Kari Ratsula, M.D.t Ilpo Huhtaniemi, M.D.t. Taru Vuorento, B.M.t Outi Hovatta, M.D.t Henri Kurunmiiki, M.D.t

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1 FERTILITY AND STERILITY Cpyright" 1990 The American Fertility Sciety Printed n acid-free paper in U.S.A. Measurements f salivary prgesterne thrughut the menstrual cycle in wmen suffering frm unexplained infertility reveal high frequency f luteal phase defects* Taru Vurent, B.M.t Outi Hvatta, M.D.t Henri Kurunmiiki, M.D.t Kari Ratsula, M.D.t Ilp Huhtaniemi, M.D.t University f Turku, Turku, and Infertility Clinic f the Finnish Ppulatin and Family Welfare Federatin, Vaestliitt, Helsinki, Finland Frty-five wmen suffering frm unexplained infertility cllected saliva fr prgesterne (P) measurements every mrning during 1 t 3 cmplete cycles. A ttal f 27 cycles analyzed were unstimulated, 19 were stimulated by clmiphene citrate (CC), and 7 with CC, human menpausal gnadtrpin (hmg), and human chrinic gnadtrpin (hcg). Cmpared with reference prfiles f salivary P frm 27 nrmal cycles, the length and magnitude f P secretin were significantly lwer in the infertility grup. Luteal P cncentratins were nrmalized during CC stimulatin and significantly elevated during the CC-hMG-hCG stimulatin. The ther abnrmalities fund in the salivary P prfiles f these patients included prevulatry P peaks, interruptin f P secretin during luteal phase, and high P level at the beginning f menstruatin. The results suggest that patients with prlnged unexplained infertility represent a hetergenus ppulatin with cmmn luteal phase defects. The disturbance is effectively crrected with treatments stimulating gnadtrpin secretin. Fertil Steril54:211, 1990 Salivary prgesterne (P) measurements prvide a practical and reliable means f fllwing crpus luteum (CL) functin during the menstrual cycle. Salivary P cncentratins crrelate well with thse f serum P and the ccurrence f vulatin, and the magnitude and length f luteal phase P secretin can be reliably evaluated. 1,2 The value f daily P measurements as a means f detecting lu- Received Nvember 14, 1989; revised and accepted AprilU, * Supprted by research cntracts frm The Finnish Life and Pensin Insurance Cmpanies and The Academy f Finland, and by grants frm the Sigrid Juselius Fundatin and the Turku University Fundatin, Turku, Finland. t Department f Physilgy, University f Turku. :j: Infertility Clinic f the Finnish Ppulatin and Family Welfare Federatin. Reprint requests: Ilp Huhtaniemi, M.D., Prfessr, Department f Physilgy, University f Turku, Kiinamyllynkatu 10, Turku, Finland. teal dysfunctin has s far nly been used fr small patient materials.3-6 In the present study, we have analyzed salivary P cncentratins during menstrual cycles in patients suffering frm unexplained infertility and related the findings with reference data frm nrmal menstrual cycles. 1 Amng infertility patients carefully examined, the incidence f unexplained infertility is apprximately 15%.7 Our hypthesis was that incidence f CL dysfunctin culd be mre frequent in these infertility patients cmpared with nrmal ppulatin. Decreased P secretin is the mst cnspicuus hrmnal finding in luteal phase defect (LPD).4,8,9 In infertility patients, vulatin is usually verified by measuring serum P cncentratin in ne t three samples taken during the midluteal phase. 8 This infrmatin may reveal the cclirrence f vulatin, but it des nt detect mre subtle abnrmal- Vurent et al. Salivary P in infertility 211

ities, e.g., in the prfile f luteal phase P secretin. Infrequent sampling is nt enugh fr accurate diagnsis f LPD, 10 especially if the length f P prductin is shrt.

