Oxygen tension in follicular fluid falls with follicle maturation

European Journal of Obstetrics & Gynecology and Reproductive Biology, 43 (1992) 39-43

39

0 1992 Elsevier Science Publishers B.V. All rights reserved 002%2243/92/$05.00 EUROBS 01239

Oxygen tension in follicular fluid falls with follicle maturation B. Fischer, W. Kiinzel, J. Kleinstein and H. Gips Frauenklinik d. JLU Giessen, Giessen, F.R.G.

Accepted for publication 18 June 1991

Summary The increase in follicular diameter during maturation is accompanied by an increase in vascular supply. The oxygen tension (Po,) in the follicular fluid of the growing follicle should therefore give an insight whether the oxygen delivery during this pertinent process is sufficient. Follicular fluid of 20 patients subjected to in vitro fertilization and embryo transfer were sampled in heparinized glass capillaries which were placed between the puncturing needle and the collecting reservoir. All patients were treated with human menopausal gonadotropin (HMG) and follicle-stimulating hormone (FSH). The PO,? Pco, and pH of the follicular fluid were measured with an automatic gas analyser (Ciba Corning). Simultaneously, the same parameters were estimated in maternal capillary blood taken from the arterialized well perfused earlobe. Follicular size was measured by ultrasound. With growing follicular size (FS), the oxygen partial pressure fell from 80 to 50-60 mmHg (PO, = 114.4-3.3 . FS; r = 0.79) while the carbondioxide partial pressure (PC-, > in the follicle increased from 35 to 50 mmHg ( Pco, = 15.1 f. 1.9 * FS; r = 0.71). In correlation to these results, the pH in follicular fluid fell from 7.40 to 7.30 (pH = 7.6 - 0.019. FS; r = 0.62). The correlation between the Pco, and the pH demonstrates a deviation from the regression line if no metabolic alterations take place (Alog Pc,JApH = -0.69). The increase in ovarian blood flow during follicular maturation is obviously not sufficient to bridge the growing diffusion distance between the capillary network surrounding the follicle and its center. It is speculated that the falling PO, could be the trigger impulse for the generation of biochemical steps, e.g., the liberation of prostaglandins within the follicular fluid and/or the increase in activity of proteolytic enzymes directed to the lysis of the follicular wall. Follicle; Oxygen tension; Follicular fluid; Follicular maturation

Introduction Follicular maturation is accompanied by an increase in follicular size, production of follicular

Correspondence: Prof. Dr. med. W. Kiinzel, Gf. Direktor d. Zentrum fiir Frauenheilkunde und Geburtshilfe, Klinikstr. 28, 6300 Giessen, F.R.G.

fluid and, finally, rupture of the membrane and ovulation. The biochemical steps of this process are well defined. The basic physiology underlying the mechanism responsible for follicle rupture remains obscure. Even less is known whether there is an increase in ovarian blood flow and whether the flow to the ovary is sufficient to bridge the growing diffusion distance between the vessels supplying the follicle and its cumulus

40

oophorus to maintain a constant oxygen supply [31. It is conceivable that the maturation of the follicle and the increase in diffusion distance is accompanied by a fall in the oxygen supply to the follicle. However, the oxygen tension in the follicular fluid of a growing follicle should give an insight into the oxygen supply during this pertinent process. The falling oxygen tension (Po,) could generate the metabolic and biochemical process responsible for ovulation.

sampling reservoir for folkular fluld.

Methods Sampling of foliicular fluid

The study was performed on 20 patients subjected to in vitro fertilization and embryo transfer. All patients were treated with human menopausal gonadotropin (HMG) (Humegon, Organon) and follicle-stimulating hormone (FSH) (Fertinorm, Serono). Ovulation was induced in each patient between the 12th and 14th day of the cycle by an injection of 10000 IU HCG. The follicular fluid was sampled in heparinized glass capillaries which were placed between the needle used to punction the follicle and the collecting reservoir for the follicular fluid (Fig. 1). In each patient all follicles of the ovary were punctured to prevent the hyperstimulation syndrome. The number of follicles punctured ranged from 2 to 10 (average 5). Follicular fluids containing flushing medium were excluded from the investigation, and only one sample from one ovary per patient was analysed. In five patients the follicular fluids from follicles of 12 mm (n = 3) and 15 mm (n = 2) in size were collected and analysed. However, most of the follicles had a size of 16 to 20 mm (n = 15). The glass capillaries were 200 mm long and had a volume of 215 ~1 (Eschweiler & Co, Kiel). Blood gases and pH in the follicular fluid and the ma ternal arterilized blood

and pH in the follicular fluid The PO,, Pq was measured with an automatic blood gas analyser (Ciba Corning). Simultaneously, the same parameters were estimated in the maternal blood. Maternal capillary blood was taken from the ear-

