Reproductive Sciences

The pituitary gonadotropin follicle-stimulating hormone (FSH) interacts with its membrane-bound receptor to produce biologic effects. Traditional functions of FSH include follicular development and estradiol production in females, and the regulation of Sertoli cell action and spermatogenesis in males. Knockout mice for both the ligand (Fshb) and the receptor (Fshr) serve as models for FSH deficiency, while Fshb and Fshr transgenic mice manifest FSH excess. In addition, inactivating mutations of both human orthologs (FSHB and FSHR) have been characterized in a small number of patients, with phenotypic effects of the ligand disruption being more profound than those of its receptor. Activating human FSHR mutants have also been described in both sexes, leading to a phenotype of normal testis function (male) or spontaneous ovarian hyperstimulation syndrome (females). As determined from human and mouse models, FSH is essential for normal puberty and fertility in females, particularly for ovarian follicular development beyond the antral stage. In males, FSH is necessary for normal spermatogenesis, but there are differences in human and mouse models. The FSHB mutations in humans result in azoospermia; while FSHR mutations in humans and knockouts of both the ligand and the receptor in mice affect testicular function but do not result in absolute infertility. Available evidence also indicates that FSH may also be necessary for normal androgen synthesis in males and females.

This brief review focuses on the functional activities of plasma hemopexin recently recognized by several authors. In particular, the protease-like activity of hemopexin in vitro is linked with downregulation of the vascular angiotensin II receptor in vivo, leading to vascular expansion. Also a potential mechanism of inhibition of hemopexin activity by extracellular adenosine triphosphate is considered.

Laser microdissection (LMD) is a selective cell isolation technique that enables the separation of desired homogenous cell subpopulations from complex tissues such as the testes under direct microscopic visualization. The LMD accompanied by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) represents an indispensable tool in quantifying messenger RNA (mRNA) expression among defined cell populations. Gene expression is temporally and spatially regulated at 3 sequential phases of mitotic, meiotic, and postmeiotic stages of spermatogenesis. The present study demonstrates a short modified LMD protocol based upon hematoxylin and eosin (H&E) staining. Stage-specific LMD success was validated by the use of mRNA profiling of “marker genes” which are conserved across species and are known to be differentially expressed during spermatogenesis. Magea4, Hspa2, Cox6b2, Tnp1, Prm1, and Prm2 are used to differentiate among the microdissected cell populations, namely spermatogonia (group I), spermatocytes (group II), round and condensing spermatids (group III), and elongated and condensed spermatids (group IV), respectively. The LMD combined with qRT-PCR is further extended to assess the cell stage-specific distribution of selected stress response genes such as Hsp90aa1, Gpx4, Ucp2, Sod1, and Sod2. The germ cell-specific mRNA profiles are suitably complemented by Western blot of the LMD samples, immunohistochemistry, and confocal localization of the corresponding proteins. The current study suggests that LMD can successfully isolate cell subpopulations from the complex tissues of the testes; and establish cell stage-specific basal expression patterns of selected stress response genes and proteins. It is our hypothesis that the baseline expression of stress response genes will differ by cell stage to create discrete stage-specific vulnerabilities to reproductive toxicants.

Objective: To test a possible neuroprotective activity of 17β-estradiol in the neonatal rat brain exposed to hypoxic-ischemia (controlled hypoxia after unilateral carotid artery ligation). Methods: Seven-day-old Wistar rats underwent ligation of the left common carotid artery followed by 80 minutes hypoxia in 8% oxygen inducing an ipsilateral brain damage. Seven days later (d14), brains were analyzed quantitatively using a macroscopic and microscopic score for structural damage, hemisphere volumes were calculated, and immunohistochemistry for cleaved-caspase-3 (marker for apoptotic cells) was performed. Animals from the study group (n = 19) received 17β-estradiol (0.05 µg/g  body weight intraperitoneally) before (−64, −40, and –16 hours) and after (+3 hours) the hypoxia (hour 0: start of the hypoxia) and the control group (n = 21) received mock treatment. Results: Of the 21 pups, 13 in the NaCl group had macroscopically a severe brain damage and 7 of 19 animals in the study group encountered only discrete to mild lesions. Microscopic brain damage in the study group was significantly lower (score 1.5 ± 0.7 vs 2.8 ± 0.8, P < .05). The determined volumes of the affected hemisphere were significantly lower in the NaCl group than in the treatment group. The numbers of apoptotic cells in both hemispheres was equal in the estradiol group, but in the control group, there were significantly more apoptotic cells in the affected hemisphere (control group: ipsilateral: 1435 ± 653 vs contralateral: 143 ± 57 cells, P < .05). Discussion: 17β-Estradiol protects newborn rat brains from hypoxic–ischemic injury, in terms of both microscopic cell injury and apoptosis.

