b:Myeloperoxidase (Endtz test) for white
blood cell measurement
The presence of white blood cells (WBC), especially
the granulocytes in semen specimens was assessed by the Endtz
test (myeloperoxidase) . A 20-uL volume of liquefied specimen
was placed in a Corning 2.0-mL cryogenic vial (Corning Costar
Corp., Cambridge, MA): 20 uL of phosphate-buffered saline
(PBS, pH 7.0) and 40 uL of benzidine solution were added. The
mixture was vortexed and allowed to sit for 5 minutes. Five
microliters of the specimen was placed on a Makler chamber
(Sefi Medical, Haifa, Israel) and counted in all 100 squares.
Peroxidase positive WBCs staining dark brown were counted.
The results, after correction for dilution, were recorded as
counts into 106/mL. Leukocytospermia was defined
as the presence of at least 1 × 106 WBC/mL. In
our study, we included both patients with and without
leukocytospermia.
c:Reactive
oxygen species
Aliquots of liquefied semen were centrifuged at 300 ×
g for 7 minutes. Seminal plasma was aliquoted and
frozen at -20°C for later measurement of total antioxidant
levels. The sperm pellet was washed twice with (PBS), pH 7.4,
and resuspended in the same medium at a concentration of 20
× 106 sperm/mL. The ROS production was measured
by the chemiluminescence assay method using luminol
(5-amino-2,3-dihydro-1,4-phthalazinedione; Sigma Chemical
Co., St. Louis, MO) as the probe . Ten microliters of 5 mM
luminol prepared in dimethyl sulfoxide (DMSO; Sigma Chemical
Co.) was added to 400 uL of the washed sperm suspension. The
ROS levels were determined by measuring chemiluminescence
with a luminometer (LKB 953, Wallac Inc., Gaithersburg, MD)
in the integrated mode for 15 minutes, and results were
expressed as 104 counted photons per minute (cpm)
per 20 × 106 sperm.
d:Total
antioxidant capacity
Total antioxidant capacity was measured in seminal
plasma with use of the enhanced chemiluminescence assay .
Aliquots of the seminal plasma stored at -20°C were thawed
at room temperature and immediately assessed for their
antioxidant capacity as follows. Seminal plasma was diluted
1:10 with deionized water (dH2O) and filtered
through a 0.20-um Millipore filter (Allegiance Healthcare
Corporation, McGaw Park, IL). Signal reagent was prepared
with a chemiluminescence kit (Amersham Life Science,
Buckinghamshire, United Kingdom). Twenty microliters of
horseradish peroxidase-linked immunoglobulin (HRP-linked Ig;
Amersham Life Science) was added to 4.98 mL of dH2O.
This was further diluted 1:1 to give a working solution with
the desired luminescence output (3 × 107 cpm).
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid), a water-soluble tocopherol analogue, was added as the
standard at concentrations between 50 and 150 uM.
With the luminometer set in the kinetic mode, 100 uL of
signal reagent and 100 uL of HRP were added to 700 uL of dH2O
and mixed. The solution was then equilibrated to the desired
level of chemiluminescence output (between 2 and 3 × 107
cpm) for 100 seconds. One hundred microliters of the prepared
seminal plasma was added to the signal reagent and HRP, and
the chemiluminescence was measured. Suppression of
chemiluminescence and the time from the addition of seminal
plasma to 10% recovery of the initial chemiluminescence were
recorded. Antioxidant capacity was expressed as molar Trolox
equivalents.
from
Fertility and Sterility 73:459-464, March 2000J
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前言 |
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子宮內膜的厚度與子宮動脈的血流量有很大的關係,而較理想的子宮內膜厚度能提高受孕率已是不爭的事實,因此,
各種可以增加動脈血流量的方法,一一的被拿出來研究,
Sildenafil citrate (Viagra)就是其中之一.至於Viagra(威而剛)的詳細說明,
請參考: http://www.lai-obs.com/page7.htm#2 |
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Sildenafil citrate (Viagra)的藥理作用 |
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Sildenafil citrate是一種
type 5-specific phosphodiesterase inhibitor,
置放於陰道內的Viagra能抑制cyclic guanosine
monophosphate(簡稱cGMP) 的分解, 這個作用可以強化Nitric
oxide(NO),使子宮動脈的平滑肌鬆弛,
進而增加子宮動脈的血流量. |
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數據 |
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以Viagra陰道塞劑來治療IVF-ET的不孕婦女,
在治療過程中, 並測其子宮動脈的博動指數(uterine artery pulsatility
index PI) , 治療7天, PI就已明顯下降(表示子宮動脈的血流量已增加),
接著以placebo 7天, 則PI又回復了.如果以Viagra及oestradiol
valerate的混合塞劑,則明顯的增加了子宮動脈的血流量及顯著的子宮內膜成長.四個接受治療的不孕婦女,
結果有三個懷孕了. |
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結論 |
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研究的數據雖然還不夠,
但這種理論應可以接受, 因此以Viagra陰道塞劑來治療不孕,
尤其對子宮內膜狀況不良的婦女,
應該深具價值. |
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from
Human Reproduction 15: 806-809, April 2000
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J |
