Primordial and pre-antral follicles are not commonly observed in IVF aspirates

Sergey I Moskovtsev1, Jeanine T Griffin1, C.Matthew Peterson2 and Douglas T Carrell1,2,3,4

1 Departments of 1Surgery (Urology), 2 Obstetrics and Gynecology and 3 Physiology, University of Utah School of Medicine, Salt Lake City, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Follicular fluid recovered from IVF patients has been proposed to be a valuable source of pre-antral and primary follicles for patient therapy and research. We evaluated the recovery of immature follicles in follicular fluid from 54 patients undergoing IVF using several techniques. METHODS: Fluid from each patient underwent several methods of follicle recovery including: filtration through a cell strainer, Ficoll–Paque density gradient, isolate density gradient, histological slide preparation, and enzymatic digestion with collagenase and DNase. RESULTS: 34 primordial and primary follicles, mean 0.63 ± 0.27/patient, and 14 pre-antral follicles, mean 0.26 ± 0.14/patient, were found in this study. The serum estradiol level on the day of HCG injection was significantly lower (P < 0.05) in patients in which immature follicles were recovered, compared with those without immature follicles in the follicular fluid (1779.9 ± 167.6 versus 2246.6 ± 153.2 pg/ml). There were no women with advanced maternal age (>39 years) who had immature follicles in the follicular fluid. CONCLUSIONS: Follicular fluid cannot be considered an efficient or reliable source of immature follicles. The presence of any immature follicles appears to be associated with cause of infertility, the random placement of the aspirating needle and may be related to the age of patient.

Key words: follicular fluid/IVF/pre-antral follicle/primary follicle/primordial follicle


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The need for alternative sources of oocytes and ovarian tissue for research and treatment is widely recognized. Practical and ethical considerations for possible sources of oocytes, such as in-vitro maturation of immature follicles, ovarian transplantation, autotransplantation, and cadaveric donation, have been widely discussed (Robertson, 2000Go). Wu et al. have reported that follicular aspirates obtained during IVF may be a new and abundant source of pre-antral follicles (Wu et al., 1998Go). This possible new source of oocytes has been widely discussed (Edwards and Beard, 1998Go; Hovatta et al., 1999Go; Wright et al., 1999Go). However, no confirmation of this research has been reported.

The purpose of this study was to analyse the feasibility of using follicular fluid aspirates obtained during IVF retrieval as a source of early immature follicles. Five protocols for the isolation of follicles were compared in pooled follicular fluid from 54 patients undergoing IVF.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient selection
This study was approved by the Institutional Review Board at the University of Utah. All participants signed informed consent documents. Follicular fluid from 54 women undergoing IVF at the University of Utah was evaluated after standard identification and recovery of all oocytes present in the fluid.

All patients underwent ovarian stimulation using GnRH agonist down-regulation (Lupron®; TAP Pharmaceutical Inc., Deerfield, IL, USA), followed by controlled ovarian stimulation using recombinant FSH (Follistim®; Organon Inc., West Orange, NJ, USA or Gonal-F®; Serono Laboratories Inc., Aubonne, Switzerland) combined with purified HMG (Repronex®; Ferring Pharmaceuticals Inc., Tarrytown, NY, USA). Serial transvaginal ultrasonograms were obtained and serum estradiol (E2) levels were determined for all patients on the day of the HCG injection. A single injection of HCG (Profasi®; Serono) was given to induce final oocyte maturation. The concentration of E2 was measured using an automated immunofluorescence assay (Immulite®; Diagnostic Products Corporation, Los Angeles, CA, USA).

Thirty-six hours after the HCG injection, aspirations were performed in an outpatient setting. All visible follicles were aspirated using one to three passes with a standard 17-gauge aspiration needle (SwemedLab, Billdal, Sweden) using transvaginal ultrasound guidance. Follicular fluids were aspirated into a tube containing 2 ml of heparinized human tubal fluid (HTF) in a 37°C warming block. An additional 5 ml of HTF was flushed through the aspiration system to clear away any cells left within the needle or connecting tubing.

After recovery of all oocytes from the follicular fluid, the fluid was transferred to the research laboratory for observation and the recovery of any immature follicles. All follicular fluid samples, whether macroscopically haemorrhagic, non-haemorrhagic, or contaminated with flushing medium, were included in the study. All recovered follicles were measured using a micrometer to ascertain the stage of maturation, then photographed.

Follicular fluid preparation
Follicular fluids from 54 IVF cases underwent several independent methods in order to recover any immature follicles. Initially, follicular fluids from 11 patients were analysed using the method of Wu et al. (centrifugation) (Wu et al., 1998Go). Since no follicles were obtained using that technique, and since the preparations were difficult to analyse due to increased levels of cellular debris and red blood cells, those 11 samples and all subsequent samples underwent either isolate or Ficoll gradient centrifugation. Additionally, filtration was included as an initial step in the preparation of any fluid with increased levels of red blood cells. Therefore, data were obtained from eleven samples using the protocol of Wu et al. (1998), and for 31 samples, a precursor step of filtration was performed prior to gradient centrifugation. The total 54 cases were divided into two groups. In group I, 27 samples had isolate gradient centrifugation and in group II, 27 samples had Ficoll preparation.

