The interaction of parameters of male and female fertility in couples with previously unexplained infertility*†

FERTILITY AND STERILITY

Vol. 54, No.5, November 1990

Printed on acid-free paper in U.S.A.

Copyright© 1990 The American Fertility Society

The interaction of parameters of male and female fertility in couples with previously unexplained infertility*t Bruce C. Dunphy, M.D.:j: Tin-Chiu Li, Ph.D. Iona C. Macleod, M.B., Ch.B.

Christopher L. R. Barratt, Ph.D. Elizabeth A. Lenton, Ph.D. Ian D. Cooke, F.R.C.O.G.

Harris Birthright Research Centre for Reproductive Medicine, Jessop Hospital for Women, Sheffield, United Kingdom

A prospective study was undertaken of the relationship between semen variables, serial endocrine and follicular ultrasound measurements in one complete menstrual cycle, and the treatmentindependent chance of conception for couples who were referred with unexplained infertility. For the 91 couples studied, the mean length of infertility was 70 months. A 100% follow-up rate was achieved. When a stepwise analysis was performed examining semen variables and ultrasonographic and endocrine variables, the mean ratio of salivary progesterone/plasma estradiol between days + 1 and +3, with respect to the luteinizing hormone surge and the Grade 2 motile sperm density, were the only variables that achieved the 5% level of significance. There was a highly significant relationship between the product of these two variables and the chance of conception. This study clearly demonstrates an interaction between parameters of male and female fertility. Fertil Steril54:824, 1990

Several studies have demonstrated that women can conceive despite their partners having a markedly "abnormal" semen profile. These include studies of fertile 1 and infertile2 •3 populations. This lack of absolute predictive power of both conventional semen analysis and other methods of assessing male fertility has been interpreted as a defect in these investigations. However, the reduced sensitivity and specificity of these investigations could at least in part be explained by either gross or subtle variation in the fertility of female partners. 3 The aim of this study was to determine whether this point could be demonstrated by identifying an interaction between parameters of male and female fertility in couples with previously unexplained infertility. Received October 11, 1989; revised and accepted June 28, 1990. * Supported by a Harris Birthright grant, Royal College of Obstetricians and Gynaecologists, London, United Kingdom. t Presented at XIII World Congress of Fertility and Sterility, Marrakesh, Morocco, October 1989. :j: Reprint requests and present address: Bruce C. Dunphy, M.D., The Department of Obstetrics and Gynaecology, Jessop H~spital for Women, Leavygreave Road, Sheffield S37RE, United Kingdom.

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Interaction of male and female fertility

MATERIALS AND METHODS Subject Recruitment and Follow-up

A prospective study was undertaken of subjects who had been investigated in other departments and were referred to us between July 1984 and April1987 with a diagnosis of unexplained infertility. Unexplained infertility was defined as couples in whom there was at least 12 months of involuntary infertility before investigation, a female partner who had regular spontaneous ovulation (a midluteal serum progesterone [P] of > 18 nmol/L), 4 and a normal pelvis at both hysterosalpingography and laparoscopy, except for the presence of minor endometriosis without concomitant pelvic adhesions, 5•6 a normal postcoital test, 7 a negative mixed antiglobulin reaction (MAR) test, 3 and a male partner with an apparently normal semen profile7 when analyzed in another laboratory. Couples were investigated according to the World Health Organization (WHO) Protocol for the Management of the Infertile Couple. 4 Those couples with a WHO clinical diagnosis4 of sexual dysfunction in either partner or ejaculatory dysfunction in the male partner were excluded from the study, as the relaFertility and Sterility

tive contribution of male and female variables in these couples would have been impossible to estimate. All female partners had been investigated by laparoscopy before referral; however, where there was a clinical suspicion of pelvic pathology or a laparoscopy had not been performed for some time, this procedure was repeated. A follow-up program was instituted by questionnaire and telephone. If a subject indicated that a conception had occurred and the pregnancy had not been managed at the Jessop Hospital for Women, the clinician responsible for the management of the pregnancy was contacted for confirmation of pregnancy details. If a subject indicated that a spontaneous abortion had occurred, then this was only accepted as a conception if reliable evidence3 was available. Semen Analysis

As a participating center in WHO multicenter studies, semen analysis has been performed in our laboratory from July 1984, as outlined in the revised WHO manual, 8 with the following modifications: pH was measured using a pH meter, viability was examined using trypan blue dye, and the immunoglobulin G MAR test with coated erythrocytes (Ortho Diagnostic Systems, Raritan, NJ) was used to detect sperm-bound antibodies. Sperm motility was reported as the percentage of spermatozoa exhibiting each of the following grades: rapid and linear progressive motility (Grade 1), slow or sluggish linear or nonlinear motility (Grade 2), nonprogressive motility (Grade 3), and immotile (Grade 4). All semen analyses were performed by one technician, as we have demonstrated that there may be considerable variation in the assessment of spermatozoal motility between technicians when examining the same semen sample, but that the intraobserver variability is very low. 9 Further Investigation of the Female Partner