Objective: To determine the value of cervicovaginal interleukin (IL)-1β, IL-6, and IL-8 in predicting intra-amniotic infection and/or inflammation (IAI) in women with preterm labor. Methods: Cervicovaginal fluid samples were collected for IL-1β, IL-6, and IL-8 measurements immediately before amniocentesis in 85 consecutive women with preterm labor. The IAI was defined as a positive amniotic fluid (AF) culture and/or an elevated AF IL-6 level (>2.6 ng/mL). Results: Receiver–operating characteristic curves demonstrated that cervicovaginal IL-6 and IL-8, but not IL-1β, predicted IAI. Cervicovaginal IL-6 had a significantly higher area under the curve (AUC) than cervicovaginal IL-8 (P = .009). However, the AUCs for the cervicovaginal IL-6 and AF white blood cell (WBC) were not significantly different. Conclusions: Among measured cytokines, cervicovaginal IL-6 is the best marker to noninvasively identify IAI in women with preterm labor. Overall, this noninvasive parameter performed as well as AF WBC count for predicting IAI.

We studied obesity-related differences in the relation of maternal levels of leptin to levels of soluble fms-like tyrosine kinase 1 (sFlt1), an antiangiogenic protein that influences placentation and risk of adverse pregnancy outcomes. In a prospective cohort of 286 gravidas, we measured maternal serum levels of sFlt1 and leptin at 5 time points across pregnancy. Analyses stratified on prepregnancy body mass index (<25 vs ≥25) were done using mixed linear models. The mean leptin concentrations were significantly higher in overweight/obese compared to normal-weight women, while mean sFlt1 levels in second and third trimester were significantly higher in normal weight compared to overweight/obese women. The relationship between sFlt1 and leptin differed between the 2 strata. After controlling for maternal weight, a 1 ng/mL increase in leptin was associated with an 19.4 pg/mL increase in sFlt1 (P = .01) in normal-weight women, while leptin was not associated with sFlt1 (β = 1.1, P = .75) in overweight/obese women. Such differences suggest that metabolic differences in overweight/obese women compared to their normal weight peers may differentially impact the physiologic changes during pregnancy.

A gradient of immunological mediators exists in the fetal membranes from the periplacental zone (PZ) to the rupture zone (RZ) at term delivery (rupture of fetal membranes [ROM]). However, it is unknown if this gradient is different in premature rupture of these tissues (premature rupture of fetal membranes [PROM]). We therefore analyzed leukocyte chemotactic activity and chemokine/cytokine production in fetal membrane zones in ROM and PROM. In ROM, leukocyte chemotactic activity increased from the PZ to the RZ; however, this did not occur in PROM. This was due to consistently elevated leukocyte chemotactic activity in PROM compared to ROM tissues. In the RZ, ROM was characterized by increased T-cell attraction and high levels of chemokine (C-X-C motif) ligand 8 (CXCL-8)/interleukin 8, and PROM by increased granulocyte attraction and high levels of granulocyte-macrophage colony-stimulating factor and CXCL-10/interferon gamma-induced protein 10. We conclude that normal and premature rupture of fetal membranes differ in regional chemotactic activity and related chemokine/cytokine production, which may represent evidence for differential mechanisms of rupture at term delivery.

We recently reported that Slit/Roundabout (ROBO) 1 pathway may be a constituent biomarker for recurrence of endometriosis, likely through promoting angiogenesis. In this study, we sought to determine as whether Slit2 overexpression can facilitate angiogenesis, increase lesion size, and induce hyperalgesia in mice with induced endometriosis. We used 30 Slit2 transgenic (S) and 29 wild-type (W) mice and cross-transplanted endometrial fragments from S to W (group SW) and vice versa (group WS), and also within the S and W (groups SS and WW, respectively), into the peritoneal cavity, inducing endometriosis. We also performed a sham surgery within both S and W mice (groups Sm and Wm, respectively). The size of the ectopic implants, microvessel density (MVD) and immunoreactivity to ROBO1, and vascular endothelial cell growth factor (VEGF) in ectopic and eutopic endometrium, along with hotplate and tail-flick tests in all mice, were then evaluated. We found that the induction of endometriosis resulted in generalized hyperalgesia, which was unaffected by Slit2 overexpression. Slit2 overexpression did increase the lesion size significantly and correlated positively with the MVD in ectopic and eutopic endometrium. Slit2 expression levels appear to correlate with the MVD, but not with VEGF immunoreactivity, in ectopic endometrium. Consequently, we conclude that Slit2 may play an important role in angiogenesis in endometriosis. The increased angiogenesis, as measured by MVD, but not VEGF immunoreactivity, likely resulted in increased lesion size in induced endometriosis. Thus, SLIT2/ROBO1 pathway may be a potential therapeutic target for treating endometriosis.

