Amihai Barash

Local injury to the endometrium doubles the incidence of successful pregnancies in patients undergoing in vitro fertilization

OBJECTIVE Exploration of the possibility that local injury of the endometrium increases the incidence of implantation. DESIGN Prospective study. SETTING Clinical IVF unit. PATIENT(S) A group of 134 patients, defined as good responders to hormonal stimulation, who failed to conceive during one or more cycles of IVF and embryo transfer (ET). INTERVENTION(S) The IVF treatment and ET were preceded by repeated endometrial biopsies, in a randomly selected 45 of a total of 134 patients. MAIN OUTCOME MEASURES Outcome of IVF-ET treatments. RESULT(S) Transfer of a similar number of embryos (3.4 +/- 1.0 and 3.1 +/- 0.9 in the experimental and control patients, respectively) resulted in rates of implantation (27.7% vs. 14.2%, P =.00011), clinical pregnancy (66.7% vs. 30.3%, P =.00009), and live births per ET (48.9% vs. 22.5%, P =.016) that were more than twofold higher in the experimental group as compared to controls. CONCLUSION(S) These results suggest that IVF treatment that is preceded by endometrial biopsy doubles the chance for a take-home baby.

Local injury of the endometrium induces an inflammatory response that promotes successful implantation

OBJECTIVE To study whether an injury-induced inflammation might be the mechanism underlying the favorable effect of endometrial biopsy on the implantation rate in in vitro fertilization (IVF) patients. DESIGN Controlled clinical study. SETTING A medical center IVF unit and a research institute. PATIENT(S) Women undergoing IVF who had previous failed treatment cycles. INTERVENTION(S) Endometrial samples were collected from two groups of patients on day 21 of their spontaneous menstrual cycle. The experimental, but not the control group underwent prior biopsy treatment on days 8 or/and 11 to 13 of that same cycle. MAIN OUTCOME MEASURE(S) Abundance of immune cells, cytokines/chemokines level, correlation between these parameters and pregnancy outcome. RESULT(S) A statistically significantly higher amount of macrophages/dendritic cells (HLA-DR+ CD11c+ cells) and elevated proinflammatory cytokines, tumor necrosis factor-α (TNF-α), growth-regulated oncogene-α (GRO-α), interleukin-15 (IL-15), and macrophage inflammatory protein 1B (MIP-1B), were detected in day-21 endometrial samples of the experimental group. A direct stimulatory effect of TNF-α on MIP-1B, GRO-α, and IL-15 messenger RNA (mRNA) expression was demonstrated. A positive correlation was found between the levels of macrophages/dendritic cells, MIP-1B expression, and TNF-α expression and the pregnancy outcome. CONCLUSION(S) A biopsy-induced inflammatory response may facilitate the preparation of the endometrium for implantation. Increased MIP-1B expression could possibly serve for prediction of implantation competence.

Endometrial biopsy-induced gene modulation: first evidence for the expression of bladder-transmembranal uroplakin Ib in human endometrium

OBJECTIVE To explore the possibility that endometrial injury modulates the expression of specific genes that may increase uterine receptivity. DESIGN Controlled clinical study. SETTING Clinical IVF unit and academic research center. PATIENT(S) IVF patients with 28- to 30-day menstrual cycles. INTERVENTION(S) Endometrial biopsies from two groups of patients were collected on days 20-21 of their spontaneous menstrual cycle. The experimental, but not the control, group underwent biopsies on days 11-13 and 21-24 of their preceding cycle. MAIN OUTCOME MEASURE(S) Global endometrial gene expression and specific analysis of uroplakin Ib (UPIb) mRNA level throughout the menstrual cycle. RESULT(S) Local injury modulated the expression of a wide variety of genes. One of the prominently up-regulated genes was the bladder transmembranal protein, UPIb, whose expression by the endometrium is shown here for the first time. Endometrial UPIb mRNA increases after biopsy in the same cycle wct 2with an additional elevation in the following cycle. Immunohistochemical analysis localized the UPIb protein to the glandular-epithelial cells. Genes encoding other membrane proteins such as adipose differentiation-related protein and mucin 1, transmembrane, were also up-regulated. CONCLUSION(S) The biopsy-induced increase in the expression of UPIb and other genes encoding membrane proteins supports the possible importance of the membrane structure and stability during implantation. The specific role of UPIb in uterine receptivity should be elucidated.