Standard and filtration procedures
All fluids were first centrifuged at 400 g for 15 min at room temperature (J6-MC; Beckman Instruments Inc., Seattle, WA, USA), then resuspended in HTF medium. In 31 cases the resuspension was filtered through a 40 µm cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ, USA) to remove red blood cells. All the cell strainers used in the procedures were rinsed with HTF media in order to recover any large follicles lodged in the filter itself. The group of cells collected from this rinsing was observed under a dissection microscope (Wild M8, Wild Heerbrugg, Switzerland).

Histological slide preparation
In 17 cases a cluster of cells pooled after filtration was fixed in a 10% neutral buffered formalin solution (Anapath®; StatLab, Lewisville, TX, USA) and processed for histological sectioning. After the tissue was dehydrated and embedded in paraffin, the tissues were cut into 2 µm serial sections and stained with haematoxylin and eosin. Slides were observed under a bright field light microscope (Olympus BH-2; Olympus Optical Co., Tokyo, Japan).

Isolate density gradient
An isolate density gradient was performed on 27 macroscopically haemorrhagic samples to remove red blood cells and recover immature follicles from the aspirates. This technique was based on the protocol of Greenwald and Moor (Greenwald and Moor, 1989Go) with slight modifications.

An isolate gradient (ISolate®; Irvine Scientific, Santa Ana, CA, USA) of 1.5 ml of 20, 35 and 90% was prepared with HTF medium. The follicular fluid was centrifuged at 400 g for 15 min, then the supernatant was removed and the pellet was resuspended in 1.5 ml of HTF and placed on the isolate column. The tubes were centrifuged at 200 g for 15 min at room temperature. Each fraction of the isolate column was collected and observed under an inverted microscope at x200 magnification using Hoffmann modulation contrast microscopy.

Ficoll–Paque density gradient
In 27 cases Ficoll density gradient centrifugation was performed, including follicular fluid rich with red blood cells. Ficoll–Paque Research Grade (Pharmacia Biotech, Uppsala, Sweden) was used according to the manufacturer's instructions with slight modifications. Briefly, 20 ml of fresh balanced salt solution (Pharmacia Biotech) were prepared for each sample from stock solution. Then 7.5 ml of Ficoll–Paque were placed in a 50 ml centrifuge tube, then a mixture of 7.5 ml follicular fluid with 7.5 ml of balanced salt solution was carefully layered on the Ficoll–Paque. The tube was centrifuged at 400 g for 40 min at 20°C and the upper layer was removed. The second layer, containing in some cases immature follicles, was washed in 3 volumes of balanced salt solution. Cells were then resuspended by gently drawing them in and out of a Pasteur pipette and then centrifuging at 100 g for 10 min at 20°C. Supernatant was removed, the same amount of balanced salt solution was added, and the procedure was repeated. The mixture was observed under x200 magnification using an inverted microscope with Hoffman contrast modulation.

Enzymatic digestion
In 14 cases, enzymatic digestion was performed after isolate gradient centrifugation in order to assure that no follicles were missed during microscopic observation due to obstruction from stromal tissue. Microscopically visible cells and pieces of tissue were removed after follicular fluid filtration. The procedure was performed as previously described (Roy and Treacy, 1993Go) with slight modification. Briefly, clusters of cells were placed in 5 ml of HTF in a 15 ml siliconized glass centrifuge tube containing 2472 units of collagenase (type I A; Sigma Chemical Co., St Louis, MO, USA) and 180 units of pancreatic deoxyribonuclease (DNase, type IV; Sigma) per ml of mixture. The tube was incubated in a water bath at 37°C for 1 h with gentle agitation. After digestion at 37°C, 5 ml of HTF with 0.5% bovine serum albumin (BSA, fraction V; Sigma) was added, and the tube was placed in a refrigerator at 4°C for 24 h to allow slow digestion. During the digestion, the suspension was periodically (every 10–14 h) agitated using a Pasteur pipette. After the slow digestion, the mixture was diluted with fresh HTF and observed under a dissection microscope.

Statistics
Statistical analyses were performed using GraphPad InStat Version 3.0a for Macintosh (GraphPad Software, San Diego, CA, USA). Analysis of variance (ANOVA) was used to determine statistical relationships. Differences in pregnancy rates and aetiology of infertility were evaluated by {chi}2 analysis. Where sample sizes were small, Fisher's exact test was used. A P value of < 0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Follicular fluid samples underwent the following techniques for recovery of immature follicles: isolate density gradient (27 cases); Ficoll–Paque density gradient (27 cases); filtration through 40 µm cell strainer (31 cases); enzymatic digestion with collagenase and DNase (14 cases); and histological slide preparation (17 cases).