2 ities, e.g., in the prfile f luteal phase P secretin. Infrequent sampling is nt enugh fr accurate diagnsis f LPD, 10 especially if the length f P prductin is shrt. Crpus luteum insufficiency is the cause f infertility in 3% t 10% f all the infertility cases,l1 and endmetrial bipsies have been the nly practical way f their diagnsis. 1O Hwever, sme pitfalls have been fund als in the evaluatin f endmetrial histlgy.11 Defects in the luteal phase ften ccur after abnrmal fllicular phase.6,8,12-16 Salivary P measurements thrughut the whle menstrual cycle are therefre imprtant because they may reveal abnrmalities als in the fllicular phase. In the present study, part f the infertility patients underwent hrmnal treatments, i.e., clmiphene citrate (CC), and/r human menpausal gnadtrpin (hmg), and/r human chrinic gnadtrpin (hcg). In these cycles, salivary P measurements were carried ut t assess their value in detecting the respnse f CL t the treatments. Subjects MATERIALS AND METHODS Frty-five wmen (age 28 t 43 years), suffering frm unexplained infertility fr 2 t 16 years, each cllected saliva samples during ne t three menstrual cycles. All the subjects were patients f The Infertility Clinic f The Finnish Ppulatin and Family Welfare Federatin (Helsinki, Finland). The cause f their infertility had remained unclear, despite careful investigatins including serum P (nrmal if >30 nml/l), luteinizing hrmne, prlactin, estradil, andrgens and sperm antibdy measurements, the investigatin f fllicle by ultrasund, pstcital test, bservatin f the cervical mucus, eliminatin fthe tubal factr, and laparscpy t find pssible endmetrisis. Als the male factr was excluded. The patients cllected saliva during a ttal f 53 cycles, 27 f which were unstimulated and 26 stimulated by hrmne(s). Nineteen f the latter cycles were stimulated by CC (50 mg tw times a day, n days 3 t 7 f the menstrual cycle), and 7 by the cmbinatin f CC (50 mg tw times a day n days 3 t 7 f the menstrual cycle), hmg (150 IV intramuscular [1M], n days 8 t 10 f the cycle), and hcg (10,000 IV 1M when the diameter fthe leading fllicle was at least 17 mm). As a cntrl grup, we used nrmal vulatry cycles f 27 healthy wmen, 24 f whm belnged t the reference ppulatin analyzed in ur earlier study.1 The cntrl grup cnsisted f 9 university students with a histry f regular menstrual cycles and n gyneclgical disrders, 15 patients f the infertility clinic (a male factr was fund t be the reasn fr infertility), and 3 healthy fertile-aged wmen wh have children. Measurement f Salivary P As described previusly in mre detai1,l 2-mL samples f saliva were cllected withut stimulatin at hme every mrning between 7:00 A.M. and 10:00 A.M. and stred in hme freezer (-17 t -20 C). All the samples were delivered at the same time t the labratry and kept there at -20 C until analyzed. Salivary P cncentratins were measured by a radiimmunassay (RIA) as described in detail earlier.1 The sensitivity f the RIA methd fr salivary P was 40 pml/l and the intra-assay and interassay cefficients f variatin were 10.3% and 15%, respectively.1 Statistical Analyses A menstrual salivary P prfile was cnstructed fr each patient using the daily P cncentratins f the cycle. Frm the prfile we calculated the length f fllicular and luteal phases as described befre.1,3 Luteal phase was designated t start n the day (day 0) that the salivary P level rse fr the first time at least 2 standard deviatins (SD) abve the mean P level f the previus days and stayed elevated fr nt <2 fllwing days. The spradic 1- day P peaks (>2 SD abve mean fllicular salivary P) that were bserved in sme wmen during the fllicular phase were excluded frm these calculatins. Starting frm day 0, the cumulative sum f daily salivary P cncentratins until the end f the cycle were measured, and a cumulative sum curve was pltted in crdinates where the day after day 0 was n the x-axis and the cumulative sum f salivary P n the y-axis. We then calculated the first day when at least 95% f the luteal phase P had been secreted and the cumulative P cncentratin (C95) fr that day. The time during which C95 was secreted (T95) was calculated in days. The length and magnitude f the luteal P secretin were incrprated int a diagram where the lgarithm f C95 was n the y-axis and T95 n the 212 Vurent et al. Salivary P in infertility Fertility and Sterility