Connexin43 in Rat Oocytes: Developmental Modulation of Its Phosphorylation

Abstract It is well established that the 43-kDa connexin (Cx43) is predominantly expressed by ovarian somatic cells, whereas the identity of the connexins contributed by the oocyte to form gap junctions with its neighboring cells is not fully elucidated. Our study aimed to examine oocytes for the expression and regulation of Cx43 throughout oogenesis. Growing and fully grown rat oocytes that were meiotically incompetent and competent, respectively, were examined. Fully grown oocytes were analyzed either before or after reinitiation of meiosis as well as at the second meiotic metaphase. Immunofluorescent analysis of zona pellucida-free oocytes using conventional and confocal microscopy demonstrated a characteristic pattern of punctuated staining of Cx43 on the oolema. Immunogold electron microscopy localized Cx43 to the oocyte surface and the microvillar processes. Reverse transcriptase-polymerase chain reaction and Western blot analysis revealed similar amounts of Cx43 gene and protein in oocytes of different developmental stages. However, a relative increase in the phosphorylated forms of the protein was observed in fully grown oocytes that had completed their maturation. Our findings demonstrate that rat oocytes express a developmentally regulated Cx43. They further suggest that homotypic gap junctions that consist of Cx43 may be present between rat oocytes and their adjacent cumulus cells.

Biopsy-induced inflammatory conditions improve endometrial receptivity: the mechanism of action

A decade ago, we first reported that endometrial biopsy significantly improves the success of pregnancy in IVF patients with recurrent implantation failure, an observation that was later confirmed by others. Recently, we have demonstrated that this treatment elevated the levels of endometrial pro-inflammatory cytokines and increased the abundance of macrophages (Mac) and dendritic cells (DCs). We therefore hypothesised that the biopsy-related successful pregnancy is secondary to an inflammatory response, and aimed at deciphering its mechanism of action. Supporting our hypothesis, we found that the pro-inflammatory TNFα stimulated primary endometrial stromal cells to express cytokines that attracted monocytes and induced their differentiation into DCs. These monocyte-derived DCs stimulated endometrial epithelial cells to express the adhesive molecule SPP1 (osteopontin (OPN)) and its receptors ITGB3 and CD44, whereas MUC16, which interferes with adhesion, was downregulated. Other implantation-associated genes, such as CHST2, CCL4 (MIP1B) and GROA, were upregulated by monocyte-derived Mac. These findings suggest that uterine receptivity is mediated by the expression of molecules associated with inflammation. Such an inflammatory milieu is not generated in some IVF patients with recurrent implantation failure in the absence of local injury provoked by the biopsy treatment.

Temporal analysis of connexin43 protein and gene expression throughout the menstrual cycle in human endometrium.

OBJECTIVE To analyze the pattern of connexin43 gene and protein expression in human endometrium throughout the menstrual cycle. DESIGN Controlled clinical study. SETTING An academic research center. PATIENT(S) Women with 28-day menstrual cycles who had mechanical infertility and failed to conceive after IVF treatment. INTERVENTION(S) Endometrial and blood samples were collected on days 8, 12, 14, 21, and 25 of spontaneous menstrual cycles. MAIN OUTCOME MEASURE(S) Endometrial expression of connexin43 protein and messenger RNA, endometrial thickness, and serum concentrations of gonadotropins and steroids. RESULT(S) The expression of connexin43 gene and protein decreased on day 12 and day 14 of the menstrual cycle and then increased on day 21 and day 25, respectively. A serum LH surge accompanied by a peak in the FSH concentration was observed on days 12-14. The progesterone concentration increased on days 21-25, but there was no significant change in the E2 concentration. The thickness of the endometrium increased between days 8 and 12 and did not change further between days 21 and 25. CONCLUSION(S) The expression of connexin43 gene and protein in human endometrium changes during the menstrual cycle in a pattern that is associated with the secretion of LH, FSH, and progesterone. This pattern may serve as a marker for implantation competence.