Fig. 1. Schematic representation of follicular fluid sampling: With a 1.2 mm gauge needle, the follicle of the ovary was punctured under ultrasound observation. With a suction device of low adjustable vacuumpressure, follicular fluid was withdrawn from the follicle. The follicular fluid in the sampling reservoir was searched for oocytes, and that in the capillary used for PO*, PC,, and pH measurements. After disconnection of the capillary the follicle was flushed with culture medium to rescue the remained oocytes in the follicular fluid.

lobe, treated with Finalgon, before the samples were taken. It has been proven that the arterialized blood of the capillaries is identical to arterial blood [5]. Estimation of follicular size

Follicular size was measured by ultrasound before the follicle was punctured. Measurements were made by taking two diameters, and the mean of both was taken for further calculations. Results

ThePO,,&I, and pH in the follicular fluid and in the maternal blood The values of the gas analysis in the follicular fluid and in the maternal arterilized blood (capillary blood) are listed in Table I. The PO, in the follicular fluid (Po2, FF) was lower than in maternal arterial blood (Poza): PO,, FF 59.8 (SD 9.8) mmHg vs. Po,a 102.0 (SD 3.4) mmHg. There was also a difference in pH between both compartments: pHFF 7.33 (SD 0.07) vs. pHa 7.41 (SD 0.04). The low pH in the follicular fluid is due to the Increase m Pco, and a result of anaerobic

41 TABLE

I

Patient

H.B. H.B. S.B. H.H. K.E. SK. B.U. R.P. P.V. M.M. B.F. N.E. B.E. V.B. P.B. K.G. H.H. M.P. F.S. M.F. Mean SD

Follicular

90.03.16 90.03.16 90.03.22 90.03.15 90.04.04 90.04.04 90.04.05 90.04.05 90.04.09 90.04.30 90.05.17 90.06.12 90.06.13 90.06.13 90.06.23 90.06.06 90.06.05 90.06.06 90.06.02 YO.06.01

fluid

Follicular

size (mm)

Maternal

arterial

blood

Paz (mmHd

Pco,(mmHd

PH

Po, (mm&)

PcoZ(mmHd

PH

65.6 46.3 61.8 66.9 56.2 50.2 53.7 58.6 80.3 50.8 50.4 50.6 59.0 53.0 76.3 59.2 62.1 59.2 56.2 79.9

44.1 48.8 46.3 46.2 41.6 47.8 62.1 56.2 34.8 48.4 47.6 54.0 47.9 46.6 35.0 47.5 48.2 46.6 48.4 39.6

7.28 7.28 7.33 7.38 7.36 7.26 7.18 7.27 7.44 7.35 7.29 7.23 7.35 7.39 7.44 7.32 7.36 7.32 7.38 7.44

18 16 11 16 15 18 17 18 12 I7 18 20 18 20 12 17 17 16 18 12

100.1 100.1 102.0 96.1 97.5 104.1 99.4 106.3 106.0 102.1 98.4 104.0 101.2 97.8 107.0 108.4 102.3 102.7 99.4 104.2

38.1 38.1 37.4 37.7 37.4 39.0 36.1 40.3 42.1 38.4 37.2 39.0 39.2 38.6 40.2 37.6 36.4 38.4 37.6 38.1

7.41 7.41 7.4s 7.40 7.41 7.39 7.50 7.40 7.40 7.38 7.38 7.37 7.36 7.41 7.43 7.42 7.36 7.34 7.46 7.42

59.8 9.8

46.9 6.3

7.33 0.07

16.6 2.3

102.0 3.4

38.3 1.4

7.41 0.04

glycolysts. The PCO, in the follicular fluid was 46.9 (SD 6.3) mmHg and in the maternal arterialized blood 38.3 (SD 1.4 mmHg). This remarkable difference between both compartments had a wide range.