Each female partner recruited to the study was investigated for one complete randomly selected menstrual cycle. Table 1 documents the timing of each observation in an "ideal" 28-day cycle. Follicular Ultrasonography

Serial follicular ultrasound examinations were commenced on day 8 of the cycle, or earlier if the subjects normal menstrual cycle was <28 days. Ultrasound observations were performed on alternate days until the follicular diameter reached 10 mm, Vol. 54, No.5, November 1990

Table 1

Timing of Investigations on the Female Partner Day of cycle

Investigation

8

10

12

13

14

15

16

17

Ultrasonography X Plasma gonadotropins X PlasmaE2 X Salivary P

X

X

X

X

X

X

X

X X

X X X

X X X

X X X

X X X

X X X

X X X

18 to 28

X

after which examinations were performed on a daily basis until 48 hours after follicular rupture had occurred. Ovulation was diagnosed by the rapid shrinking or disappearance of the follicle. The appearance of fluid in the Pouch of Douglas or an endometrial ovulation ring were considered to be supportive evidence only. Ultrasonography was performed on subjects who had a full bladder, initially using a Sonoline 3000 mechanical sector scanner with a 3 mHz output (Siemens, Erlanger, West Germany), and subsequently using a Kretz Combison 320 scanner (Kretz Technik, Zipf, Austria). We assessed the intra- and interobserver coefficients of variation (CVs) during follicular ultrasonography, and found that no CV exceeded 10%. 10 Plasma Endocrine Estimations

On those days that ultrasonography was performed, 10 mL of venous blood was taken for analysis. The plasma concentration of gonadotropins was determined by a combination of conventional radioimmunoassay (RIA) 11 and radiometric assay. Plasma estradiol (E 2 ) was measured in a direct RIA using commercially available reagents. 11 Salivary P Estimations

Each patient expectorated 3 mL of saliva into a small collecting tube at approximately the same time each day. The collection was started from approximately 2 days before the luteinizing hormone (LH) peak and continued until the 1st day of menstruation. The samples were stored in a box in the patients domestic freezer and returned to the laboratories within 1 week of the end of the collection. Progesterone concentration in the saliva was determined by a simple direct steroid assay employing reagents available from Seranti Research Laboratories (Seranti Research Ltd., St. Albans, United Kingdom). 12 These comprised P 125 -I-tyramine glucuronide (code number ER 205) and an antibody against P glucuronide 11 fJ hemisuccinate (code number ER 125), which was covalently Dunphy et a!.

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bound to a solid phase support. Solutions of P, initially dissolved in ethanol and then progressively diluted in assay buffer (phosphate/gelatin, pH 7.0), were used as standard solutions (covering a range of 10 to 2,600 pmol/L). Quality controls were prepared from male saliva by the addition of P (high quality control [QC], 732 pmol/L; low QC, 380 pmol/L). Before analysis, all samples of saliva were frozen, then thawed and centrifuged at 900 X g to precipitate mucins. Reagent volumes employed in the assay were: antibody, 50 J,LL; 1251-P, 50 J,LL; saliva or standard solution, 150 J,LL; giving a total volume of 250 J.LL. Assay tubes were incubated overnight at room temperature, then centrifuged at 1,400 X g for 20 minutes. The supernatants were decanted and the precipitate (containing the bound fraction) was counted in a gamma-counter for 1 minute. For reasons of economy, all unknown saliva determinations were performed in singlet only. Sensitivity was 20 pmol/L and interassay CVs of high and low QCs were 8.0% and 7.5%, respectively. 12 Statistics

Frequency distributions and descriptive statistics were performed using the Statistical Package for Social Sciences (SPSSX). 13 The contribution of variables and combinations of variables to the time to conception was assessed using a modification of Cox's Regression Model 14 of life table analysis, as implemented by the Statistical Analysis System (SAS), 15 from which xi and P were derived. Relative risks (RRs) were calculated as outlined by Polansky and Lamb. 16 These are estimated multiplication factors for the conception incidence rate for unit change for the parameters of interest. V ariables were analyzed as continuous variables unless otherwise stated. For the purposes of life table analysis, follow-up was calculated from the outset of investigation at our clinic. RESULTS