Forty-eight immature follicles were found in the 54 cases, mean 0.88 ± 0.31 follicles per case (Figure 1Go). Of the 48 follicles found, 34 were in the primordial or primary stage with a range of 0–10 per case (mean 0.63 ± 0.27), 14 of the follicles were of the pre-antral stage, ranging from 0–7 per case (mean 0.26 ± 0.14). None of the follicles were attached to stromal tissue. The size ranges for primordial/primary and for pre-antral follicles were 28–51 µm and 58–193 µm respectively.



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Figure 1. A pre-antral follicle, 135 µm in diameter, recovered from follicular fluid aspirates of a 31 year old patient by filtration through a 40 µm cell strainer (original magnification x400). Scale bar = 50 µm.

 
Follicles were recovered using three techniques. No follicles were found through either enzymatic digestion or the histological slide preparation. The two density gradient techniques used for recovery of immature follicles were compared (Table IGo). There was a significant difference (P < 0.05) in the total number of immature follicles found between Ficoll gradient (1.29 ± 0.54) and isolate gradient (0.11 ± 0.06). No significant differences were observed in the number of follicles recovered when comparing filtration with the other techniques.


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Table I. Summary of comparing different techniques for recovery immature follicles from follicular fluid aspirates
 
The recovery of small primordial or primary follicles was significantly different (P < 0.05) between Ficoll gradient (1.18 ± 0.53) and isolate gradient (0.07 ± 0.05). Filtration yielded no primordial or primary follicles. There were no significant differences observed in the number of pre-antral follicles retrieved by the three techniques used. There were no significant differences in the demographics of the populations for the three techniques.

All IVF cases were divided into two groups, based on the presence or absence of any immature follicles in follicular fluid aspirated during IVF. Group I consisted of cases with any immature follicles; group II consisted of cases without any immature follicles (Table IIGo). The two groups were compared by several different criteria. No significant differences were observed in the age of patients, days of ovarian stimulation prior to IVF retrieval, number of recovered oocytes or pregnancy rate. The serum E2 level on day of HCG injections was significantly lower (P < 0.05) in group I (1799.9 ± 167.6) than in Group II (2246.6 ± 153.2).


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Table II. Summary of data for patients with and without immature follicles recovered from follicular fluid aspirates
 
There was a significant difference in the aetiology of infertility. Male factor was a significantly higher cause of infertility in group I than in group II, (P = 0.0318). There was no significant difference in the diagnostic categories of endometriosis, unexplained or tubal factors of infertility between the two patient groups.

The techniques used in the study yielded 12 oocytes (eight of them in MII stage) (from 0–2 per case; mean 0.23 ± 0.07), which were missed during oocyte recovery after IVF procedure.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Wu et al. reported the discovery of a new source of immature human follicles by using follicular fluid aspirated during oocyte retrieval in an IVF procedure (Wu et al., 1998Go) . In their study, fluid from 16 women from age 29–39 years was centrifuged and the resulting pellets were rinsed twice and examined under an inverted microscope. Between 20 and 150 pre-antral follicles were reportedly found in each follicular fluid sample (mean 51.8). Our study does not confirm that finding, but rather indicates that IVF follicular fluid is not an efficient source of immature follicles, at least utilizing oocyte retrieval methods in our centre. We believe that our experimental data are not only accurate, but also supported by other studies and theoretical considerations.

Roy and Treacy isolated intact pre-antral follicles from human ovaries by digesting ovarian tissue with collagenase and DNase (Roy and Treacy, 1993Go). From 25% of a human ovary they collected a consistent yield of 120–160 class 1 follicles with an inner diameter <90 µm (consistent with classification of Gougeon, 1986Go) and 15–30 class 2 follicles ranging from 90–220 µm in diameter. The yield varied with the age of the women and almost no pre-antral follicles could be harvested when ovarian pieces came from women >40 years old. Thomas and Shaw reported even fewer pre-antral follicles collected with the same enzymatic digestion (Thomas and Shaw, 1995Go). Approximately 5% of one ovary was treated and yielded a mean of 20 pre-antral follicles per tissue sample in age group 26–30 years, 17.5 follicles in ages 31–35 years, 2.8 for ages 36–40 years, and 0.2 for ages 40+ years.