(pH = 7.6 - 0.019 . FS, r = 0.62) (Fig. 4). The fall of the pH in the follicular fluid was due to anaerobic glycolysis. The PCO, on a logarithmic scale is related to the pH according to SiggaardAnderson nomogram for fluids not containing hemoglobin (Fig. 5). The correlation between

Follicular size and PO,, Pco, and pH in the follicular fluid

Follicular fluid has been gained from follicles of various size. The size of the follicles ranged from 12 to 20 mm. The PO,, PcO, and the pH was related to the follicular size (FS) (Figs. 2-4). At a follicular diameter of 12 mm, the PO, in the fluid was about 80 mmHg. With increasing follicular size the PO, in the fluid decreased to 60 mmHg at a size of 18-20 mm (PO, = 114.4 - 3.3 - FS); r = 0.79) (2cx < 0.01) This was accompanied by an increase in the PO, difference between the maternal arterial blood and the center of the follicle (Fig. 2). In parallel to the fall in PO, with follicular maturation, an increase in PCO, from 40 to 50 mmHg ( PCs2 = 15.1 f 1.9. FS; Y= 0.71) (Fig. 31, and a fall m pH from 7.40 to 7.30 occurred,

5

-

y = 114.3986

3.3081~

R = 0.79

20 8

a

OJ-ff.

8 12

8

14 Follicular

16

I

18

t

20

size (mm)

Fig. 2. Correlation between the Po, of the follicular fluid and the follicular size. For comparison the arterialized maternal blood PoZ is shown. With increasing follicular size, the PO2 in the follicular fluid fell, showing that the arterial follicular fluid PO, difference rises. This rise in PO2 difference could be the trigger impulse for a biochemical process finally leading to rupture of the follicular wall and to ovulation.

42 PC02 (mm Hn) too 90 80

60 I 791

’

’

’

12

14

16

.

’

.

18

’

’

20

22

Follicular size (mm)

Fig. 3. Correlation between the pH in the follicular fluid and follicular size. With the increase in follicular size, the pH fell from 7.40 to 7.30.

PH

o&J/.

1

’

’

8

12

14

16

16

.

8

’

20

22

Follicolar sire (mm)

Fig. 4. Correlation between Pco, and follicular size. With the increase in follicular size, the Pco, in the fluid rose, indicating that the fall in pH is partly due to respiratory acidosis.

P co2 and pH demonstrates

a deviation from the regression line where no metabolic alterations take place (Alog P&ApH = - 1.18). The dotted line shows the calculated regression for the measured data. The correlation coefficient is Alog Pcol/A pH = - 0.69 and gives evidence for anaerobic glycolysis. Discussion

The present investigations demonstrate convincingly and for the first time that follicular growth and maturation is paralleled by a fall in the PO, in the follicular fluid from 80 to 50-60 mmHg.

Fig. 5. The correlation between the Pco2 and the pH in the follicular fluid plotted in a Siggaard-Anderson nomogram (log pcoz = 6.75 -0.69.pH; R = 0.83 (2a < 0.01) (solid line). The solid line shows the relationship for fluids not containing hemoglobin (log Pco, = 10.36- 1.18.pH). The regression coefficient for the measured data shows a slight deviation from the one for hemoglobin free solutions, indicating an anaerobic glycolysis with falling pH.

This fall in PO, is accompanied by a decrease in the pH and a rise in Pco,.These changes in the follicular fluid show evidence for anaerobic glycolysis, as demonstrated in Fig. 5. The fall in PO, in the follicular fluid with growing size gives rise to speculations concerning its cause and its impact on a biochemical process ultimately leading to ovulation. Continuous measurements of ovarian blood flow have been performed by the implantation of crossed-thermo-couples in the ovaries of female rabbits [4]. Following HCG administration, ovarian blood flow increased and reached a maximum of 150% after 2-4 h. It declined again to 110% after 8 h. This rise was paralleled by a dilatation of the perifollicular tissue capillaries. However, the oxygen transport calculated from the time to reach the maximal follicle PO, after the start of 100% 0, inhalation

43

was decreased [4]. The PO, measurements in the follicular fluid during follicular maturation in the human support the assumption that despite an increase in ovarian blood flow the increase in flow is not sufficient to maintain a constant 0, supply to the core of the follicle. Gosden and Byatt-Smith [3] calculated the oxygen concentration across the ovarian follicular epithelium. From this model they concluded that most dissolved oxygen entering the follicle by diffusion is consumed in the outer layer of the cells and that only a small amount would reach the oocyte. They assumed that the follicular core is virtually anoxic. These theoretical assumptions are not in agreement with the present findings. The basis for the calculations has been the oxygen consumption of granulosa cells in vitro under normoxic conditions. There is, however, evidence that 0, consumption in cells as proved in fetal skeletal muscle cells is reduced if the PO, fell below 80 mmHg [l]. Concerning the follicle, it may be speculated that in each ‘shell’ from the outer layer towards the follicular core, the oxygen consumption of the cell layer adjusts to the low

oxygen partial pressure finally ending in the enzymatic liquidization of the follicle. Further investigations are necessary to evaluate the impact of the falling PO, in the core of the follicle on hormone and prostaglandin synthesis. References Braems

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