined at 1, 6, 12, and 24-27 hours with differential interference contrast and confocal laser-scanning microscopy. MAIN OUTCOME MEASURE(S): Demonstration of adherence and invasion of endometrial cells through peritoneal mesothelium. RESULT(S): At 1 hour, there was adherence of the ESCs, EECs, and Myos on the perimeter of PMCs. There was no invasion by the Myos. By 6 hours, ESCs and EECs spread over the surface of the PMCs and extended cell processes through PMC junctions. Extension of pseudopodia under the PMCs followed. By 12 hours, there was vacuolization and lifting of PMCs that had been undermined by endometrial cells. CONCLUSION(S): This is the first time-phase study to demonstrate adherence and the process of invasion of endometrial cells through the mesothelium. The application of three-dimensional confocal laser-scanning microscopy is a novel technique that can be used to further examine mechanisms involved in the pathogenesis of the early endometriotic lesion.

Culture of menstrual endometrium with peritoneal explants and mesothelial monolayers confirms attachment to intact mesothelial cells.

BACKGROUND: To evaluate adhesion of menstrual endometrium (ME) to intact peritoneal mesothelium. METHODS: Explants of peritoneum were cultured for 1 h with ME (n = 6). Specimens were serially sectioned for haematoxylin and eosin stain and immunohistochemistry using an anti-cytokeratin antibody to label mesothelium. Confocal laser scanning microscopy (CLSM) was performed to identify an intact layer of mesothelial cells (MC) underlying sites of ME attachment. Also, ME and MC were labelled with Cell-Tracker dyes. ME was cultured with mesothelial monolayers for 1 h (n = 10). Cultures were examined with differential interference contrast and CLSM. Optical sections were taken and a three-dimensional model was constructed. RESULTS: In the peritoneal explants, ME adhered to intact mesothelium. There was no evidence of transmesothelial invasion. CLSM of sections of the explants demonstrated an intact monolayer of cytokeratin positive cells below the sites of ME implantation. Cytokeratin negative and positive ME cells adhered to mesothelial cells. Likewise, the ME attached to cultured mesothelium. Orthogonal sections and three-dimensional reconstruction confirmed an intact monolayer of mesothelium underlying ME attachment sites. CONCLUSIONS: This study confirms that ME adheres rapidly to intact peritoneal mesothelium. Further studies are needed that characterize the mechanisms of ME adhesion to, and migration through, mesothelial cells.

The alpha(2)beta(1) and alpha(3)beta(1) integrins do not mediate attachment of endometrial cells to peritoneal mesothelium.

OBJECTIVE: To evaluate the possible role of mesothelial alpha(2)beta(1) and alpha(3)beta(1) integrins in the attachment of endometrial stromal cells (ESCs) and endometrial epithelial cells (EECs). DESIGN: In vitro study. SETTING: University medical center. PATIENT(S): Women of reproductive age (n = 26). MAIN OUTCOME MEASURE(S): Mesothelial cells were grown on collagen IV. Endometrial stromal cells and EECs were plated on mesothelial cells for 1 hour. Before plating, mesothelial cells or endometrial cells were incubated with antibodies to alpha2, alpha3, and beta1 integrin subunits. The effect of these antibodies on ESC and EEC binding to collagen IV and collagen I was also examined. The expression of collagen I, collagen IV, fibronectin, and laminin by cultured ESCs and EECs was examined. RESULT(S): The anti-integrin antibodies had no effect on endometrial binding to mesothelium. The beta1 integrin antibody decreased binding of ESCs and EECs to the collagen matrices. In culture, ESCs and EECs expressed collagen I, collagen IV, fibronectin, and laminin to varying degrees. CONCLUSION(S): The initial adhesion of ESCs and EECs to mesothelium is not mediated by beta1 integrins. In contrast, ESC and EEC attachment to collagen IV and collagen I, which are present in the submesothelial extracellular matrix, is mediated by beta1 integrins.

An in vitro model to study the pathogenesis of the early endometriosis lesion.

OBJECTIVE: To determine if whole fragments of endometrium can adhere to peritoneum with intact mesothelium. DESIGN: Tissue culture and immunohistochemical study. SETTING: University Medical Center. PATIENTS: Reproductive-age women undergoing surgery for benign conditions. INTERVENTIONS: Whole explants of human peritoneum from the anterior abdominal wall and the posterior surface of the uterus were cultured with whole fragments of mechanically dispersed endometrium. MAIN OUTCOME MEASURES: Adhesion of endometrial fragments to the surface of the peritoneum was evaluated. Adherent fragments of endometrium were identified using the dissecting microscope and by performing serial sections of the peritoneum explants for light and confocal laser-scanning microscopy. Immunohistochemical staining of the mesothelium with antibodies to cytokeratin and vimentin was used to ensure an intact layer of mesothelium beneath the endometrial implants. Transmission electron microscopy was also used to evaluate the adhesion of endometrium to the mesothelium. RESULTS: Endometrium was identified attached to the surface of the peritoneum. After 18-24 hours of culture, the majority of implants did not have identifiable mesothelium beneath them, but most had intact mesothelium running up to the point of attachment. Approximately 10% of the endometrial implants had intact mesothelium at the site of attachment. After 1 hour of culture, both endometrial stromal and epithelial cells were attached to intact mesothelium in nearly all cases. Early transmesothelial invasion involves endometrial stromal cells. CONCLUSIONS: Endometrial stromal and epithelial cells can attach to the intact mesothelial surface of the peritoneum. Endometrial stromal cell invasion through the mesothelium occurs in less than 18-24 hours.

Endometriosis and infertility: is there a cause and effect relationship?

An association between endometriosis and infertility has long been noted. Endometriosis affects approximately 5% of the general population. In infertile women, the prevalence may be as high as 30%. Multiple studies, the majority of which are retrospective, indicate that the monthly fecundity of patients with endometriosis may be decreased by half compared to women without the disease. The precise cause-effect relationship between endometriosis and infertility remains controversial. In advanced cases of endometriosis, with distorted pelvic anatomy, the mechanism of infertility is more easily explained. Recent evidence suggests that treatment of early-stage endometriosis may increase pregnancy rates. Many etiologies of infertility in early-stage endometriosis have been proposed. These include endocrine dysfunctions such as luteal phase defect and luteinized unruptured follicle syndrome. In the last 15 years, alterations in the local pelvic immune environment have been the subject of multiple basic science investigations. Unfortunately, there is no satisfactory hypothesis that unequivocally explains the association of early stages of endometriosis with infertility.

Pathogenesis of endometriosis.

Various theories have been promulgated to explain the pathogenesis of endometriosis. Interest in the genesis of the endometriotic lesion has been a focus since the earliest investigations. More recently, investigators have addressed aspects of the immune system and local peritoneal factors that may be involved with both the histogenesis of endometriosis as well as its sequelae. This review will consider evidence for different theories of histogenesis and will discuss our current understanding of the contribution of the immune system to the etiology of endometriosis. Data will be presented regarding recently described models of the early endometriotic lesion. The interaction of endometrial cells with the peritoneal mesothelium seems critical to our understanding the formation of the early endometriotic lesion. Evidence of rapid transmesothelial migration and invasion of the peritoneum will be considered. As well, candidate adhesion molecules that may facilitate the initial binding of endometrium to the peritoneum will be discussed.