3 4.0 ~ 3.5 ~ ~ , _ T95 (days) 1 cycle was anvulatry, i.e., withut elevatin f salivary P (Table 1). Thirty-three percent f the patients had P peaks in the fllicular phase (Table 2 and Fig. 2). Three f them had therwise nrmal P secretin. Only 7% f the 27 patients had a cmpletely nrmal P secretin prfile during the menstrual cycle (Table 2). The respective percentage in the reference ppulatin was 85% (P < 0.01), and prevulatry P peaks were the nly abnrmality identified in this grup. "' '" 0.Q '" I--I _+-+--I l T95 (days) Figure 1 The parameter pairs T95/lg C95 f the patients n the diagram where the reference area is indicated by the slid circle. Each pint represents ne cycle. (A), The clsed dts depict the unstimulated cycles f the infertility patients. Sixtysix percent fthe cycles are within the reference area. (B), The pen symbls represent the cycles treated with CC alne. The clsed triangles are the cycles treated with CC, hmg, and hcg. Eighty percent f the treated cycles are within the reference area. x-axis. Our previus data n nrmal menstrual cyclesl were used t cnstruct the reference area f the diagram (Fig. 1). The salivary P secretin parameters fthe unexplained infertility grups, with and withut stimulatin, were cmpared with the respective data f the reference grupl and the significance f differences was verified by unpaired tw-tailed Student's t-test. The prprtins f different abnrmalities in the grups were cmpared by Pearsn x 2 test. RESULTS The Unstimulated Cycles The parameters T95 and lg C95 f the 27 unstimulated cycles were significantly (P < 0.05) lwer than in the reference ppulatin (Table 1); the ther parameters measured were nrmal. In the diagram lg C95 versus T95, 66% f the infertility cycles were within the 95% reference area (Fig. la). In five patients, the length f P secretin was t shrt, in ne case it was t lng (17 days), and The Stimulated Cycles Nineteen cycles were stimulated by CC. The treatment nrmalized the parameters T95 and lg C95, which in the unstimulated cycles indicated a shrt and/r lw luteal salivary P prfile (Table 1). The peak luteal P cncentratin was significantly higher than in the cntrl and unstimulated cycles (P < 0.05). In the diagram lg C95 versus T95, 80% f the cycles were nw lcated in the reference area, and the rest f them were either abve the reference range r the duratin f P secretin was prlnged (Fig. IB). This 80% did nt differ significantly frm the respective prprtin (66%) fthe unstimulated cycles lcated in the nrmal area. In all f the CC-stimulated cycles, a significant P level elevatin was detected. Thirty-ne percent f the CC-stimulated cycles were nrmal if the prevulatry P peaks were cnsidered abnrmal (Table 2). Seven cycles were stimulated by CC, hmg, and hcg. The mean values f the lg C95 and the luteal peak salivary P cncentratin were significantly higher than the nrmal mean values (P < 0.01, Table 1). Tw f the cycles were within the reference area in the diagram lg C95/T95, and in the remaining 5, ttal P secretin was cnsiderably elevated (Fig. IB). All parameters were nrmal in tw f the seven cycles. Pregnancies Within a year after the saliva cllectins, 40% (n = 18) f the 45 patients became pregnant. Sme f the pregnancies began withut any stimulatin either spntaneusly (n = 2) r with intrauterine inseminatin (n = 1) r artificial inseminatin with a dnr (n = 1). The treated cnceptin cycles were as fllws (methd f inseminatin in parentheses): six were stimulated with CC, hmg, and hcg (intrauterine inseminatin, in vitr- fertilizatin [IVF), r direct intraperitneal inseminatin), five Vurent et al. Salivary P in infertility 213

Table 1 Lengths fthe Cycles and the Salivary P Parameters in the Cntrls and in the Unstimulated and Stimulated Cycles f the Patients Unexplained infertility Cntrls Unstimulated CC CC-hMG-hCG Length f

4 Table 1 Lengths fthe Cycles and the Salivary P Parameters in the Cntrls and in the Unstimulated and Stimulated Cycles f the Patients Unexplained infertility Cntrls Unstimulated CC CC-hMG-hCG Length f cycle (d) Fllicular phase (d) Luteal phase (d) T95 (d) LgC95 Luteal peak P (pml/l) Anvulatry cycles (%) N ± ± ± ± ± ± ± ± ± ± 0.48 b 3.22 ± 0.04b 350 ± ± ± ± ± ± ± 55 b ± ± ± ± ± 0.09 c 1,033 ± 162 C 7 Values are means ± SEM. b P<0.05. c P < 0.01 versus cntrls. were stimulated with CC (nly the stimulatin r intrauterine inseminatin), tw were stimulated with buserelin acetate, hmg, and hcg (IVF), and ne with CC and brmcriptine (nly the stimulatin). DISCUSSION A number f abnrmalities were detected in salivary P prfiles f the infertility patients studied, and a cnsiderable interindividual variatin amng the findings was typical. Hwever, the cmpiled mean values f each parameter tended t fall within the nrmal range because f the hetergeneus nature f the abnrmalities bserved (Table I). The unstimulated cycles f the wmen with unexplained infertility, as a grup, had significantly lwer mean cumulative cncentratins f luteal phase salivary P, and the duratin f P secretin was shrter. This suggests that LPD is cmmn in the grup studied and culd be the explanatin t infertility in many fthe cases. Accrding t earlier estimatins, almst half f the wmen having unexplained infertility may have lw P secretin during the luteal phase.8 Until nw, many f the LPDs may nt have been diagnsed because n practical means has been available fr thrugh mnitring f P secretin thrughut the cycle.7,8 In many studies, serum P levels have been nrmal even if clear signs f LPD were fund by endmetrial bipsy,6,17,18 which has been the mst reliable means f diagnsing LPD. Li et al.19 have recently studied the crrelatin between salivary P prfile and endmetrial develpment. They fund that in half f the cases f retarded endmetrial develpment, the salivary P prfile was nrmal, suggesting an abnrmal respnse f endmetrium t P. Therefre the endmetrial histlgy can be abnrmal even if the CL Table 2 Frequencies f Sme Abnrmalities Fund in the Cycles Unexplained infertility Cntrls Prevulatry P peaks 15 P secretin interrupted b 0 High P level in the beginning f the menstruatin d 0 Cycle cmpletely nrmal 85 Cycle nrmal if prevulatry P peaks cnsidered nrmal 100 N. 27 Frequencies are given in percents. b i.e., P declined temprarily under 100 pml/l fr 1 r 2 days during the luteal phase. c P < 0.05 versus cntrls. Unstimulated CC CC-hMG-hCG c ' 31 t 29 19' d i.e., P was ver 100 pml/l fr 3 days during the menstrual bleeding., P < 0.01 versus cntrls. t P < 0.05 versus unstimulated cycles. 214 Vurent et al. Salivary P in infertility Fertility and Sterility