Of the 105 subjects referred for the investigation of unexplained infertility, 14 (13%) were excluded from the study as they were found on further investigation in our department to have significant female pelvic pathology, or sexual or ejaculatory dysfunction. Eighteen subjects had minor endometriosis without the presence of pelvic adhesions. These subjects were retained in the study as we have previously demonstrated that minor endometriosis doe's not significantly alter the fertility outcome for women attending our infertility clinic. 4 •5 826

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The mean female age was 32.5 years (range, 22 to 43; SD, 3.9). The mean male age was 35.3 years (range, 26 to 64, SD, 5.9). The mean length of involuntary infertility before investigation was 70 months (range, 15 to 172, SD, 33.9). Follow-up was terminated for 24 (25.3%) couples at the point of therapeutic intervention, all couples receiving in vitro fertilization. These couples were retained in the analysis as censored observations. Follow-up ranged from 1 to 32 months with a mean of 16 months (SD, 9.6), and 100% follow-up was achieved. Of the 91 women remaining in the study, 15 conceived independent of treatment to either partner. When semen variables were entered into a stepwise analysis, there was a significant relationship between the Grade 2 motile sperm density3 and the chance of conception (xi = 7.39, P = 0.0066, RR = 1.69 per 50 X 106/mL). Five women were demonstrated to have a luteinized unruptured follicle, and one of these women subsequently conceived. The luteal phase length was not significantly related to the chance of conception, with a mean value of 13.1 days (SD, 1.6; range, 8 to 15). The following variables did not achieve the 5% level of significance: the day of the cycle on which the LH surge occurred; the minimum, mean, and maximum LH values; the area under the LH curve; the skewness and kurtosis of the LH surge; the minimum, maximum, and mean follicular diameter; and the rate of follicular growth. Progesterone:E2 ratios were also examined as there is a suggestion that an optimal ratio is required for endometrial development. 17 When a stepwise analysis was performed on ultrasonographic and endocrine variables from the female partner, the mean ratio of salivary P:plasma E 2 between days LH + 1 and LH + 3 was the only variable that achieved the 5% level of significance (xi = 8.02, P = 0.0046, RR = 1.81/U). This ratio ranged from 0.07 to 4.58 U (mean, 0.95, SD, 0.89). When the latter ratio and the Grade 2 motile sperm density were analyzed together, the predictive value of each variable was increased: ratio (x12 = 8.52, P = 0.0035 RR = 2.0/U), Grade 2 motile sperm density xi= 7.64, P = 0.0057, RR = 1.77 per 50 X 106/mL). The product of the female endocrine ratio and the Grade 2 motile sperm density was entered into life table analysis. There was a highly significant relationship between the latter variable and the chance of conception (xi = 14.09, P = 0.0002 RR = 1.95/50 U). The latter variable ranged from 0.26to 203 U (mean, 24.59; SD, 38.11). Finally, to determine whether there was a statistical interaction (synergism) between the female enFertility and Sterility

docrine ratio and the Grade 2 motile sperm density, the product of these two variables was forced into an analysis with them. The product was significantly related to the chance of conception (xi = 8.25, P = 0.0041). However, the Grade 2 motile sperm density and the female endocrine ratio were not, confirming an interaction (synergism). DISCUSSION

This is the first study to clearly demonstrate an interaction (synergism) between male and female parameters of fertility in couples with previously unexplained infertility. Further predictive information was obtained when these variables were combined, which was peculiar to the combination of these variables. There are two important implications of this finding. First, it is unlikely that any investigation of one sex will produce absolute predictive power. This conclusion could explain at least a portion ofthe loss of predictive power of current methods of assessing male fertility. Second, future research might focus on providing an index ofthe fertility of couples and not exclusively on one sex. We have previously demonstrated and explained the predictive value of the Grade 2 motile sperm density. 3 Previous authors have demonstrated a significant relationship between a ratio of E 2 :P and fertility outcome. 18 This relationship might be related to the quality and or maturity of the follicle and oocyte, tubal motility, and endometrial development. 17 We are currently examining the relationship between P:E 2 ratios and data obtained from the morphometric examination of endometrial biopsies. 19 The predictive power of the couple index would appear to be immense, with a marked increase in the chance of conception with each increment of 50 U. Furthermore, the distribution ofthis variable would appear to be skewed to the left, in keeping with the subfertility of the population being studied. Therefore, a further study is required to examine its distribution in a fertile population. In summary, we have clearly demonstrated that there is interaction between parameters of male and female fertility in couples with previously unexplained infertility. Further research is required to determine whether such a relationship exists in a fertile population. Acknowledgment. We are grateful to Richard Kay, Ph.D., of S4effield Statistical Services Ltd., Sheffield, United Kingdom, for his statistical advice and for his help in the preparation of the manuscript. Vol. 54, No.5, November 1990

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