We hypothesized that chronic hypoxia disrupts mitochondrial function via oxidative stress in fetal organs. Pregnant guinea pig sows were exposed to either normoxia or hypoxia (10.5% O₂, 14 days) in the presence or absence of the antioxidant, N-acetylcysteine (NAC). Near-term anesthetized fetuses were delivered via hysterotomy, and fetal livers, hearts, lungs, and forebrains harvested. We quantified the effects of chronic hypoxia on cytochrome oxidase (CCO) activity and 2 factors known to regulate CCO activity: malondialdehyde (MDA) and CCO subunit 4 (COX4). Hypoxia increased the MDA levels in fetal liver, heart, and lung with a corresponding reduction in CCO activity, prevented by prenatal NAC. The COX4 expression paralleled CCO activity in fetal liver and lung, but was unaltered in fetal hearts due to hypoxia. Hypoxia reduced the brain COX4 expression despite having no effect on CCO activity. This study identifies the mitochondrion as an important target site in tissue-specific oxidative stress for the induction of fetal hypoxic injury.

MicroRNAs (miRNAs) act as important epigenetic posttranscriptional regulators of gene expression. We aimed to gain more understanding of the complex gene expression regulation of endometrial receptivity by analyzing miRNA signatures of fertile human endometria. We set up to analyze miRNA signatures of receptive (LH + 7, n = 4) versus prereceptive (LH + 2, n = 5) endometrium from healthy fertile women. We found hsa-miR-30b and hsa-miR-30d to be significantly upregulated, and hsa-miR-494 and hsa-miR-923 to be downregulated in receptive endometrium. Three algorithms (miRanda, PicTar, and TargetScan) were used for target gene prediction. Functional analyses of the targets using Ingenuity Pathways Analysis and The Database for Annotation, Visualization and Integrated Discovery indicated roles in transcription, cell proliferation and apoptosis, and significant involvement in several relevant pathways, such as axon guidance, Wnt/β-catenin, ERK/MAPK, transforming growth factor β (TGF-β), p53 and leukocyte extravasation. Comparison of predicted miRNA target genes and our previous messenger RNA microarray data resulted in a list of 12 genes, including CAST, CFTR, FGFR2, and LIF that could serve as a panel of genes important for endometrial receptivity. In conclusion, we suggest that a subset of miRNAs and their target genes may play important roles in endometrial receptivity.

Severe inflammation has been shown to induce a shedding of the endothelial glycocalyx (EGX). Inflammatory cytokines, such as tumor necrosis factor α (TNF-α), impede the thickness of the EGX. While a controlled inflammatory reaction occurs already in normal pregnancy, women with hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome had an exaggerated inflammatory response. This study investigates the shedding of the glycocalyx during normal pregnancy and in women with HELLP syndrome. Glycocalyx components (syndecan 1, heparan sulfate, and hyaluronic acid) were measured in serum of healthy women throughout pregnancy (4 time points, n = 26), in women with HELLP syndrome (n = 17) before delivery and in nonpregnant volunteers (n = 10). Serum concentrations of TNF-α and soluble TNF-α receptors (sTNF-Rs) were assessed once in all 3 groups. Syndecan 1 serum concentrations constantly rose throughout normal pregnancy. Immediately before delivery, a 159-fold increase was measured compared to nonpregnant controls (P < .01). Even higher amounts were observed in patients with HELLP prior to delivery (median 12 252 ng/mL) compared to healthy women matched by gestational age (median 5943 ng/mL; P < .01). Relevantly, increased serum levels of heparan sulfate, hyaluronic acid, and sTNF-Rs were only detected in patients with HELLP (P < .01). These findings suggest that considerable amounts of syndecan 1 are released into maternal blood during uncomplicated pregnancy. The HELLP syndrome is associated with an even more pronounced shedding of glycocalyx components. The maternal vasculature as well as the placenta has to be discussed as a possible origin of circulating glycocalyx components.

Our objectives were to (1) quantify the relationship between daily swallowed volume and amniotic fluid volume (AF volume) in late gestation ovine fetuses and (2) use the resulting regression equation to explore the role of swallowing in regulating AF volume. Daily swallowed volume ranged from 36 to 1963 mL/d while experimental AF volume ranged from 160 to 6150 mL (n = 115). Swallowed volume was near zero when AF volume was far below normal, a maximum of 635 ± 41 (standard error) mL/d when AF volume was 1682 ± 31 mL and did not increase further with higher AF volumes. Computer simulations predicted that fetal swallowing would (1) return AF volume to normal in 5 to 6 days following an acute volume change in the absence of changes in other amniotic inflows or outflows and (2) stabilize AF volume in 4 to 8 days following sustained alterations in amniotic inflows or outflows other than swallowing. Conclusions: The volume of AF swallowed each day by the fetus is a strong function of AF volume and reaches a maximum when mild polyhydramnios develops. With deviations in AF volume from normal, changes in fetal swallowing protect against oligohydramnios and polyhydramnios because the changes in swallowing over time reduce the extent of the AF volume change. However, with experimental changes in AF volume stabilizing in 1 to 2 days, it appears that swallowing is not the major regulator of AF volume.