During IVF retrieval, it is doubtful that >0.1% of ovarian tissue from both ovaries is typically aspirated. A simple calculation provides the approximate density of pre-antral follicles in both ovaries of one patient. Based on the data of Roy and Treacy, 1080–1520 pre-antral follicles between 90 and 220 µm could theoretically be obtained by the enzymatic digestion of two whole ovaries (Roy and Treacy, 1993Go). Thus, only 1.0–1.5 pre-antral follicles would be obtained in 0.1% of ovarian tissue. Likewise, using the data of Thomas and Shaw, a total of 112–800 pre-antral follicles could be expected from the enzymatic digestion of two human ovaries (Thomas and Shaw, 1995Go). Using this calculated density only 0.1–0.8 pre-antral follicles would be obtained in 0.1% of ovarian tissue. Our data revealed 0.26 ± 0.14 pre-antral follicles per IVF retrieval, which is consistent with the pre-antral follicular density proposed by Thomas and Shaw.

The aetiology of infertility was significantly related to whether immature follicles were obtained. In group I, in which an immature follicle was recovered from patients, the major cause of infertility was male factor (83%). Group II also contained a number of male factor patients, though significantly lower than in group I. But group II also included patients with advanced maternal age (19%) and endometriosis (24%). While the incidence of advanced maternal age (>39 years of age) and female factor infertilities were not statistically increased in group II, the data clearly indicate a trend of follicles being more readily recovered in young, fertile females. This observation is supported by the fact that male factor infertility, as opposed to any form of female infertility, is more commonly seen as the primary aetiology in those patients whose follicles were recovered than those without any recovered follicles (Table IIGo).

No follicles were obtained from a patient with advanced maternal age, which could be explained by decreased follicular density in the ovarian tissue. Lass et al. developed a method of assessing follicular density in ovarian biopsies in women undergoing infertility evaluation (Lass et al., 1997Go). Their results indicate that infertile women >=35 years of age have a decreased ovarian reserve, with a median 8 follicles/mm. The majority of the follicles were primordial (88%) with very few primary (8%) and secondary (4%). Women >35 years of age had only a third of the concentration of follicles of younger women. Vital et al. have also reported decreased numbers of primordial, primary and secondary follicles in infertility patients with premature ovarian failure (Vital et al., 2000Go). Our data, supported by these studies, indicates a low recovery of immature follicles from IVF patients in general, but particularly in patients of advanced maternal age.

Most of the follicles in the adult human ovary are unilaminar primordial or primary follicles 30–50 µm (Hovatta et al., 1996Go) and reside in the ovarian cortex. We expected to see a higher number of primordial and primary follicles in ovarian fluid aspirates. The minimal yield of this type of follicles, 0.63 ± 0.27 per fluid, could be highly dependent on the location of the aspiration needle placement in the ovarian cortex. Since the aspiration needle usually only penetrates the cortex from one to three times per ovary, several antral follicles would be aspirated through the same needle penetration. This correlates to studies of ovarian tissue biopsy, where the pieces of tissue obtained are bigger, but may not be fully informative in some cases. In fact, the low follicular density in many ovarian biopsies may simply be due to the fact that the particular ovarian slice cultured was void of follicles (Abir et al., 2001Go).

Our results are supported by multiple studies. Several investigators have analysed sources of human oocytes from unstimulated IVF cycles (Trounson et al., 1994Go; Russell et al., 1997Go; Thornton et al., 1998Go) and from excised ovaries (Cha et al., 1991Go) by multiple penetration of the ovarian cortex by the aspiration needle. Lastly, Enien et al. analysed 63 follicular aspirates with purification of the granulosa cells by Ficoll density centrifugation and employing immunomagnetic beads (Enien et al., 1998Go). None of these studies reported the recovery of immature follicles.

Based on our data, IVF follicular fluid cannot be considered a reliable or an abundant source of immature follicles. Immature follicles were found in only 25.9% of cases (mean 0.88 ± 0.31 follicles per aspiration). Pre-antral follicles, which are the most valuable source for possible maturation, were found in just 9.3% of patients (0.63 ± 0.27/patient, range 0–7) and primordial or primary in 16.6% of patients (0.26 ± 0.14/patient, range 0–10). The most effective method we found for primordial or primary follicle recovery was Ficoll–Paque density gradient (94% of recovered follicles). We did not find filtration significantly different from the two other techniques for recovering pre-antral follicles, however, this method seems to be useful based on its cost efficacy, time saving and ease of use. The presence of any immature follicles varied from case to case and appeared to depend on the cause of infertility. It seems likely that age is also a factor, since no patient with advanced maternal age yielded immature follicles. Lastly, given the low number of follicles recovered, it is possible that a factor is the chance passing of the needle through a follicle. In any event, the data indicate that aspirated IVF follicular fluid is not an abundant source of immature follicles.


    Notes
 
4 To whom correspondence should be addressed at: Division of Urology, University of Utah School of Medicine,50 North Medical Drive, Salt Lake City, UT 84132, USA. E-mail: dcarrell{at}med.utah.edu Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on January 7, 2002; accepted on March 1, 2002.





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