700 600 :J'... 500 "0 E.e. 400 0.. 300 ~ D <!: "0 200 U) 100 0 0 5 10 15 20 25 DAY OF CYCLE Figure 2 Tw examples f abnrmal salivary P prfiles.

5 :J' "0 E.e ~ D <!: "0 200 U) DAY OF CYCLE Figure 2 Tw examples f abnrmal salivary P prfiles. One prfile (0--0) has a prevulatry P peak, the level increasing t 200 pml/l fr 1 day during the fllicular phase. In the same prfile, P secretin is interrupted during the luteal phase, P level declining under 50 pml/l fr 2 days. In the ther prfile (.--.) there is a sufficient rise in the luteal P level, but the length f P secretin is abnrmally shrt (7 days). Als, the whle cycle is shrt. functin is nrmal. Salivary P directly reflects the functin f CL itself. The mst frequent abnrmality f the unstimulated cycles was a shrt T95, i.e., the length f the luteal P secretin, which was fund in 38% f the cycles (P < 0.05). Stimulatin with CC r CChMG-hCG crrected mst f these abnrmalities because this parameter remained shrt in nly 11 % f the stimulated cycles. The fllicular phase was abnrmal in 33% f the unstimulated cycles, 19% being t lng and 14% t shrt. It has been reprted that a lng fllicular phase impairs fertility, particularly when assciated with a shrt luteal phase.14,15 In the present study, three f the fur cycles having an abnrmally lng fllicular phase als had an abnrmally shrt luteal phase (belw 11 days). The imprtance f this finding remains pen because there are als studies suggesting that in vulatry cycles with lng fllicular phase, the fllicular maturatin is delayed but adequate and des nt affect fertility.12,13 Nne f the stimulated cycles had a t lng fllicular phase. In CC-hMG-hCG stimulated cycles, n disturbances were fund in fllicular phase except a P peak in ne f the cycles. Fllicular P peaks were identified in 16% f the CC cycles (Table 2). The significance f the prevulatry P peaks in fertility is uncertain because such peaks were als fund in 15% fthe nrmal cycles. Sixty percent f all the spradic P peaks were bserved n day -3 (i.e., 3 days befre the significant rise f salivary P), hwever the mean (±SD) day f the spradic P peak was -6.6 ± 4.3, suggesting a large variatin. The frequency f the P peaks was duble in the unstimulated infertility cycles cmpared with the reference grup, suggesting that the P peaks may be assciated with infertility. Clmiphene citrate stimulatin crrected the rati t be cmparative with the nrmal cycles. The results suggest that the hrmne stimulatins used make the vulatin ccur earlier and lengthen the luteal phase, withut necessarily affecting the P secretin itself. Stimulatin with CC, hmg, and hcg was in this respect mre effective than CC alne, which is als supprted by the fact that mean lg C95 and the mean luteal peak P cncentratin during CC-hMG-hCG stimulatin were clearly higher than the cntrl mean values. Only the latter parameter was higher in the CC grup (Table 1). The cycles stimulated by CC had significantly (P < 0.05) less abnrmalities (31 % cmpletely nrmal) than the unstimulated cycles (7% cmpletely nrmal). Cnsidering the higher risk f side effects-hyperstimulatin syndrme and multiple pregnancies-f CC-hMG-hCG stimulatin, the CC treatment alne shuld pssibly be tried first.20 The pregnancy rates fund in ur study suggest that CC and CC-hMG-hCG bth are effective, althugh the stimulatin alne is enugh nly in a few cases. Hwever, ur results demnstrate that the luteal defect is s cmmn amng patients with unexplained infertility that sme kind f stimulatin treatment is indicated with them. In summary, ur salivary P measurements revealed that a luteal phase defect is cmmn in patients with unexplained infertility and may prvide an explanatin fr the prblem in a great prprtin f these patients. Salivary P measurements prvide a reliable methd fr diagnsing an bscure LPD. The luteal dysfunctin is crrected by treatments stimulating gnadtrpin secretin in a large prprtin f the cases. The treatments increase the P secretin and balances the lengths f the phases f the cycle. REFERENCES 1. Vurent T, Hvatta 0, Lahti A, Huhtaniemi I: Daily measurements f salivary prgesterne reveal a high rate f anvulatin in healthy students. Scand J Clin Lab Invest 49: 395, Adekunle AO, Kim JB, Cllins WP, Whitehead MI: Prgesterne in saliva as an index f varian fynctin. Int J Gynaecl Obstet 28:45, Walker RF, Wilsn DW, Truran PL, Read GF, Richards Vurent et ai. Salivary P in infertility 215