Comparison of sperm parameters, in vitro fertilization results, and subsequent pregnancy rates using sequential ejaculates, collected two hours apart, from oligoasthenozoospermic men

OBJECTIVE To evaluate the effect of second consecutive ejaculate collected 2 hours after the first one from infertile men on sperm quality and fertilization and pregnancy rates (PRs) in IVF. DESIGN A prospective case-control study. SETTING In vitro fertilization unit of a university hospital. PATIENTS Thirty-nine consecutive infertile patients with oligoasthenozoospermia scheduled for IVF-ET. MAIN OUTCOME MEASURES Two consecutive ejaculates were obtained 2 hours apart and were assessed for volume, sperm count, motility, morphology, and quality of swim-up fraction. The subsequent fertilization, cleavage, and PRs (as defined by the appearance of intrauterine gestational sac) were compared between the two ejaculates. RESULTS In 28.2% of the individuals the semen analysis of the first ejaculate precluded proceeding with IVF. A statistically significant improvement was shown in sperm cell motility (31.9% +/- 20.7% versus 15.6% +/- 15.3%) and in motile count after swim-up (4.9 +/- 4.5 versus 2.6 +/- 3.1 x 10(6) sperm). No improvement could be demonstrated in sperm density or morphology. The volume of the second ejaculate was decreased significantly as compared with the first one. The fertilization rate, the cleavage rate, and PR were all increased when oocytes were exposed to sperm from the second ejaculate compared with oocytes exposed to sperm from the first ejaculate. The overall PR in our series was 25.6%. CONCLUSIONS We suggest that in the group of infertile men with oligoasthenozoospermia, whose partners are scheduled for IVF-ET, if on the day of retrieved oocytes insemination, the ejaculate is of unacceptable quality, a second ejaculate collected 2 hours after collection of the initial ejaculate may produce a sample that exhibits improvements in both semen parameters and reproductive potential.

Tamoxifen Treatment in Women with Failure of Clomiphene Citrate Therapy

Summary: Fourteen pregnancies were achieved with tamoxifen therapy in 12 women who failed to conceive with clomiphene citrate. There were no side‐effects and fewer treatment cycles were required than with clomiphene citrate treatment. Ovulation and cervical score with tamoxifen therapy were significantly higher (p< 0.005).

Endometrial morphology and hormonal profiles in in vitro fertilization patients

Endometrial biopsy was performed in 27 infertile women participating in the IVF program. Their mean age was 31.8 years, 33% of the women being over 35 years old. The average duration of infertility was 6.9 years. The superovulation protocol consisted of hMG/hCG in 5 cases, of combined GnRH analog/gonadotropin therapy in 20 women, and 2 patients received combined contraceptive pill/gonadotropin treatment. Judging by hormonal profiles, follicular growth rate and number of oocytes retrieved, the response to stimulation was normal. The mean estradiol (E2) levels increased from 132.7 pg/ml on day -5 (SEM = 9.67) to 1272 pg/ml (SEM = 103.7) on the day of hCG administration and to 1813 pg/ml (SEM = 209.6) 1 day later. One day before the hCG application, the mean progesterone and LH levels were 1.34 ng/ml and 8.38 IU/ml, respectively. Only one patient had clinical hyperstimulation syndrome. Ova were harvested in all women, the mean number of oocytes being 7.7 (SEM = 0.83) per patient. In all 27 cases lack of fertilization or faulty ovum cleavage were observed. Thus, an endometrial biopsy (EB) was performed 72 h after oocytes retrieval. The mean estrogen and progesterone levels on the EB day were 610.9 pg/ml (SEM = 78.44) and 45.4 ng/ml (SEM = 7.53), respectively. Histologic examination of the endometrium showed normal secretory endometrium consistent with day 16-17 of spontaneous ovulatory cycle. Two women who received combined contraceptive pills/gonadotropin therapy showed inactive endometrium with subnuclear vacuoles and decidual reaction in the stroma similar to that observed in women on estrogen-progestin birth control medication.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of Human Embryos in the Presence of a Copper Intrauterine Device

This study was designed to examine the teratogenic potential of copper releasing intrauterine contraceptive devices on the developing embryo. The tissues and organs of 11 whole embryos between 7 and 12 weeks of gestation were histologically examined for copper absorption after exposure to a copper intrauterine device in utero. The findings were not significantly different when compared with a control group of 7 embryos. No malformation or copper aggregates were observed in the various organs and placentas. The observations in this small sample would suggest that the copper-releasing intrauterine device has no deleterious effects on fetal development. The question remains whether malformations reported in some cases are associated with copper deposits in the tissues or with free copper ions in the fetal circulation. The implications are discussed.