6 G, Walker SM, Riad-Fahmy D: Characterizatin f prfiles f salivary prgesterne cncentratins during the luteal phase f fertile and subfertile wmen. J Endcrinl 104: 441, Lentn EA, Landgren BM, Sextn L: Nrmal variatin in the length f the luteal phase f the menstrual cycle: identificatin f the shrt luteal phase. Br J Obstet Gynaecl 91: 685, Zrn JR, McDnugh PG, Nessman C, Janssens Y, Cedard L: Salivary prgesterne as an index f the luteal functin. Fertil Steril41:248, Khan-Dawd FS, Cai H-Y, Dawd MY: Luteal phase salivary prgesterne cncentratins in vulatin-induced cycles. Fertil Steril49:611, Mghissi KS, Wallach EE: Unexplained infertility. Fertil Steril 39:5, Burslem RW, Osbrn JC: Unexplained infertility. Br Med J 292:576, Aksel S: Spradic and recurrent luteal phase defects in cyclic wmen: cmparisn with nrmal cycles. Fertil Steril33: 372, Jnes GS: The luteal phase defect. Fertil Steril27:351, Shepard MK, Senturia YD: Cmparisn f serum prgesterne and endmetrial bipsy fr cnfirmatin f vulatin and evaluatin f luteal functin. Fertil Steril 28:541, Brm TJ, Matthews CD, Cke ID, Ralph MM, Seamark RF, Cx LW: Endcrine prfiles and fertility status fhu- man menstrual cycles f varying fllicular phase length. Fertil Steril36:194, Aksel S: Hrmnal characteristics f lng cycles in fertile wmen. Fertil Steril 36:521, Smith SK, Lentn EA, Cke ID: Plasma gnadtrphin and varian sterid cncentratins in wmen with menstrual cycles with a shrt luteal phase. J Reprd Fertil 75: 363, Rdriguez-Rigau LJ, Sheni PN, Smith KD, Steinberger E: The relatinship between the lengths f the fllicular and luteal phases f the menstrual cycle and the fertility ptential f the female. Fertil Steril 39:856, Ayers JWT, Birenbaum DL, Menn KMJ: Luteal phase dysfunctin in endmetrisis: elevated prgesterne levels in peripheral and varian veins during the fllicular phase. Fertil Steril47:925, Balasch J, Creus M, Vanrell JA: Luteal functin after delayed vulatin. Fertil Steril45:342, Cheesman KL, Cheesman SD, Chattertn RT, Jr, Chen MR: Alteratins in prgesterne metablism and luteal functin in infertile wmen with endmetrisis. Fertil Steril 40:590, Li TC, Lentn EA, Dckery P, Rgers A W, Cke ID: The relatin between daily salivary prgesterne prfile and endmetrial develpment in the luteal phase ffertile and infertile wmen. Br J Obstet Gynaecl 96:445, Pepperell RJ: A ratinal apprach t vulatin inductin. Fertil Steril40:1, Vurent et al. Saliuary P in infertility Fertility and Sterility

INFERTILITY DIAGNOSIS

INFERTILITY DIAGNOSIS INFERTILITY Infertility is the inability t cnceive after 12 mnths f unprtected intercurse. There are multiple causes f infertility and a systematic way t evaluate the cnditin. Let s lk at sme f the causes.